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Proceedings of the 41st annual scientific session of the american rheumatism association a section of the arthritis foundation december 911 1976 miami beach florida.

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105
Proceedings of the 41st Annual Scientific Session of the
AMERICAN RHEUMATISM ASSOCIATION
A Section of The Arthritis Foundation
December 9-11, 1976
Miami Beach, Florida
Abstracts of Papers Presented
Chondromalacia Patellae as a Separate Entity
Peter J . A bernethy. Paul R . Townsend, Robert M . Rose, and Eric L. Radin, Edinburgh, Scotland and Boston, Massachusetts
Chondromalacia can be defined as cartilage fibrillation that
does not progress to exposure and eventual eburnation of the underlying bone. It is often asymptomatic. Most authors feel it is a precursor of osteoarthrosis. It has been suggested that the progression of
cartilage lesions is related to the state of its underlying subchondral
bone.
In order to investigate the relationship between cartilage fibrillation, its progression, and the relative state of the underlying subchondral bone, 24 patellas obtained at autopsy were examined. Meachim’s India ink technique was used to grade their cartilage damage by
the Collins scale. The quantitative density of the underlying subchondral bone of various regions of the patella was measured. This
parameter has been shown to be directly related to the relative stiffness
of the bone specimens. Previous studies of the contact surface area of
the patello-femoral joint at various angles of flexion had shown that
the periphery is basically non-weight-bearing, the “odd (extreme medial) facet” is in contact above about 135” of flexion, and the central
medial and lateral facets are in contact through much of knee motion.
It was found that the earliest cartilage fibrillation occurred along the
unloaded periphery and habitually loaded central medial areas. These
changes were age-related and tended to be nonprogressive. The bone
underlying these areas was relatively osteopenic. The cartilage changes
on the habitually loaded lateral facet tended to occur in older individuals and tended to be progressive. The bone in this area was denser
and stiffer than the bone of the medial facet. An “odd facet” was
observed at 30% of specimens. There was no correlation between the
changes on this facet and those of the rest of the patella. When the
“odd facet” was present it was crossed by a synovial fringe 40% of the
time.
I t was concluded that nonprogressive chondromalacic
changes occur in both loaded and unloaded areas of the patella.
Erosion of cartilage to bear bone (Collins grade IV) was unlikely over
relatively osteopenic bone and was more likely over stiffened bone.
The distribution of stiffness in the underlying bone related well to both
the degree and rate of cartilage damage. The observations were consistent with the hypothesis that the symptoms of chondromalacia
patellae could well be due to synovial irritation rather than cartilage
fibrillation. This series clearly needs to be enlarged.
Antibodies to an Acidic Nuclear Antigen in Connective Tissue Diseases
M . Akizuki, M . J . Boehm-Truitt, S. S. Kassan. A . D. Steinberg. and T . M.Chused, Bethesda. Maryland
The soluble extract of mammalian cell nuclei contains several
distinct antigens against which patients with connective tissue diseases
produce antibodies. Determination of the specificity of such antibodies is important because of increasing evidence of correlations
between antibody specificity and the clinical characteristics of patients
with autoimmune disease. At present the major problem in the identification of antibodies to soluble nuclear antigens is the lack of purified
preparations of the individual antigens, which would allow specific
and quantitative titration of antibody.
In this study an acidic nuclear protein, distinct from the Sm
and RNP antigens and tentatively named the Ha antigen, was purified
approximately I ,000-fold and a specific, sensitive assay for detecting
antibodies was developed.
The antigen was purified from calf thymus nuclear extract by
ammonium sulfate fractionation (60-8070 saturation), DEAE Sephadex chromatography (eluted between 0.25 and 0.38 M NaCl in 0.05
M Tris buffer, pH 7.4), and affinity chromatography utilizing DEAE
purified IgG from a patient with a high titer of antibody to the antigen
covalently bound to Sepharose. This preparation was iodinated by the
Arthritis and Rheumatism, Vol. 20, No. 1 (January-February 1977)
method of Bolton and Hunter and finally purified by gel filtration
through a Sephadex GI00 column. On polyacrylamide gels the antigen is homogeneous, has an apparent molecular weight of approximately 53,000, and is composed of two components of approximately
33,000 and 20.000 daltons. It is heat labile, resistant to nucleases, but
destroyed by trypsin treatment.
Antibody to Ha antigen in 20 pl serum was assayed by the
ammonium sulfate method, by using 5 ng of I-Ha. Binding greater
than 24%, two standard deviations above normal, was observed in 73%
(32/44) of patients with Sjogren’s syndrome (SS) without rheumatoid
arthritis (RA). and in 85% (17/20) of patients with SS and systemic
lupus erythematosus (SLE). Only 6% (1/18) of patients with the
combination of SS + RA, and only 3% (2/58) of patients with SLE
had anti-Ha, results suggesting that they represent distinct populations. Patients with other connective tissue diseases and healthy
subjects did not have anti-Ha antibody.
This rapid and accurate method of measuring antibody to Ha
may be useful in the diagnosis of SS and in the recognition of subpopulations of patients with the sicca complex.
ARA ABSTRACTS
106
Circulating Immune Complexes in Acute Uveitis
Brian S. Andrews, Valerie Petts. Jill Mclntosh, and Ronald Penny, La Jolla, California and Sydney, Australia
The aim of the study was to determine if circulating immune
complexes (IC) were present in 39 consecutive referred patients presenting with acute uveitis. Of 15 patients (38%) who were HLA-B27
positive (827 (+), 12 were males (80%). 12 (80%) had radiologic
evidence of sacroiliitis, but systemic disease was uncommon. In the 24
HLA-B27 (B27 (-) negative patients (62%), 16 were females (67%).
sacroiliitis was present in I (4%). and systemic disease, including
polyarthritis, was common.
IC were sought in serum by the following techniques: a )
complement (C) activation (plasma C3, C4 levels, C 3 fragments in
plasma, Clq precipitins, and anticomplementary activity in serum); b)
rheumatoid factor ( R F ) in serum; c ) monoclonal R F precipitins; d )
presence of cryoglobulins; e ) inhibition of EA (IgG) rosette formation
by blockage of Fc receptors on human peripheral blood lymphocytes
(PBL); f) increased numbers of PBL with surface membrane bound Ig
(Smlg); g ) spontaneous neutrophil chemotactic activity in plasma. The
mean f 2 SD for normals in all of the above tests was determined and
values outside this range were regarded a s abnormal.
Thirty-six percent of uveitis patients had evidence of C activation, in particular anticomplementary activity in serum (P < 0.005);
14% had cryoglobulins; 33% inhibited EA rosette formation (P <
0.05); 32% had increased numbers of Smlg positive PBL; and 47%
showed neutrophil chemotactic plasma activity (P < O.Ol), while R F
precipitins were uniformly absent. Anticomplementary activity was
more common in the 827 (-)than in the 827 (+)group (30% vs loo/,),
as was neutrophil chemotactic activity (63% vs 21%). One o r more
tests were positive in 60% of B27 (+) and 96% of B27 (-) patients,
while 2 or more were positive in 27% of B27 (+) and 67% of 827 (-)
patients.
Thus we have demonstrated that when a variety of techniques
is employed, circulating IC are frequently detected in patients with
acute uveitis. In addition, acute uveitis in the B27 (-) group is more
frequently associated with circulating IC, a fact suggesting that B27
( - ) patients differ from the 827 (+) patients either in their immune
response to antigen o r in their handling of circulating IC.
HLA-A28 in Patients with B27-Associated Rheumatic Diseases
Frank C. A rnett. Jr., Bernice Z . Schacter, Marc C. Hochberg, and Wilma B. Bias, Baltimore, Maryland
Tissue typing for HLA-B27 has become a widely applied test
in various rheumatic disorders. However the association of B27 with
the spondylitic group of diseases remains an unexplained biologic
phenomenon, and other factors determined by the HLA region may
play a role in disease susceptibility. HLA-A2 has been reported to be
increased in B27-positive patients with disease; however the high prevalence of this antigen in control populations (50%) makes statistical
confirmation difficult. HLA-A28 is a cross-reactive antigen with A2
and may be technically difficult to define if A2 is also present. Our
studies indicate that A28 may be increased in white B27-positive
patients with rheumatic disease, but not in white B27-positive normal
individuals.
Thirty-eight unrelated, white 627-positive patients were studied with careful attention directed toward A locus antigens. Thirteen
had ankylosing spondylitis (AS), 21 Reiter’s syndrome (RS), and 4
juvenile chronic polyarthritis (JCP). The A28/B27 phenotype was
found in 9 (23.7%). Of 38 age, sex, and race matched controls, 4
(10.5%) had 827, 2 (5.3%) had A28, but none had A28/B27. In 210
normal, healthy, unrelated white controls, 29 (13.6%) had B27,
11(5.2%) had A28, but only I (0.5%) possessed the A28/B27 phenotype. The difference between the incidence of A28/B27 in the B27positive disease (9/38 o r 23.7%) and B27-positive control (1/29 o r
3.4%) groups is significant (P 5 0.05). In addition, calculations based
on actual gene frequencies would predict an A28/B27 phenotypic
frequency of 3.8%, irrespective of whether their alleles were on the
same or different haplotypes. The A28/B27 phenotype was found in
31% of AS, 51% of JCP. and only 10% of RS patients.
The A2 antigen was present in 53% of B27-positive disease
patients, 41% of matched controls, 37% of B27-positive normal controls, and 45% of the 210 white population controls (nonsignificant
differences). In addition to these Caucasian patients, studies of 11
blacks (RS 7, AS 3, and JCP 1) revealed 827 in 10 (90%). None had
A28.
These results suggest that A28 may be an additional marker
for 827 disease or may be a contributing factor in pathogenesis.
Further determinations of multiple gene products determined by the
HLA region in disease states are needed.
HLA-B7 Cross-ReactiveAntigens in B27-Negative Reiter’s Syndrome
Frank C. Arnett, Jr.. Bernice Z . Schacter, Marc C . Hochberg. and Wilma B. Bias, Baltimore, Maryland
Reiter’s syndrome (RS) is strongly associated with HLA-B27,
yet there is good evidence for infectious agents in pathogenesis. The
“molecular mimicry” and “cellular surface receptor site” theories of
histocompatibility antigen and infectious agent interaction to produce
disease have been demonstrated in animals and proposed as possible
explanations for HLA and disease associations in man. The finding of
HLA antigens cross-reactive t o B27 could lend indirect support for
these concepts when the HLA antigen is itself involved and make the
“linked immune response gene” hypothesis less likely.
Thirty-one patients with RS have been studied with respect to
clinical features and HLA phenotypes. A clinical diagnosis of RS was
made prior to tissue typing in all. The complete triad of nonspecific
urethritis (NSU), conjunctivitis, and typical arthritis was present in 13
(42%). Eighteen (58%) had incomplete Reiter’s syndrome (IRS). Of
these, 8 (26%) had NSU and arthritis (IRS-I) and the remaining 10
(32%) had arthritis alone (IRS-11). Twenty-four were white, 6 black,
and I tri-racial. Only 2 were female, and both had the complete triad.
HLA-B27 was present in 25 (80%), including all 10 with IRS11. Six patients did not have 827; 3 had complete RS and 3 IRS-I
(including the I black patient without B27). Of these 6 B27-negative
patients, 5 had HLA-B locus antigens cross-reactive with B27 (HLAB7 cross-reactive group). In the complete RS group, 1 had B7, I had
BW22, and 1 had no 8 7 cross-reactive antigens. In the IRS-I group, 2
had BW22 and the 1 black patient had an unidentified B7 crossreactive antigen.
There were no significant clinical differences between the 827
positive and negative groups, except for the absence of uveitis and
radiographic sacroiliitis in the latter. Two B27 negative families have
been studied, and a 10-year-old daughter of the black patient has
unilateral sacroiliac pain, heel pain, and an elevated sedimentation
rate, but as yet normal X rays. She does possess the same B7 crossreactive antigen.
Therefore further studies of HLA phenotypes and genotypes
in R S should direct special attention t o B27-negative patients.
ARA ABSTRACTS
107
A Recently Recognized Familial Arthropathy in Children
Balu H . A threya, H . Ralph Schumacher. and Peter Vanace, A tlantic City, New Jersey, and Philadelphia, Pennsylvania
Three siblings with a recently recognized form of familial
a r t ~ o p a t h yhave been studied clinically and by light and electron
microscopy of the synovium. T w o of these children were girls and I
was a boy. All of them had symmetric swelling, effusion, and limited
motion of large joints starting within the first 6 months of life. All of
them also had flexion deformity of the PIP joint of one of the fingers
from early infancy and resembled the patients reported briefly by
Jacobs ef al (Pediatrics 57:696, 1976).
One of these children was studied extensively at age 3. CBC,
urinalysis, sedimentation rate, rheumatoid factor, and antinuclear factor were all normal or negative. The synovial fluid from one knee was
straw-colored, had good viscosity, formed a good m u c h clot, and
showed 300 WBC/m13 with predominantly mononuclear cells. Only
soft tissue swelling was seen o n roentgenograms of the knee and wrist
joints.
Needle synovial biopsy was done o n this girl. Synovium was
available from synovectomy in the 2 siblings. All of them showed
synovial hyperplasia, necrotic synovial villi alternating with normal
villi, deposition of eosinophilic and PAS-positive material in the subsynovial layer, and large numbers of multinucleated giant cells with
pink cytoplasm. There was n o vasculitis and there were only occasional lymphocytes o r polymorphonuclear leukocytes. Alcian blue and
amyloid stains were negative
Electron microscopic study of synovium in I patients revealed many connective tissue cells with dilated rough endoplasmic
reticulum. Some phagocytic cells contained degenerated collagen in
vacuoles. Acid phosphatase was normally distributed and was prominent in the giant cell lysosomes. Giant cells also had profuse rough
endoplasmic reticulum. Ruthenium red stains identified normal
amounts of mucopolysaccharide. There was granular and fibrin-like
interstitial material but no amyloid.
This symmetric noninflammatory arthropathy now reported
in 2 families appears to have a characteristic histologic appearance of
synovium that allows its identification as distinct from other joint
diseases.
Kinetics of Amyloid Protein SAA in Casein-Induced Amyloidosis
Merrill D. Benson. Morton A . Scheinberg, Tsuranobu Shirahama, Edgar S . Cathcart, and Martha Skinner, Boston. Massachusetts
and Indianapolis, Indiana
Secondary amyloid protein (AA) is derived from a circulating
alpha globin (SAA) which is elevated in chronic diseases such as
rheumatoid arthritis. SAA is also elevated in acute diseases, including
bacterial and viral infections, and in cancer, in which n o association
with secondary amyloid is known. T o study the relationship between
SAA production and amyloid deposition and to determine factors
involved in the cause of secondary amyloid in chronic inflammatory
diseases, the murine model of casein-induced amyloidosis was used.
Amyloid was induced in CBA/J mice given daily casein injections for 28 days. SAA levels were measured throughout the course by
radioimmunoassay (RIA), by using antiserum to protein AA. SAA
levels were also monitored in casein-treated A/J mice, bovine serum
albumin (BSA) treated CBA/J and A/J mice, and casein-treated nude
(athymic) mice. SAA levels were reported in microliter equivalents of
a serum standard.
SAA levels rose within 3 hours after a single casein injection
and peaked by I2 hours (I350 & 350 units vs normal 7 f 2 units). SAA
remained elevated throughout the preamyloid and amyloid phases,
but dropped rapidly after cessation of casein injections. N o significant
difference was found between the SAA response of CBA/J mice t o
injections of casein and A/J mice, a strain relatively resistant t o the
induction of amyloid. Also, treatment with BSA, which has a low
potential for inducing amyloid, gave high levels of SAA. Athymic mice
were shown to produce levels of SAA as high as those produced by
CBA/J mice (1860 & 401 units 18 hours after a single casein injection).
These data show that T lymphocytes are not necessary for the
production of SAA. Although the production of SAA may be necessary for the formation of secondary amyloid. other factors must be
involved in the deposition of the fibril protein. These factors may be
important in the degradation of SAA by the reticuloendothelial system,
and certain substances, such as casein, may interfere with this process
so as to accelerate the formation of amyloid. Similar mechanisms may
be involved in the production of secondary amyloid in chronic inflammatory diseases, such as rheumatoid arthritis, ankylosing spondylitis, and granulomatous colitis.
In Vitro Culture of Articular Cartilage Stimulates Collagen Synthesis and Maintains the Differentiated Collagen Phenotype
Paul D. Benya and Marcel E. Nimni. Los Angeles. California
Thin slices of articular cartilage from rabbit hip and shoulder
were incubated in a 10% CO,, 90% air environment in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum and 1%
(v/v) penicillin-streptomycin. Duplicate dishes were cultured for 0, 1,
2, 3, and 5 weeks and fed every third day. Radioactive collagen was
synthesized during the last 24 hours of culture by incubating the slices
in media supplemented with tritiated proline (50 pCi/ml), B-aminoproprionitrile (25 mM), and ascorbate (100 pg/ml). The medium and
slices were lyophilized together and extracted at 4" with 1 M NaCI, 50
mM Tris, pH 7.5. The extracted collagen was treated with pepsin and
purified. The collagen types were fractionated by SDS-polyacrylamide
gel electrophoresis on 5% gels t o give discrete peaks identified by cyanogen bromide peptide analysis as: Type Ill. X (a new collagen
chain), a1 (11). and a2.
In the first 2 weeks of culture, collagen synthesis increased
approximately 5-fold; by the third week, 20-fold. N o significant
changes were detected in the number of viable cells per milligram of
cartilage. In the first 24 hours of culture the cartilage slices synthesized
1.0% Type 111, 3.2% X. 95% Type I I , and 0.6% a2. At 5 weeks cells
synthesized 1.7% Type 111,0.6% X,87.8 Type 11, and 2%a2. Thus the
differentiated collagen phenotype of these cells remained relatively
unchanged during long-term culture, in contrast t o what we have
previously observed for subcultured chondrocytes.
This finding emphasizes the importance of some as yet undefined part of the cell environment for maintaining the proper phenotype. The behavior of these chondrocytes t o organ culture should
provide a phyisiologic model t o elucidate further the factors that
control the synthesis and turnover of macromolecules in normal and
diseased cartilage.
ARA ABSTRACTS
108
Auranofin: An Oral Chrysotherapeutic Agent for the Treatment of Rheumatoid Arthritis
Frans-Erik Bergliif: Kerstin Bergliif: and Donald T. Waltz, Ostersund, Sweden and Philadelphia, Pennsylvania
This study was undertaken to evaluate the safety and clinical
efficacy of auranofin (SK&F D-39162) (2,1,4,6-tetra-O-acetyl-l-thioP-D-glUCOpyranOSat0-S) (triethylphosphine) gold, in the treatment of
rheumatoid arthritis (RA). Eight patients with established R A by the
ARA criteria were treated with auranofin at an oral dose of 3.0 mg,
b.i.d. for 3 months. Oral absorption of auranofin was evidenced by the
blood and urine gold levels. The mean blood gold level of the 8
patients the first week of treatment was 0.26pg/ml, which increased to
0.70 pg/ml at week I2 of treatment. Improvement in the biochemical,
immunologic, and objective/subjective clinical parameters were seen
in the 5th-7th week and continued throughout the study. Single episodes of auranofin-related diarrhea (1- o r 2-day duration) were seen in
3 patients. A fourth patient withdrew from auranofin treatment (week
10)after four episodes of drug-related diarrhea (1 t o 4 days duration).
The efficacy of auranofin treatment was evident by alterations of the
biochemical parameters toward normal levels as evidenced by the
following observations: increases in albumin and albumin/globulin
ratios; decreases in alpha-2-globulin, rheumatoid factor titers, sedimentation rate, and in the level of immunoglobulin G. Clinically,
effects were evident with auranofin treatment by the reduction of the
number of painful and swollen joints, knee and finger joint size, and
morning stiffness, by a decrease in walking time, and by an increase in
grip strength. The data are summarized in the table.
~~
Parameters (units)
Painful joints (no.)
Swollen joints (no.)
Knee joint size (cm)
Finger joint size (mm)
Morning stiffness (Hrs)
Walking time (Sec)
Grip strength (kg/cm*)
Clinical Mean Values
Pre-Drug
Week 12
32
39
38
74
4
52
0.2 I
8
14
36
68
1
33
0.29
No. RA Patients
Responding/Treated
7/7
8/8
8/8
7/8
8/8
6/8
7/8
Reflex Neurovascular Dystrophy in Childhood
Bram Bernstein. James Kent. Berne Singsen, Karen Koster King, Helen Kornreich, and Virgil Hanson. Los Angeles, California
Reflex neurovascular dystrophy ( R N D ) has been reported
only in adults, usually as a complication of trauma o r surgery. During
the past 7 years we have observed 20 instances of R N D in 19 children
who ranged in age from 9 to 15 years (median: 12 years). There were
15 girls and 4 boys. A recognizable traumatic event preceded the onset
of symptoms by 2 to 24 months in 9 of 20 cases. The duration of illness
before diagnosis ranged from 1 week to 24 months (median: I month),
and previous diagnoses included collagen-vascular diseases, gout, tendinitis, spinal cord tumor, and other neurologic diseases. Previous
treatments included casting, corticosteroids, other antiinflammatory
agents, antibiotics, acupuncture, and narcotic analgesics, but symptoms persisted in all cases. Twelve children had ankle and foot involvement, 4 had knee involvement, and 4 had the hand-shoulder
syndrome. All complained of severe pain, and there was exquisite
tenderness of the involved area in 18 of 20 cases. Changes at the involved
site included swelling in 16 of 20, decreased temperature in 15, skin
mottling o r discoloration in 8, decrease in pulses in 5 , and asymmetry
of perspiration in 2 cases. The CBC, creatine phosphokinase, antinuclear antibody. and rheumatoid factor tests were negative. In 19 of 20
cases the erythrocyte sedimentation rate was normal. Radiographs
showed diffuse demineralization of the involved site in 8 of 19. Seventeen cases were hospitalized (mean duration: 22 days) and 3 were
treated as outpatients. All children received physical therapy. Emphasis was placed o n mobilization and weight bearing, in spite of pain. It
was strongly suggested that improvement would follow participation
in this program. Fifteen children underwent psychologic evaluation
and treatment. They were found t o be accepting of responsibility
beyond their years, to have difficulty in expressing anger, a n d to show
little anxiety about their illness or in its outcome. There was a high
frequency of long-standing family conflict; the illness appeared t o
allow the child to avoid this. At the time of discharge 5 of 19 were well,
1 I showed marked improvement, and 3 showed moderate improvement.
R N D occurs in children; it should be considered in the child
with an exquisitely tender and painful extremity. Vigorous physical
therapy, in spite of pain, appears effective. The high frequency of
family conflict suggests that psychologic intervention is also important.
Formation of Single-Strand Regions in Native DNA by Lupus Sera
Ronald N . Berzofsky. Carole A . Dorsch, and Mary Betty Stevens. Baltimore, Maryland
Although antibodies t o multiple nuclear antigens have been
described in several connective tissue diseases, antibodies specific for
native DNA have become the hallmark of SLE. In an attempt to
determine the nature of the association of native DNA and antinative
DNA antibodies, we observed that sera from patients with SLE were
capable of generating single-strand regions in highly purified preparations of native T 7 DNA under conditions normally employed for
binding assays. That this observation might be due to the perturbation
of the double helix by the binding of antibody was further investigated.
The formation of single-strand regions was observed in the
following manner. Native T7 DNA, after incubation with the appropriate serum or serum factor, was subsequently incubated with the
single-strand specific endonuclease from Pseudornonas Bal 31, and the
ARA ABSTRACTS
109
reaction products were analyzed by velocity sedimentation through
alkaline sucrose gradients. Although native T7 D N A is resistant to the
Bal endonuclease, the combined action of SLE serum and the Bal
enzyme results in a dramatic decrease in the sedimentation of the
DNA. Surprisingly, the incubation of T7 DNA and serum alone also
caused a decrease in the sedimentation of the DNA, but never as
dramatic an effect as seen with a combined digest. This latter observation suggests the presence of intrinsic serum nucleases capable of
degrading native D N A .
To determine the role of antibody to DNA in the synergistic
effect of lupus serum and Bal enzyme, an IgG fraction of lupus serum
still possessing DNA-binding activity was tested in the above assay.
N o shift in the sedimentation of DNA was observed with this fraction
either alone o r with the Bal enzyme. Furthermore, normal serum
possessing no detectable D N A binding activity, but with significant
levels o f serum nucleases, was capable of further decreasing the sedimentation of D N A in conjunction with the Bal enzyme. These observations suggest that intrinsic serum nucleases are responsible for most
of the single-strand regions produced by serum and not antibody
binding. In support is the finding that a lupus serum with high DNA
binding activity but with n o detectable serum nuclease, as well as
normal sera lacking nuclease, was unable t o shift the sedimentation of
DNA.
The role and effect of serum nucleases on the measurement of
anti-DNA antibodies are under investigation. The mechanism of binding of antibody to D N A remains t o be determined.
Antineuronal Activity in Systemic Lupus Erythematosus (SLE) Serum
Harry G. Bluestein, San Diego, California
SLE patients with central nervous system (CNS) disease have
increased titers of lymphocytotoxic antibodies that bind to human
brain. This fact suggests that antineuronal antibodies may be responsible for some C N S manifestations in SLE. SLE sera were tested on a
neuronal cell line, SK-N-SH, established from a human neuroblastoma, for complement-dependent neurocytotoxicity and for their effect on Veratridine-induced sodium fluxes across the cell membrane.
At a 1 . 2 dilution 25/32 S L E sera were cytotoxic (> 10%
5'Cr-release) t o SK-N-SH cells. The mean 6*Cr-releasefor the entire
group was 31.3%. Only 5/16 seropositive rheumatoid arthritis (RA)
sera were cytotoxic, mean cytotoxicity 8.2% (P < 0.001). Almost all
of the cytotoxic activity in SLE sera was recovered in the exclusion
peaks from Sephadex G-200. Anti-IgM (p-chain specific) completely
inhibited the killing. Absorption of S L E sera with SK-N-SH cells
removed all neurocytotoxicity; lymphocytotoxicity was reduced but
not eliminated. After absorption with a human lymphoblast cell
line (WIL-2). most of the neurocytoxicity remained. When compared
in I8 SLE sera. antineuronal and antilymphocyte cytotoxicity titers
were weakly correlated ( r = 0.5; P < 0.05).
Veratridine stimulates z2 N a incorporation in cells with elec-
trically excitable membranes. Cell line SK-N-SH responds to lo-' M
Veratridine with 4-fold increased uptake of 22 Na. Addition of 26 SLE
sera to the cultures during the period of Veratridine stimulation significantly reduced 22 Na. Addition of 26 SLE sera to the cultures during
the period of Veratridine stimulation significantly reduced 22 N a uptake to 56% of the amount of sodium incorporated with a standard
normal human serum. In contrast, the Veratridine response with sera
from 21 RA patients and 8 healthy volunteers was 92 and 99% of the
standard. In 13/26 of the SLE sera, the Veratridine response was less
than 50% of the standard. None of the RA or control sera were less
than 50%. Veratridine stimulation is not reduced by the addition of a
rabbit anti-human brain antiserum to the cultures. The suppressive
factor(s) in SLE sera are nondialyzable, precipitate with 50% (NH,)Z
SO,, and their activity is not blocked with antiimmunoglobulin serum.
SLE sera contain at least 2 factors that can interfere with
neuronal function: a ) IgM antibody cytotoxic t o neurons in the presence of complement, and b ) a nonantibody factor that blocks the
opening of sodium channels in the neuronal membrane. These observations support the hypothesis that attributes C N S manifestations of
S L E to circulating antineuronal factors.
Acute Monocytic Arthritis
Arthur D . Brawer and Edgar S . Carhcart, Boston, Massachusetts
Five patients presented with a triad of fever, skin rash. and
acute polyarthritis. The fever was low grade, continuous, and without
spikes: the skin rash was papular in 4 and urticaria1 in I ; the arthritis
involved small and large joints with knees and ankles affected in all
cases. One patient had transient renal involvement, but none received
medications prior to the illness. Acute rheumatic fever, viral hepatitis,
infectious mononucleosis, and bacteremia were excluded as possible
diagnoses. Synovial membrane biopsy was performed in Case No. 5
and skin biopsies in Cases 2, 3, and 5. All showed vasculitis with a
predominant mononuclear infiltrate in small blood vessel walls.
Multiple joint aspirations were performed in all cases. The
initial synovial fluid analyses revealed inflammatory effusions with
marked monocytosis and low percentages of polymorphonuclear cells
and lymphocytes (Table). Other synovial fluid findings included normal C3 (complement) levels and negative aerobic and anerobic cul-
tures.
Mononuclear cells from the synovial fluid of Case No. 1 were
purified on a Ficoll-Hypaque gradient and demonstrated 20% E (erythrocyte) rosettes, less than 5% E A C (erythrocyte, amboceptor, and
complement) rosettes, and 80% EA (erythrocytes coated with 1gG
amhoceptor) rosettes. This last finding, plus the observation that most
of the mononuclear cells were phagocytic for latex particles, confirmed
the monocytic origin.
In summary. we have attempted t o describe a new and distinct syndrome characterized hy synovial fluid monocytosis. I t appears
t o be caused by more than one etiologic agent and bears striking
similarities to certain viral arthritides. Although synovial fluid analyses have not been previously reported in systemic vasculitis. it also
deserves consideration as a form of acute hypersensitivity angiitis.
Synovial Fluid Analyses in Acute Monocytic Arthritis
Vol. Aspirated
Case No.
(ml)
WBC/mms
% Poly
1
25
15
15
4,400
12,600
I
13,000
2,300
3
4
18
5
0
2
3
4
5
10
1
,om
% Lymphs % Monocytes Mucin Test
14
4
0
15
35
83
92
82
80
65
Good
Fair
Fair
Fair
Fair
ARA ABSTRACTS
110
Antibody-Forming Cells in the Peripheral Blood of Patients with Active SLE
D. R . Budman, E. B. Merchant, B. Doft, M . E. Gershwin, E. Lizzio, J . P. Reeves, and A . D . Steinberg. Bethesda, Maryland
Thirty-seven patients with systemic lupus erythematosus
(SLE) and 18 normal controls were studied for spontaneous background IgM plaque forming cell number per 10' cells (PFC) to 5
specific chemical haptens (Dansyl, T N P , DNP, NIP, Sulfonate) in 2
separate series of experiments (Table). Active SLE patients had significantly more PFC in their peripheral blood t o these chemical determinants than did patients with inactive disease or controls (P< 0.005).
The greatest increase was in PFC, with specificity for the Dansyl and
T N P haptens. The increased PFC number correlated with depressed
serum C3, but not with other laboratory measures by Spearman rank
order analysis.
The finding of elevated numbers of spontaneous PFC to
chemical haptens supports the concept that in active SLE there is a
widespread increase in B-cell activity toward a variety of antigens
consistent with a generalized loss of immune regulation.
Group 2
Group 1
Patients
Active SLE
Inactive SLE
Controls
No.
PFC (log,, f SE)
No.
PFC (log,, f SE)
9
13
10
838 (2.92 f 0.11)
174 (2.24 f 0.23)
146 (2.16 f 0.13)
8
7
8
6140 (3.79 f 0.28)
216 (2.33 f 0.38)
184 (2.26 f 0.32)
Connective Tissue Activation: Stimulationof DN A and Glycosaminoglycan Synthesis by a Platelet Factor
C . W . Castor, M . E. Scott, J . C . Ritchie, a n d S . L. Whitney, Ann Arbor, Michigan
Earlier studies identified a platelet factor or factors that stimulated glycosaminoglycan (GAG) synthesis and glycolysis in human
synovial cell cultures. The connective tissue activating peptide (CTAP111) clearly differed from CTAP-I (of lymphocyte origin) in its lability
to thiols and in the added capacity t o stimulate G A G synthesis in
human dermal fibroblast cultures.
Current data indicate that CTAP-I11 is an acid-ethanol extractable basic protein with a molecular weight of 9,300 daltons containing one or more disulfide groups essential for its biologic activity.
CTAP-111 is mitogenic in that it stimulates incorporation of ['HI
methyl thymidine into DNA in cell cultures. CTAP-111 stimulated
DNA synthesis in human synovial cells, cartilage cells, skin and thyroid fibroblasts, and N R K (rat) cells, but not in continuous cultures of
human epithelial or lymphoid cells. Mitogenic activity was retarded by
concurrent addition of
M dithiothreitol, cysteine, and glutathione. but not by cystine, ascorbic acid, or cortisol.
CTAP-I11 stimulated increased hyaluronate synthesis and
glycolysis not only in human synovial cells and dermal fibroblasts, but
also in thyroid fibroblasts and cartilage cells. G A G stimulating capacity of CTAP-Ill was not blocked by cytosine arabinoside, but it was
inhibited by cycloheximide and actinomycin D. Both PGE, and
dibutyryl cyclic A M P potentiated the G A G stimulating action of
C T A P- I I I.
Platelets aggregated by bovine thrombin extruded CTAP-111
into the aqueous suspending medium. Much of the fibroblast mitogenic activity of serum appeared t o be derived from the clotting
process, because plasma supported less [9H]methyl thymidine incorporation into synovial cell DNA than did serum. The diverse
stimulatory properties of CTAP-III are appropriate for a mediator
designed to initiate the connective tissue component of the inflammatory response.
Systemic Lupus Erythematosus: Failure to Detect Endogenous Baboon Leukemia Virus or Mammalian Type C Virus Interspecies
Antigens in Tissue Extracts
Howard P. Charman and Paul E. Phillips, Frederick, Maryland and New York. New York
Recent studies suggest enhanced type C virus expression in
systemic lupus erythematosus (SLE). We have examined human SLE
tissues for evidence of mammalian type C virus interspecies antigens
using sensitive radioimmunoassays (RIAs). Eight patients with definite, and 1 each with probable or possible SLE (ages 25-58, duration
0.5-15 years, 3 active, 5 on prednisolone and/or lmmuran) provided
14 tissues including 5 placentas, 5 spleens, and 4 kidneys. Thirty
percent (w/v) extracts were homogenized, solubilized with Triton X100, clarified, and examined for mammalian type C virus p30 and an
antiserum prepared by sequentially immunizing with several purified
p30s. This RIA detects currently known isolated type C viruses with
equivalent sensitivity. These tissues were also tested for BaLV p30 in a
heterologous interspecies RIA employing 12s1-labeled BaLV p30 and
an antiserum to the highly related endogenous cat virus, RD-114. All
extracts but one were unreactive in both assays at levels of negativity
of less than 0.1 ng RD-I 14 p30 per mg of soluble protein. Despite the
incorporation of phenylemethanesulfonylfluoride in all buffers and
assay conditions restricting incubation at 37°C to 1-1.5 hours, one
kidney extract degraded labeled BaLV p30 to produce false positive
results in both assays. This is a well-known complication of RIAs.
Interestingly, although 3 of the tissues contained structures
resembling type C viruses, type C virus isolation studies on 6 tissues
were negative as previously reported. These results strongly suggest
that the structures observed in SLE are unrelated either to the
endogenous BaLV or to other known type C viruses. Thus we are
unable to confirm previous reports of type C virus p30 expression
in human SLE.
ARA ABSTRACTS
111
Serum Complement Values During Pregnancy in Patients with Systemic Lupus Erythematosus
Sherwood M . Chetlin, Thomas A . Medsger. Jr., and Steleanos N . Caritas. Pittsburgh. Pennsylvania, and Anthony G. DiBartolomeo.
Morgantown, West Virginia
It is well recognized that clinical manifestations of S L E may
exacerbate o r first appear during pregnancy or the early postpartum
period. Because immunologic alterations frequently antedate the expression of SLE, serum complement values in pregnant patients with
lupus may be an important guide to disease activity.
Serum complement determinations (C, and C, by radial immunodiffusion) were performed o n over 400 women in various stages
of uncomplicated pregnancies and during the postpartum period. The
mean C, level rose progressively from a baseline of 145 mg% in
nonpregnant women t o 194 mg% a t 39-40 weeks gestation (Table). At
6-8 weeks postpartum, the mean level had dropped to 172 mg%.
Eight patients with SLE were followed serologically and clinically through ten pregnancies. The mean pre-pregnancy serum c, level
was 121 mg%, which is significantly lower than for normals. C, increased during pregnancy but, in contrast to normals, peaked a t 32-36
weeks and declined thereafter. Four patients developed exacerbation
of SLE, and in all cases the symptoms were preceded by a fall in serum
complement. In 2 instances these changes occurred in the third trimester and in the remaining 2 during the postpartum period. Three of
these 4 patients had serum C, values less than 100 mg%. N o individuals developed renal involvement associated with pregnancy. Increased doses of corticosteroid therapy were employed only as clinically indicated and not as therapy for hypocomplementemia alone. c,
levels generally paralleled the C, levels.
It is concluded that mean serum complement levels are significantly reduced during the entire course of pregnancy in SLE patients
compared with normal women. A declining serum complement level is
often the harbinger of increased disease activity. As in nonpregnant
SLE patients, values of less than 100 mg% most commonly antedated
new symptoms.
Weeks of Gestation
Normals C, (mg%)
SLE C, (mg%)
Nonpregnant
8-12
16-20
24-28
32-36
39-40
6-8 Weeks Postpartum
145
121
162
108
I62
I24
I68
135
I82
I58
194
I39
I72
I37
Plasma Renin Activity (PRA) in Progressive Systemic Sclerosis (Scleroderma) with Malignant Hypertension
Sherwood M . Chetlin, Alvin P. Shapiro, Gerald P. Rodnan. Thomas A . Medsger, Jr.. Sanford F. Tolchin. Daniel E. Leb, and
Peter Cohen, Pittsburgh, Pennsylvania
The onset of malignant hypertension in progressive systemic
sclerosis is most often associated with rapidly advancing renal failure.
In 8 recently observed patients with renal involvement by scleroderma,
the peak levels of PRA by immunoassay ranged from 81.4 to 1000
ng/ml (angiotensin released in 3 hours incubation). These values are
16-200 times the upper limit of normal for this laboratory (5.0 ng/ml
in supine subjects). In 2 of these patients, PRA had been measured as
recently as 6 months before the onset of this complication and was
found to be normal. In 32 normotensive scleroderma patients, the
mean was 5.6 ng/ml (range: 0.8-16); in this group patients with the
CREST variant had significantly lower values.
Six of the 8 patients died despite hemodialysis in all and
bilateral nephrectomy in 3. The remaining 2 have survived 6-8 months
on dialysis after nephrectomy. T w o additional scleroderma patients
with hypertension and azotemia had P R A values elevated to only 31.7
and 54.1 ng/ml. Both have survived with intensive antihypertensive
therapy, and P R A has subsequently fallen t o normal levels.
All 8 patients were admitted with the onset of renal failure
between the months of October and March. It is possible that this
vascular complication of scleroderma may begin with a Raynaud’slike phenomenon in the kidney vessels followed by marked renin
release and resulting in accelerated arteriolar necrosis.
Musculoskeletal Manifestations of Bacterial Endocarditis (BE)
Melvin A . Churchill, Jr., Joseph E. Geraci, and Gene G. Hunder, Rochester, Minnesota
Although musculoskeletal (MS) symptoms are recognized in
BE, they have been poorly studied. To determine the frequency, character, and relationship of these manifestations t o the onset of disease,
we reviewed the histories of 192 patients with BE seen between 1965
and 1975. Eighty-four of the 192 patients (44%) exhibited MS findings.
Arthralgias were most common (34 cases) and tended t o
involve larger joints asymmetrically, but were occasionally monoarticular or migratory. Symmetric arthralgias were less common (8
cases), but in 2 instances the combination of shoulder and hip pain
and stiffness, anemia, and elevated ESR led to a diagnosis of polymyalgia rheumatica until blood cultures were obtained. Synovitis was
present in 26 patients and was monoarticular in 16 and polyarticular in
10 (usually 2 or 3 asymmetrically involved joints). The ankle and knee
joints were affected most often, but others-including
the sternoclavicular and acromioclavicular joints-were sometimes involved.
One patient had migratory polyarthritis for 3 months before the
onset of fever. The two synovial fluids aspirated were sterile. Low buck
pain occurred in 28 cases and was the presenting complaint in 15. The
pain was frequently unilateral, mimicking sacroiliitis or ruptured disk.
A disk space infection was found in 5. Diffuse myalgias (16 cases) were
usually related to the onset of high fever. In 10 other cases myalgias
were limited to the thigh o r calf and at times were severe and incapacitating. In 1 right thigh myalgia antedated fever and the diagnosis by 3 months. Less common findings included nail clubbing,
Achilles tendinitis, avascular necrosis (hip), and hypertrophic osteoarthropathy.
In 52 of the 84 patients MS complaints were the first o r
among the first manifestations of BE. In 20, M S complaints were the
only presenting symptom and preceded fever in 16 of these 20. Fortyeight of the 84 were tested for rheumatoid factor and 16 (33%) were
ARA ABSTRACTS
112
positive. Antinuclear antibodies were found in the serum of 5 of 25
patients tested. Only 1 of 35 patients tested had low serum complement (migratory polyarthritis and focal glomerulonephritis).
The relatively high incidence, frequent distinctive character,
and tendency to occur early in the course of the disease emphasize the
importance of MS manifestations in BE. An awareness of these various symptoms may aid in early diagnosis.
Sjogren's Syndrome Associated with the Lymphocyte-Defined Antigen HLA-DW3
Thomas M . Chused, Stuart S . Kassan, Haralampos M . Moutsopoulos, Gerhard Opelz, and Paul Terasaki, Bethesda, Maryland and
Los Angeles, California
An increased frequency of the HLA-BB antigen has been
observed in Sjogren's syndrome (SS) as well as in other organ-specific
autoimmune diseases. Regulation of the ability of the immune system
to respond t o antigens in the salivary and lacrimal glands might play a
role in triggering the immunologically mediated destruction of these
secretory tissues in SS. In mice it has been shown that the Ia antigens
are probably the product of immune response genes and that they
determine the intensity of the mixed lymphocyte reaction (MLR), i.e.,
they are lymphocyte-defined histocompatibility antigens. Although
immune response genes have not yet been described in man, by analogy with the mouse they might be associated with the human lymphocyte-defined antigens of the HLA-D locus. For this reason we
asked whether SS is more closely associated with HLA-B8 or with
HLA-DW3. the lymphocyte-defined antigen known to be in strong
linkage disequilibrium with HLA-B8.
Twenty-five unrelated patients with SS were typed for HLAA and HLA-B antigens by the microcytotoxicity method. HLA-DW3
typing was done by determining the M L R responsiveness of the subject's peripheral lymphocytes t o homozygous HLA-AI, HLA-B8, and
HLA-DW3 stimulator cells. Lymphocytes bearing the HLA-DW3
antigen gave a low response. The Haldane method was used for
statistical analysis.
Of 25 SS patients, 14 were B8+ (corrected P = (0.003) and 17
were DW3+ (P = 0.00009). Of 19 patients with SS in the absence of
another connective tissue disease, 14 were B8+ (corrected P =
0.00009) and 16 were DW3+ (P = 0.00001). Of 6 patients with SS and
rheumatoid arthritis, none was B8+ (P = 0.25) and 1 was DW3+ (P
= 0.84). By dividing the patients with SS alone into subgroups positive
and negative for the HLA-B8 o r HLA-D3 antigens and then testing
for association of SS with the other antigen, the interference from the
linkage disequilibrium can be removed. This technique showed that
the primary association of SS is with DW3 (P = 0.002) rather than
with B8 (P = 0.17)
These results suggest a) that there are two subpopulations of
SS patients, those with SS alone, 84% DW3+, and those with SS and
RA, 17% D W 3 f (compared to 24% in normals); and b ) that in the
first subpopulation the primary association of SS with DW3 is consistent with the involvement of an immune response gene in the pathogenesis of their disease.
A Prospective Clinical Analysis Comparing Polycentric and Geometric Knee Replacement with 2 to 4 Years Follow-Up
Andrea Cracchiolo. 111, Harlan C . Amstutz, and Gerald A . M . Finerman, Los Angeles, California
A prospective comprehensive analysis of 100 polycentric and
75 geometric replacements with a follow-up of 2 to 4 years has demonstrated the effectiveness and pitfalls of surface replacement arthroplasty in rheumatoid and osteoarthritic knees. Standard treatment
regimens and operative techniques were used in both groups. Both the
polycentric and geometric groups were similar, except that the polycentric patients were an average of 10 years younger and 58% had
rheumatoid arthritis while 59% of the geometrics had osteoarthritis.
Pain was the prime indication for surgery.
Data were collected by using six standardized proformas with
480 entries, which were transferred to a specially designed computer
program for in-depth analysis. A successful result was maintained in
187 knees, and pain relief showed the most improvement-being comparable in both groups-as was the improvement in stability, alignment, and motion. Functional motion increased because the preoperative flexion contracture was significantly reduced in most knees.
The improvement in walking and overall function was related to the
specific disease and involvement of other joints.
Results appear to correlate best with the type of disease, the
degree of preoperative knee pathology, and the presence of complications. Twenty-one knees (20 patients) failed. Of this group 80% were
osteoarthritics, more were men, and knees previously operated upon
failed more frequently. The most common causes of failure were
component loosening, ligamentous instability, and postoperative
varus alignment; sepsis occurred in only four knees.
Thus, following this intermediate evaluation period, we can
determine the results of treating various types of osteoarthritic and
rheumatoid knee deformities and establish more specific indications
for knee arthroplasty. Specific patient management procedures that
have improved our results are the use of antibiotics, anticoagulation,
proper wound drainage, and early physical therapy.
Prostaglandin Production by Synovial Cells: Stimulation by a Human Lymphocyte Factor
Jean-Michel Dayer, Dwight R. Robinson, and Stephen M . Krane, Boston, Massachusetts
Prostaglandins (PG ) have been implicated in the bone resorption of rheumatoid arthritis (RA) and some neoplasms. Culture media
from R A synovial explants as well as isolated adherent synovial cells
(ASC) produce large quantities of PGE, and collagenase. Dexamethasone (< IOnM) inhibits production of PGE, and collagenase >90%,
whereas indomethacin (IOpM), while inhibiting PGE,, stimulates collagenase production (PNAS 73:945, 1976). We reasoned that products
of lymphocytes, which we have previously shown to increase produc-
tion of collagenase, would also influence P G production.
Normal and R A peripheral blood lymphocytes separated by
Ficoll-Hypaque gradients were further cultured (after removing adherent monocytes) at 37°C in Dulbecco's medium with 10% fetal calf
serum at 2X l(P cells/ml for 3 or 4 days. The supernatants at dilutions
1.5 to 1:60 were incubated with ASC and PGE, and assayed by
radioimmunoassay (using specific antisera); the identity of PGE, was
confirmed by thin-layer chromatography. After 3-7 passages of ASC,
ARA ABSTRACTS
when levels of PGE, and collagenase in culture media had decreased
markedly (normalized to cell number), PGE, release was increased
5-10-fold by stimulating factor (SF) from lymphocytes (e.g.. from
(-)SF, -50 ng to (+)SF -500 ng/lCP cells/d). In the same cultures
collagenase (assayed by using labeled reconstituted collagen fibrils)
was increased 10-100-fold. In some cultures the levels of PGE, and
collagenase produced by ASC in the presence of SF approached those
found in early primary cultures of ASC. Indomethacin blocked the
effect of SF and PGE, production by ASC but potentiated the effect of
113
SF on collagenase production. A similar although not identical dose
response for both PGE, and collagenase was observed; enhancement
of production of SF by phytohemagglutinin was found and was evident at all dilutions tested. Similar apparent MW by gel filtration
(-8000-12,000) was obtained for the factor that stimulated both
PGE, and collagenase. PG and collagenase production by connective
tissue cells stimulated in vivo by lymphocytes could play a role in
connective tissue resorption of RA, as well as in other pathologic
processes such as neoplastic invasion.
Sympathetic or Reflex Footpad Swelling Due to Crystal-Induced Inflammation in the Opposite Foot
Charles W . Denko. Cleveland, Ohio
Although patterns of inflammatory joint swelling in rheumatic disorders are well documented, their immediate causes remain
obscure. Symmetric joint swelling is characteristic in patients with
rheumatoid arthritis, whereas asymmetric joint swelling is characteristic in patients with gout. Joint swelling due to deposition of various
calcium salts can mimic that of rheumatoid arthritis or that of gout.
Joints previously involved are sites of subsequent attacks.
Techniques were devised to investigate patterns of footpad
swelling in rats due to inflammation induced by several crystals known
to be human pathogens. Swelling (inflammation) was quantified by
measuring the foot diameter by a mechanical spring dial. Initial or
primary inflammation was induced in adult rats by injecting the footpad of a rear foot with fixed amounts of crystals in suspension.
Monosodium urate (MSU), xanthine (X).calcium oxalate, and hydroxyapatite were used to induce primary or initial bouts of inflammation
as well as subsequent episodes.
MSU and X induced swelling in the opposite uninjected
footpad as well as in the footpad for which they were primary irritants.
MSU injected initially into one foot induced marked swelling in the
opposite foot that had previously been inflamed by MSU, calcium
oxalate, or hydroxyapatite as the primary inflammogen. These effects
appear dose-related. Larger inocula produce larger swelling. When the
initial inflammation had not completely subsided, the injection of a
second inflammogen in the opposite foot was accompanied by a pronounced swelling of the initially inflammed foot. The foot receiving
the injection always had more swelling than the opposite uninjected
foot.
The sympathetic swelling is thought to be due to elaboration
of a humoral factor locally following reflex or overlapping nervous
stimuli initiated by the crystal injection to the opposite foot. This work
is an experimental model for reflex dystrophy.
Role of Calcium in the Phenotypic Expression of Rabbit Articular Chondrocytes in Culture
Kalindi Deshmukh, Indianapolis, Indiana
Rabbit articular cartilage synthesizes Type I I collagen, comprised ofa,(II) chains, in vivo or in vitro. Chondrocytes from thesame
tissue have the ability to produce Type I collagen [Za,(I).a,],which is
normally present in skin, bone, tendon, etc., or Type II collagen,
depending upon the culture system. The cells synthesize Type I collagen in monolayer cultures. Upon transfer from monolayer to suspension cultures, they synthesize Type I1 collagen in the medium with no
CaCI, and Type I collagen in the complete medium. Incubation of the
cells with ionophore A23 I87 increases the transmembranous flux of
Ca+, into the cells, intracellular levels of c-AMP, and causes the cells
to synthesize Type I collagen in the medium containing no CaCI,.
Similar results were obtained by addition of dibutyryl c-AMP to the
medium.
Each type ofcollagen was identified by incubation of the cells
with the medium containing aH-proline, ascorbic acid and @-aminopropionitrile; by pepsinization and salt-precipitation of labeled collagen; and by study of the composition of the chains and their CNBr
peptides with CM-cellulose chromatography.
The change in the synthesis of collagen by normal chondrocytes from Type I1 to Type I collagen in the presence of extracellular calcium is of great significance in light of the fact that osteoarthritic joints show signs of remodeling of subchondral bone and
ectopic calcification. Increase in the pyrophosphate concentration and
deposition of calcium phosphate in the synovial fluid and the extracellular matrix of the cartilage of osteoarthritic joints have been recently reported in the literature.
Crystal-Enhanced Membrane Permeability in Liposomes: Dissolved Urate Reverses the Protective Effect of Albumin
Charles A . DiSabatino. Mary G. Breitenstein, and Stephen E. Malawista. New Haven. Connecticut
Anionic liposomes have been used successfully to show the
disruptive effects of silica and of monosodium urate crystals on membranes that are not protected by protein in the ambient media. Thus
urate crystals within digestive vacuoles, when plasma-derived proteinaceous material has been enzymatically degraded, are thought to cause
perforation of the vacuolar membrane and rapid cell death, which may
in turn contribute to the violence of gouty inflammation. We have now
found that urate in solution can reverse the protective effect of protein
on liposomes challenged by crystals. We used anionic liposomes composed of lecithin, cholesterol, and dicetyl phosphate (7:1:2), with I T glucose trapped in their aqueous phase. In the representative experiment shown, quadruplicate aliquots of liposomes were incubated in a
standard salt solution at 37°C for 30, 60. and 90 minutes, with or
without additives. The mean percent release of their total radioactivity, f 1 SEM. is recorded in the table on the next page.
Silica crystals, 15 mg/ml (used rather than urate crystals
because the former do not dissolve) enhanced membrane permeability.
Human serum albumin, 0.5 mg/ml, significantly protected the liposomes from silica-induced leakage. Urate in solution, 8.4 mg%, reversed the protective effect of albumin. Soluble urate alone had no
effect on the permeability of liposomes (not shown in the table).
We conclude that urate in solution enhances membrane permeability by reversing the protective effect of albumin. Thus the
increased concentrations of dissolved urate characteristic of gout may
hasten crystal-associated disruption of the phagolysosomal membrane, and thereby contribute to gouty inflammation.
A R A ABSTRACTS
114
Mean Percent Release of Total Liposome Radioactivity
* 1 SEM
Time of Incubation (minutes)
No silica
Silica
Silica + albumin
Silica + albumin
+ urate
30
P
60
P
90
6.5% f 0.9
38.8% f 1.5
20.3% f 0.4
34.0% f I . I
<.001
<.01
<.001
9.4% f 0.6
41.2% f 1.3
22.0% f 0.6
36.1% f 1.2
<.001
<.Ol
< .01
43.4% f 1.4
P
12.2% f 0.8
24.6% f 0.4
38.4% f 1.0
<.001
<.01
< .oo I
Clinical Significance of Antibodies to Extractable Nuclear Antigen (ENA) in Systemic Lupus Erythematosus (SLE)
Carole A . Dorsch, Edward J . Feinglass, and Mary Betty Stevens, Baltimore, Maryland
Although antibodies to ENA, especially antiribonucleoprotein (RNP), have been described in other connective tissue disorders,
they have been associated primarily with the mixed connective tissue
disease (MCTD) syndrome. The significance of these antibodies in
patients with SLE, selected only on the basis of clinical diagnosis, has
not been fully established. Antibodies to ENA were determined in 81
patients with SLE by means of a combination of Ouchterlony immunodiffusion and hemagglutination of tanned, ENA-sensitized,
sheep red blood cells before and after ribonuclease digestion. Both
techniques were required to detect optimally the incidence and specificity of antibodies present. Patients' histories were independently
reviewed for clinical and laboratory features of SLE.
Forty-three of 81 sera (53%) were positive for anti-ENA: 6
had anti-RNP alone, 13 anti-Sm alone, and 4 both anti-RNP and anti-
Sm. Raynaud's phenomenon, myositis, and sicca symptoms were significantly more frequent in the 30 patients having anti-RNP antibodies. These features were absent in the 13 patients having anti-Sin
alone, with the exception of 1 patient who had Raynaud's phenomenon. Vasculitis and leukopenia occurred more often in anti-ENA
positive patients and were significantly associated with the presence of
anti-Sm. There was no difference between anti-ENA positive or negative groups with respect to renal disease, hypocomplementemia, and
the presence of antibody to denatured DNA. It thus appears that
antibodies to components of ENA are associated with specific disease
features in SLE. For anti-RNP these represent features common to
MCTD. However, unlike MCTD, renal involvement remains a prominent feature in anti-RNP positive SLE.
Periosteocytic Demineralization : A Phase of Bone Erosion in Rheumatoid Arthritis
Howard Duncan, C . H . E. Mathews, Donna Witzgall. Detroit, Michigan
At the level of cellular activity, bone erosion, juxtaarticular
demineralization, and joint destruction in rheumatoid arthritis have
not been wholly understood. In many rheumatoid patients, a paucity
of multinucleated osteoclasts suggests that these bone-resorbing cells
are not exclusively the cause of bone erosion. Most rheumatoid inflammatory cells lining bone surfaces are mononuclear and do not
share the staining features of osteoclasts. Some bone damage is
thought to depend upon an initial demineralization that exposes the
collagen matrix to the numerous enzymes synthesized by rheumatoid
synovitis. The means by which demineralization could occur have not
been clear.
In bone removed at surgery, we observed a periosteocytic
demineralization that exposes the underlying matrix. Fully mineralized serial sections, 5 p thick and appropriately stained, were used.
We observed areas close to the invading rheumatoid inflammation
where individual osteocyte domains demineralize beyond the matrix of
their osteocytic lacuna to a distance of 3 to 4 p . Coalescence with
adjacent demineralizing osteocytes occurs, and the closer to the inflammatory tissue, the more frequent is the confluence of adjacent cell
domains. Occasionally the osteocyte lacuna itself is enlarged, but the
initial process appears to be a demineralization rather than a simultaneous collagen disruption. There is subsequent invasion by inflammatory tissue, which replaces the collagen with a typical rheumatoid vascular inflammatory reaction.
A mechanism for bone erosion independent of osteoclasts is
identified in some areas of active rheumatoid joints removed at surgery. This occurrence is due to the amalgamation of the rheumatoid
inflammatory tissue.
(Supported by a grant from the Michigan Chapter of The
Arthritis Foundation)
Nonconstrained Metal to Plastic Total Elbow Arthroplasty in Rheumatoid Arthritis
Frederick C. Ewald. William H . Thomas, Richard D. Scott, Clement B. Sledge, and Robert Poss. Boston, Massachusetts
A nonconstrained metal to plastic total elbow replacement
fixed with methyl methacrylate has been developed, and clinical trials
are in progress. This paper is a report of the design concept and a
review of the first 50 replacements in patients with rheumatoid arthritis. The maximum follow-up is 2 years 6 months, and minimum
follow-up 6 months.
Results have been encouraging, with a significant increase in
postoperative flexion (X = 135" P <0.005) and pronation (X = 71"
P<0.0025) but no significant increase in postoperative extension or
supination. Pain-relief and functional improvement have been uniformaly excellent. Preoperative evaluation based on a predetermined
rating system was 37% of normal compared to 97% of normal postoperatively. There has been no loosening of components or avascular
necrosis of the olecranon process, but two procedures have failed. The
first failure was for peptococcus sepsis and the prosthesis was removed. The second failure was in a patient with a previous fascia1
arthroplasty with no ligaments or capsule, and this elbow had to be
converted to a hinge type of replacement. The two other serious
complications were caused by previously unrecognized severe cubitus
valgus in the rheumatoid elbow. Two elbows with cubitus valgus of
32" and 27' dislocated at time of surgery and stable reduction could
not be achieved. One elbow was salvaged by revision with a 15"
ARA ABSTRACTS
115
cubitus valgus fixation stem in the humeral component instead of the
standard 5" stem. Other complications have been two ulnar nerve
palsies (one transient, one permanent), two single dislocations with
stable reductions and no further problem, two small posterior skin
flap losses secondary to splint pressure, and one new bone formation.
Contraindications to the procedure include previous fascia1
arthroplasty or loss of collateral ligaments or capsule, previous sepsis,
or excessive loss of bony stock as in giant rheumatoid bone cysts.
There has been no experience with post-traumatic arthritis, and at this
writing the prosthesis is not recommended as salvage for these problems.
Early Clinical Features That Predict Outcome in Rheumatoid Arthritis (RA) :Results of a Prospective Study Using Multivariate
Analytic Techniques
Seth L. Feigenbaum, Stanley B. Kaplan, Alfonse T. Masi and Robert W. Chandler, Memphis, Tennessee
In an ongoing follow-up study of newly diagnosed RA in
younger adults, entry variables were analyzed to identify predictors of
the subsequent course in 52 patients closely observed for a mean
interval of 5 years. All satisfied ARA criteria for at least probable RA.
These cases included 42 females and 10 males, with 28 blacks and 24
whites. Outcome was classified initially by the presence (N=26) or
absence (N=26) of joint swelling during the last 2 years of follow-up.
I n addition, patients without swelling were subdivided into outcome
groups with (N=19) or without (N=7) pain or tenderness (P/T).
Similarly, patients with joint swelling were subdivided into those who
did (N=9) or did not (N= 17) develop bone erosions on hand films
during the course of follow-up.
Stepwise discriminant function analysis identified entry varia-
Entry Variables
Predicting Outcome
Total number ofjoints involved
Weight loss around onset (&3 mos)
Serum complement (C3, mg/dl)
Sex (male)
Rheumatoid factor positive
Lymphadenopathy on examination
Raynaud's phenomenon
bles which correlated significantly with outcome. Seven entry variables
correctly predicted the outcome into swelling and no swelling groups
in 48 (92%) of the 52 cases. These variables are ranked in the table
according to their predictive value, with mean results of percentage of
frequency at entry.
With few substitutions these variables correctly predicted
classification into the four subgroups with 80% accuracy. R F was
raised to second rank in that analysis because 8 of the 9 patients who
developed erosions had R F at entry (P<O.OOl). Thus the number of
initially involved joints is the best predictor of continued joint swelling
in RA, with R F predicting erosive disease. Multivariate analytic techniques are valuable in sorting out complex interactions and can offer
new insights into disease relationships.
Entry Results in Outcome Groups
Rank
Order
N o Swelling
Swelling
I
2
3
4
5
6
7
7.7
19%
183.1
30%
15%
4%
0%
18.1
58%
166.2
8%
50%
27%
15%
Univariate
Significance
P
P
P
P
P
P
<
<
0.001
0.05
NS
< 0.05
< 0.01
< 0.05
< 0.05
Cellular Infiltrates in Scleroderma Skin
Raul Neischmajer and Jerome S . Perlish, Philadelphia, Pennsylvania
. The purpose of this study was to estimate the incidence and
nature of cellular infiltrates i n scleroderma skin. Skin biopsies, including the subcutaneous tissue, were taken from 65 patients with
systemic scleroderma (SS) and 43 with localized scleroderma (LS).
Ten SS and 4 LS skin specimens were processed for electron microscopy (EM). 'H-thymidine labeling of vascular and extravascular cells
was estimated by in vitro autoradiography in 14 SS and 5 LS patients.
Most cell infiltrates were perivascular, although they were also noted
around nerves and skin appendages. By EM we identified blast cells
(T-blasts and plasmablasts), plasma cells, mature lymphocytes, macrophages, and fibroblasts with marked RER. T-blast cells were noted
within the wall of some capillaries. Cell contact between macrophages
and lymphocytes or plasma cells was frequently noted. Capillaries
showed marked alterations of endothelial cells (vacuolization. nuclear
degeneration, release of organelles to the lumen), whereas other endothelial cells showed a well-developed RER and numerous microfilaments. There was a marked increase in the 'H-thymidine labeling
index of endothelial cells i n SS (5.50+0.92; normal: 0. I6-+0.12;
P<0.0005), whereas increased labeling of extravascular cells was
noted in both SS and LS. This study suggests that B and T cells
participate in the tissue immune response in scleroderma skin and
probably play a role in the mechanism of the fibrovascular alterations.
The Actions of IgM Rheumatoid Factor (RF) on an IgG-Mediated Reverse Passive Cutaneous Arthus Reaction in the Rat
Michael Floyd and Joseph T. Tesar, Chicago, Illinois
Vasculitis and extraarticular complications of rheumatoid
arthritis appear more frequently in patients with high titers of RF. We
have directly investigated the actions of an RF on an experimental
immune vasculitis in rats. This rheumatoid factor was obtained from
the serum cryoglobulin of a patient with symptoms of purpura, arthralgia, and hepatospenomegaly. The cryoglobulin was IgM-IgG, and
after purification by multiple cold precipitations and washing at 4°C.
the IgM antiglobulin component was obtained by acid dissociation
and preparative ultracentrifugation in a 10-25% sucrose dentity
gradient in pH 2.8, 0.2 M tris HCL buffer, using an IEC ultracentrifuge with an SBI 10 rotor. The pure 19s fraction was obtained after
extensive dialysis with sterile phosphate-buffered saline, pH 7.4. I t was
concentrated to 4 mg/ml for use. Intravenous (IV) injection of the
antiglobulin induced a prompt hypocomplementemia in the rat only
ARA ABSTRACTS
116
after the administration of noncomplement fixing reduced and alkylated heat-aggregated human IgG. A complement-dependent vasculitis was induced by the intradermal injection of 20 pg of rabbit IgG
antibody to BSA (AB-BSA) followed 40 minutes later by IV BSA 10
mg/100 g. This lesion was characterized by a striking local dermal
leakage of simultaneously IV administered '*'Iodinated rat serum
albumin marker, and histologically by infiltration of dermal vessels
with neutrophil granulocytes. Decomplementation with cobra venom
factor totally inhibited the appearance of this vascular injury.
After incubation with R F the injected AB-BSA induced a
significantly greater leakage of isotope marker than that generated by
AB-BSA preincubated with normal human IgM (P<O.001). Intradermal antibody alone or after incubation with R F and normal
human IgM did not induce a measurable vasculitis in the absence of
parenteral BSA. Further decisive evidence of a role for R F in this
experimental inflammation was obtained with IgG antibody rendered
noncomplement fixing by carbamylation. Thus treated, the antibody
alone or with normal human IgM failed to induce vasculitis following
intravenous BSA. In contrast, carbamylated antibody preincubated
with R F consistently gave a marked lesion characterized by extensive
leakage of isotope marker and perivascular infiltration with neutrophil
granulocytes.
Automated Quantitative Particle Aggregate Assay (QPAA) for Rheumatoid Factor ( R F ) with the Coulter Counter
Eric Gall, David Templin. and William Dito. Tucson, Arizona
In an attempt t o automate the R F assay, a method utilizing
the Coulter Channelyzer (H-4) with attached Coulter CountercR'has
been devised. These instruments electronically determine particle size.
R F causes agglutination of IgG-coated latex beads. As the concentration of R F rises, aggregate particle volume increases. Histograms of
populations of particle size are displayed, and an arbitrary unit-the
MPRF-is derived from the median size of a dilution that produces a
histogram meeting predetermined criteria. The M P R F is curvilinearly
related to the concentration of the RF. Previous work in this laboratory has confirmed the precision of this method. One hundred and
twenty-eight tests were run on patients with a variety of rheumatic
DiseaseGroup
RA-poss, prob
RA-Def
RA-class
JRA
Vasculitis
Gout-chondrocal
DJD
Nonartic rheum
Lung fibrosis
Rheum variants
diseases. IgG-coated latex particles obtained from Behring (Beh) and
Hyland (Hyl) laboratories were utilized to determine R F by using
standard latex tube dilutions and the QPAA. Correlation coefficients
comparing the standard tube assay to the QPAA were 0.89 with both
Hyl and Beh particles, P < 0.00001. Mean f SE results of the R F
assay for each disease group tested are disulayed in the table.
classic RA
chronic fibrotic lung
Patients with definite
disease are statistically distinct from the other disease groups by the
QPAA (Duncan's multiple range test). The QPAA provides a rapid,
reliable R F assay, comparing favorably with techniques in use at this
time but with the added advantage of automation.
+
+
No. of Cases
Beh Tube Titer
Beh QPAA M P R F
Hyl Tube Titer
HylQPAA M P R F
15
0 f.0
795 f 356
I254 f 701
20 f 20
10f 10
23 f 23
OkO
Of0
1892 f 780
1214 282
20 f 20
80 f 80
II fII
Of0
Of0
448 f 234
Of0
3fl
676 f 188
703 f 190
89 f 89
33 f 32
13 f 12
1 f .5
Of0
193 f 62
5
216 f 120
2 f 1
663 f 157
646 f 161
4 f 3
55 f 54
I f 1
I f .6
6 f 4
766 f 693
13
Of0
Of0
29
29
4
8
7
13
9
Of0
*
Of0
Histidine Inhibition of the Oxidation of Serum Protein Sulfhydryl Groups: A Possible Explanation for the Low Serum Sulfhydryl
Concentration in Rheumatoid Arthritis
Donald A . Gerber and Kee-Yee Shum, Brooklyn, New York
The concentration of serum protein sulfhydryl groups is low
in patients with rheumatoid arthritis and this abnormality, like the low
free serum histidine concentration in patients with rheumatoid arthritis, is proportional to the activity of the arthritis. Cystine reacts with
serum protein sulfhydryl groups by means of a copper-catalyzed sulfhydryl-disulfide interchange reaction. Because histidine is an excellent
copper ligand, and because patients with rheumatoid arthritis have a
low serum histidine concentration, we have studied the effect of Lhistidine on the interaction between L-cystine, copper, and serum
protein sulfhydryl groups.
Serum sulfhydryl groups were measured by using 5,5'-dithiobis (Znitrobenzoic acid) in the presence of EDTA. Reactions were
carried out in 0.1 M phosphate buffer, p H 7.4, NaCl = 1 M. Serum
was diluted 1:37. In the presence of 2 pM cystine and 25 pM cupric
ion, 16.2% of the serum protein sulfhydryl groups disappeared after
incubation at 37°C for 1 hour. When, in addition to these reagents, 2.8
mM L-histidine was also present, only 3.4% of the protein sulfhydryl
groups disappeared after incubation for 1 hour. Other amino acids did
not have this effect.
In 85 sera from patients with rheumatoid arthritis, there was
a correlation ( r = 0.4, P = 0.0005) between the free serum histidine
concentration and the free sulfhydryl group concentration.
These results suggest that the low serum histidine concentration of rheumatoid arthritis may contribute t o the low serum protein
sulfhydryl concentration found in this disease by allowing a coppercatalyzed interaction between serum sulfhydryl groups and some disulfide, perhaps cystine.
ARA ABSTRACTS
I17
HLA-D and Ia-Type Alloantigens in Juvenile Rheumatoid Arthritis (JRA)
M.Eric Gershwin. Gerhard Opelz, Paul I. Terasaki, James J . Castles, and Theresa Gorman, Davis and Los Angeles. California
Study of the murine histocompatibility system (H-2) led to
the realization that the H-2 complex has broad implications for the
genetic control of immune responsiveness and disease susceptibility.
Analogous human studies identified a comparable complex, the HLA
system and its four major series: HLA-A, B, C, and D. Subsequently
numerous studies of the HLA supergene identified several significant
associations between HLA-B alleles and seronegative arthropathies
and autoimmune diseases. The explanation for these associations is
unclear but appears due to the close linkage of HLA-B with Ir genes
(immune response genes) and/or Ir gene products.
Although identification of Ir associated (Ia) antigens in inbred mice has been relatively simple, their definition in humans is
considerably more complex. Nevertheless, several la-type alloantigens
have been described by means of platelet-absorbed sera from multiparous women in cytotoxic reactions against purified B cells. Thus, in
parallel with the use of homozygous stimulator cells in MLC reactions
to define HLA-D loci of responders, it is possible to genetically characterize B-cell markers.
Because of the association of an MLC-defined locus and
adult RA, and a questionable association of HLA-B27 with JRA, we
have prospectively examined 46 consecutive unrelated children, 28
female and 18 male, for HLA-A, B, D, and la-type antigens. The data
were analyzed according to mode and age of onset, as well as extraarticular features. Patients with JRA had an elevated frequency of
HLA-B27 (23% versus 8% of controls, P < 0.01). However the most
striking associations were apparent when patients were divided into
either a monoarticular or polyarticular (including Still's) group.
Patients with polyarticular disease had an HLA-DW3 frequency of
64% compared to 22% in normals and 21% in monoarticular disease (P
< 0.001). Furthermore, of 12 polyarticular patients thus far la typed,
I I possessed the la allele B5. This latter association and its significance
will require further typing and more refined definition of the genetic
region involved before definitive conclusions are possible. However
the associations of the loci HLA-DW3 and B5 (Ia) underscore the
relationship between genetic markers and susceptibility to JRA. Further definition may aid both in understanding the mechanism of
pathogenesis and in the classification and prognosis of childhood
onset arthritis.
Age-Dependent Loss of Morphologic and Functional Maturation of New Zealand Mouse Thymic Epithelium
M . Eric Gershwin. Floyd Wilson,and Moshe Shifrine. Davis, California
Numerous immunologic abnormalities have been catalogued
in New Zealand mice, including development of autoantibodies. immune complexes, and progressive loss of T-cell function with age.
Although several plausible theories have been advanced to explain the
spontaneous occurrence of immunopathology, the precise mechanism
remains to be elucidated. Nonetheless, present evidence indicates that
one of the most critical features in the development of autoimmunity is
a progressive imbalance of thymic function. Several previous studies
of thymic histology from both New Zealand mice as well as patients
with SLE have disclosed premature thymic involution, with a predominant degeneration and vacuolization of epithelial cells. Because
of recent evidence that thymic epithelial cells are critical for both
development of the thymus in utero as well as the continued maintenance of thymic dependent function, an attempt was made to study the
growth and immunologic characteristics in vitro of isolated cultures of
murine thymic epithelial cells.
Thymic explants were cultured in vitro and serially observed
in 2-week to I-year-old NZB, BALB/c, DBA/2, and N:GP(S) mice.
Within 48 hours of culture, control mice demonstrated a proliferation
of large PAS positive epithelial cells in a mosiac-like arrangement.
Such cells contained prominent nucleoli and intense granular material
and were both relatively radioresistant and extremely adherent. They
could be readily separated from fibroblast contamination by selective
treatment with trypsin-EDTA. In contrast, proliferation of epithelial
cells was noted only in young NZB mice. Indeed, clones of epithelial
cells derived from these young NZB mice underwent vacuolization
and degeneration with age, an occurrence suggesting a preprogrammed defect. Furthermore, isolated thymic epithelial cells from
control strains, but not NZB mice, induced T-cell surface markers on
spleen cells derived from congenitally athymic (nude) mice. These
abnormalities of thymic epithelial cells in NZB mice occur before the
loss of tolerance to foregn proteins (BGG) or the appearance of
thymocytotoxic antibody. Although a considerable amount of immunocytotology remains to be performed, we suggest that functional and
morphologic alterations of thymic epithelial cells, perhaps genetically
or virally induced, may be critical in the primary development of
autoimmunity in New Zealand mice.
DNA Binding to Normal Skin Connective Tissue as a Localizing Factor for DNA: Anti-DNA Complexes
James N . Gilliam. Dallas, Texas
There is a considerable body of evidence indicating that Clq
and basement membrane collagen have similar chemical, structural,
and binding characteristics. It is also well known that Clq efficiently
binds DNA. In systemic lupus erythematosus (SLE) DNA.anti-DNA
complexes often accumulate in the connective tissue (CT) structures of
the upper dermis, glomerulus, choroid plexus, and vasculature. This
study was undertaken to examine the possible role of DNA binding to
skin CT as an explanation for the localization of immune complexes to
the subepidermal region in SLE.
Cryostat sections of normal human skin were incubated for
60 minutes in moist chambers at room temperature, with either calf
thymus DNA in 0.15 M Tris buffer or buffer alone, and then washed
extensively in 0.01 M PBS. Sections on glass slides were left untreated
or were treated with DNAase, RNAase, or sodium metaperiodate.
The enzyme-treated and nontreated tissues were then incubated with
normal human serum or sera containing DNA or RNP antibodies in
high titer. After washing, fluorescein-conjugated goat-antihuman-yglobulin was used to detect tissue bound antibody by fluorescence
microscopy. Strong fluorescence of dermal CT was observed in the
specimens that were preincubated with DNA and reacted with serum
containing DNA antibodies. The fluorescence was most intense in the
subepidermal zone. Preincubation with normal serum and RNP-positive serum gave negative results. DNAase digestion of the tissue or
removal of DNA antibodies from the serum abolished the CT staining. Digestion of tissues with RNAase and sodium metaperiodate
failed to interfere with the CT staining.
This in vitro demonstration of DNA binding to skin CT in a
location that corresponds to the usual area of immunoglobulin deposi-
ARA ABSTRACTS
118
tion in SLE is strong evidence for a localizing effect o n DNA:antiDNA complexes by DNA-CT binding in vivo. C T binding of DNA
may also account for the tendency in SLE for DNA-containing im-
mune complexes to accumulate along basement membranes at other
sites.
Increased Sensitivity of Platelets and Neutrophils to Aggregated IgC in the Presence of the Lipid Moiety of Bacterial Endotoxin
Mark H. Ginsberg. Peter M . Henson. and David C. Morrison, La Jolla. Cali$ornia
In the course of study of the interaction of human platelets
with highly purified bacterial endotoxins (Epopolysaccharides, LPS)
we noted that mixtures of y-globulin preparations and LPS of S
minnesofa Re595 were potent stimuli for platelet serotonin release.
LPS or y-globulins alone were orders of magnitude less potent platelet
activators. It seemed likely that the Re595 activity resided in the lipid
moiety (lipid A ) of this LPS, because lipid A is the major constituent
of Re595, is part of all bacterial LPS, and has been shown t o account
for much of the biologic activity of LPS. This hypothesis was confirmed by acid hydrolysis of several different LPS preparations to
produce isolated lipid A fractions, all of which were active in this
system. Sephadex (3-200 gel filtration chromatography of the y-globulin preparations demonstrated that the active constituent migrated in
the void volume, a finding suggesting that it was not monomeric IgG.
Gentle heating of previously inactive unaggregated 1gG produced
platelet serotonin releasing activity in the presence of LPS Re595.
Velocity sedimentation analysis of heated IgG o n sucrose density
gradients indicated that the newly acquired activity was found in
material of sedimentation coefficient greater than 7s. Material sedimenting at 9 s (IgG dimer) and above was active. In the presence of
25pg/ml LPS Re595, significant platelet secretion was induced by as
little as 0.5 pg aggregated IgG/ml. Platelets were at least 25-fold more
sensitive to aggregated IgG-induced serotonin release in the presence
of LPS Re595 than in the absence of LPS. Induction of neutrophil
lysozyme secretion by IgG aggregates was also enhanced by LPS
Re595.
Because IgG aggregates share many of the properties of immune complexes, these data indicate a possible mechanism of amplification of immune complex activation of mediator cells by a product of
gram-negative bacteria. Further, these data suggest that platelet serotonin secretion in the presence of the lipid moiety of LPS may be
used to detect minute quantities of IgG aggregates or immune complexes.
Altered Numbers of Circulating and Pokeweed-Mitogen-Induced Immunoglobulin Secreting Cells in SLE
William W . Ginsburg. Peter E. Lipsky, Fred D. Finkelman, and Morris Z i x Dallas, Texas
The number of immunoglobulin secretingcells (ISC) found in
peripheral blood and the number generated by in vitro stimulation
with pokeweed mitogen (PWM) were determined in 5 normals and 6
patients with active, untreated SLE. Peripheral blood mononuclear
cells (PBM) prepared by Ficoll-Hypaque centrifugation were assayed
for 1SC immediately or after culture for 7 days with or without PWM.
1SC were identified by a reverse hemolytic plaque assay that allowed
quantitation of total ISC or cells secreting lgG, IgM, or IgA. Sheep
erythrocytes coated with rabbit anti-human immunoglobulin (RAHI)
were mixed with PBM in agarose and incubated for 1 hour at 37°C.
After development with RAHl or class-specific anti-p, anti-y, or anti(Y and complement, ISC were detected as hemolytic plaques. Such
plaques represented active ISC and not passive carryover of Ig into the
assay because they were a ) abolished by cyclohexamide, b ) unaffected
by 37OC preincubation and washing of the cells, and c) not found in
PBM from patients with X-linked agammaglobulinemia.
In normals, 945 ISC/IV PBM (815-1,IOO)were found,60.4%
secreting lgG, 14.6% IgM, and 38.5% 1gA. Five of 6 SLE patients had
markedly increased numbers of circulating ISC, with 11,166/KP
(5.250-20.424) found. 74.5% lgG, 7.2% IgM, and 22.1% IgA. Stimulation of normal PBM with PWM for 7 days generated 9,894 ISC/IV
(5.900-16.484). 61.8% IgG. 45.3% IgM, and 13.3% IgA. Similar incubation without PWM yielded only 534f141 ISC/lW. Culture of
SLE PBM for 7 days yielded strikingly different results in that incubation without PWM resulted in a 7-fold decrease in ISC (1,683
352/1V) compared to the number found in fresh SLE PBM. Moreover, incubation with PWM failed to increase significantly the number
of 1SC (2,095*731/1V) in 5 of 6 SLE patients.
These data indicate that SLE is characterized by both a
marked increase in the number of circulating ISC in vivo and a
significantly diminished capacity of PBM t o generate ISC when stimulated with PWM in vitro.
*
Computer Analysis of Factors Influencing Frequency of Infection in SLE
Ellen Ginzler. Herbert Diamond, David Kaplan. Max Weiner. Michael Schlesinger, and Mitchel Seleznick. Brooklyn, New York
Infection has become an increasing cause of both morbidity
and mortality in patients with SLE. Among 182 patients followed for
an average of 3.2 years (0.1-lo), 334 infections occurred in 129
patients. Bacterial or fungal infections occurred 168 times; 166 viral
syndromes were diagnosed. O f 4 8 total deaths, infection was the cause
or a major contributing factor in 26 (54%). Data from a prospective
study of clinical course in SLE was analyzed by computer t o determine
the influence on frequency of infection of a ) dose of adrenocorticosteroid, b) treatment with an immunosuppressive agent (azathioprine),
and c) three separate parameters of disease activity: new disease exacerbations, low serum C3, and elevated ESR.
Results were as follows. a ) The frequency of all infections
increased progressively with increasing steroid dose. The increase was
even more striking when only bacterial and fungal infections were
considered (Table). b) Patients both receiving and not receiving
azathioprine had similarly increased rates of infection with increasing
steroid dose, azathioprine itself not accounting for an increased risk of
bacterial, fungal, or nonspecific viral syndromes (183 observed vs 191
expected). Azathioprine was, however, independently associated with
a greater than 3-fold increase in the frequency of herpes zoster; steroids were not. c) New disease flares, low C3, and high ESR were
associated with a 20-100% increase in infection rate. However few
patients with inactive disease received high-dose steroids, and when
patients taking comparable steroid doses were compared, the difference in frequency of infections was no longer observed. Disease activity itself did not predispose to infection.
ARA ABSTRACTS
119
We conclude that the risk of bacterial and fungal infection in
patients with SLE is directly related to the dose of steroids they
receive. In view of the associated high morbidity and mortality, studies
designed to evaluate the possible steroid-sparing action, as well as
efficacy of drugs used in combination with steroids appear to be
warranted.
Steroid dose (mg/day)
0
1-10
1 1-20
21-40
41-60
61-100
All infectiondpt-yr
Bacterial and fungal
in fections/pt-yr
0.32
0.44
0.61
0.95
1.06
2.28
0.10
0.13
0.35
0.57
0.68
I .64
Accelerated Intravascular Coagulation in Scleroderma
Geoflrey Gratwick, Ronald S. Klein. John S . Sergent, and Charles L. Christian, New York, New York
Fibrin has been demonstrated by electron microscopy and
specific fibrin staining in the intima of renal arterioles and along the
glomerular basement membrane in scleroderma. The current study
was designed to determine whether the coagulation system is activated and fibrin consumed more rapidly in scleroderma patients than
in normals.
Eleven patients (9 women and 2 men) with a clinical diagnosis
of scleroderma have been studied with either autologous or heteroOur 6 normal controls had a
logous fibrinogen labeled with
fibrinogen half-life of 88.3 hours SD 6.5 hours, while our 11 patients
had a fibrinogen turnover of 59.5 hours SD 12.4 hours. Using identical
techniques to study patients with systemic lupus erythematosus, Sergent et al (Arthritis Rheum 19:195, 1975) found a fibrinogen half life
of 60.5 SD I2 hours, with their normal controls running 90.1 SD 1 I
hours.
In the present study the usual parameters of intravascular
coagulation including platelet count, staphylococcal clumping, partial
thromboplastin, and prothrombin times were normal. There was no
correlation between the fibrinogen half-life and the ESR, complement,
creatinine clearance, pulmonary function tests, or upper GI series. Six
patients with severe disease (3 have died) had a half-life of 55.8 SD
10.9 hours, while 6 patients with less severe disease had a fibrinogen
half life of 63.0 f 13.8 hours. These observations support the hypothesis that there is ongoing disseminated intravascular coagulation in
scleroderma contributing to vascular injury. Preliminary results suggest that heparin may prolong the survival of autologous fibrinogen in
patients with scleroderma.
Relative Roles of Receptors for Complement Components, Clq and C3 in Binding of Immune Complexes to Raji Lymphocytes
Ramesh C . Gupta, Frederic C. McDufie, Gerhard Tappeiner, and Robert E. Jordon, Rochester, Minnesota
Theofilopoulos, Wilson, and Dixon (J Clin Invest 57:169,
1976) have developed a radioimmunoassay for measuring soluble
immune complexes (IC) which depends on binding of IC via receptors
for complement (C) on Raji cells, a B lymphocyte culture cell line
derived from Burkitt’s lymphoma. Though these authors have cited
evidence that the binding of such complexes occurs via receptors for
C3b and C3d, we found that Raji cells were capable of binding IC and
aggregated human gammaglobulin (AHG) after incubation in normal
human serum (NHS) to the same extent in the presence or absence of
EDTA, so that a role for C3b or C3d in binding seemed unlikely.
AHG incubated in Clq-deficient NHS at 37°C for 30 minutes
failed to bind to Raji cells but did if purified Clq was added. Raji cells
were found to possess receptor sites for Clq capable of binding 5.4 X
I @ molecules of lallClq to Raji cells that were specifically inhibited by
unlabeled Clq. Another cell culture, MOLT,a T-lymphocyte cell line
derived from acute lymphoblastic leukemia and lacking C3b receptors,
possessed a comparable number of Clq receptors. MOLT cells were
capable of binding IC to the same extent as Raji cells, and when
substituted for Raji cells in a radioimmunoassay, gave results comparable to those of Raji cells on the sera of 20 patients with immune
complex diseases. Results with both assays were similar to those found
with the 1311 Clq precipitin test.
To further assess the possible role of C3b receptors in the
binding of IC by Raji cells, these receptors were blocked by incubating
Raji cells with inulin-converted C3 in Clq-deficient NHS, and then
further reacted with erythrocyte antibody complement complex
(EAC) and AHG. There was a marked inhibition of EAC-Raji cell
rosettes, but no significant change in uptake of AHG.
We conclude that Clq receptors on Raji cells play a major
role in binding to soluble immune complexes. C3b receptors on lymphocytes probably play a significant role in the binding of particulate
complexes, such as EAC.
Nonvasomotor Postinjection Reactions Associated with Chrysotherapy
James T.Halla, Joe G. Hardin, and Julius E. Linn. Birmingham, Alabama
Transiently increased rheumatic symptomatology after individual gold injections is an acknowledged, but uncharacterized, reaction. This study was designed to determine the frequency of nonvasomotor postinjection reactions (PIR) and to define their nature and
significance. In addition to record review, a partially structured interview was used to elicit a history of all untoward gold responses from
100 patients who had received regular gold within a year prior to
evaluation, but who were otherwise unselected. Care was taken not to
suggest to the subject the reaction being investigated.
N o postinjection symptoms or typical vasomotor reactions
occurred in 85 subjects. Thirteen described a PIR predominately of
new or increased rheumatic symptoms, all initially occurring after gold
sodium thiomalate (GST)injections and beginning within 1 hour
postinjection in I , and within 6-24 hours postinjection in 12 subjects.
Reactions persisted from 1/2 to 5 days (mean: 2) and consisted of new
or increased stiffness (l3), arthralgia (12). myalgia (8), constitutional
symptoms (5) and joint swelling (3). Initial reaction occurred during
the first month of therapy in 5 subjects, during the second month in 4,
ARA ABSTRACTS
120
and after the third month in 4; and the PIR had recurred > 5 times in
10 and >I0 times in 4 subjects. Therapeutic manipulation had been
felt to be warranted in 6 patients because of the severity of the
reaction. For these 6 gold thioglucose (GTG) was substituted for GST
with no subsequent PIR in I and with decremental decreases in
reaction severity in the remaining 5, so that the reaction had disappeared by the fourth GTG injection in 4 patients. The milder PIR
continued predictably in the remaining 7. A predictable nonrheumatic
PIR-consisting only of constitutional symptoms beginning 12-24
hours after GST injection and lasting 3-5 days-was described by 2
additional subjects. Other untoward gold reactions, besides the PIR,
occurred in 43% of the 85 subjects without, and in 41% of the 15 with
PIRs, a result suggesting no tendency for the PIR to occur in a
reaction-prone group.
Fifteen percent of 100 unselected subjects experienced new or
increased symptoms, primarily rheumatic, within hours of GST injection. Contrary to prior beliefs, these reactions may begin late as well as
early in the course of therapy, tend to persist, and may be severe
enough to require intervention, in which case GTG substitution may
result in waning of the PIR. It is suggested that these PIRs are
significant and deserve further study.
Application of the Radioimmunoassay for Fibrinopeptide A to Serial Evaluation of Patients with SLE
John A. Hardin, Malcolm Cronlund, Edgar Haber, and Kurt J . Bloch, New Haven, Connecticut and Boston, Massachusetts
We have previously reported the development of a radioimmunoassay for fibrinopeptide A (FPA) which is useful for detecting
activation of the clotting mechanism. Application of this assay to the
study of patients with systemic lupus erythematosus (SLE) demonstrated higher FPA levels in patients with active disease than in
patients with inactive disease or in normal donors. Elevated levels
correlated directly with the presence of antibodies to native DNA and
inversely with depression of serum C3 concentrations. These findings
suggested that measurements of FPA might be useful for assessing
disease activity in patients with SLE.
To test this hypothesis, serial determinations of FPA levels
were carried out in 21 patients who were followed for a mean of 18
months. In individual patients it was found that FPA levels fluctuated
in parallel with disease activity; exacerbations were accompanied by a
rise in the FPA level and improvement was accompanied by a fall.
FPA levels were always elevated when clinical evidence of inflammation was present, and the extent of elevation correlated with
the extent of inflammation. Some patients with inactive disease also
had elevated levels of FPA, a finding suggesting the possibility that
subclinical inflammation was present (Table).
These data indicate that activation of the clotting mechanism
occurs in association with the inflammatory response in SLE. Measurements of FPA appear to provide a sensitive method for evaluating
these patients.
Correlation of FPA Values with Number of Organ Systems Involved Clinically with SLE
Determinations
FPA ng/ml (mean f 1 SD)
Number of Systems Involved
Normal
Donors
Clinically
Inactive SLE
One
Two
Three or More
20
0.9 f . 3
36
2.1 f 1 . 1
25
5.2 f 4. I
20
7.5 f 2.5
12
12.1 f 2.6
Costs of Monitoring Chrysotherapy
Benjamin K . Harris, De Witt W . Englund, H . Arlene Ross, and Sanford H . Roth, Phoenix, Arizona
Cost and potential toxicity are two factors that may deter
both physician and patient from considering gold therapy for rheumatoid arthritis. In 1975 Liang and Fries presented a cost analysis of
alternative monitoring strategies in administering gold. Their data,
based on a survey of 10 rheumatologists and 3 hospitals in northern
California, indicated that the cost of an initial course of gold therapy
was approximately $800. A number of cost-reducing measures were
suggested, including the use of a physician-assistant and a decrease in
the frequency of certain laboratory tests.
Since 1970 our approach to monitoring chrysotherapy has
included many features advocated by Liang and Fries. We have re
viewed our experience with gold therapy administered in 1975 to find
the average cost to initiate therapy and to determine whether our costreducing measures in any way increased toxic reactions to gold. Gold
therapy was begun in 64 patients in 1975. Forty-nine patients (77%)
completed the initial course with a favorable response; in 8 patients
(13%) gold was discontinued because of toxicity. Five patients were
lost to follow-up, and 2 patients were considered treatment failures.
Our monitoring plan called for an average of 5 office visits, 21 gold
injections, 22 urinalyses, and 9 complete blood counts with platelet
estimates. Average cost to the patient was $315. Major savings resulted from utilizing a physician-assistant to screen for toxicity and
administer gold, and thus to reduce physician visits from weekly to
monthly.
If, as indicated by our data, alternative approaches do not
increase risk to the patient or detract from gold effectiveness, further
exploration for an optimal monitoring regimen appears warranted.
HLA-B27-Associated Arthritis in 6 Families: Homozygosity, Patterns, and Severity of Disease
Marc C. Hochberg, Frank C . Arnett. Jr., Bernice Z . Schacter. and Wilma B. Bias, Baltimore, Maryland
HLA-B27 is strongly associated with ankylosing spondylitis
(AS), Reiter’s syndrome (RS), and juvenile chronic polyarthritis
(JCP). Although these disorders share certain clinical similarities,
there are differences between them, most notably the predominance of
ARA ABSTRACTS
121
peripheral arthropathy in RS and J C P and axial skeletal involvement
in AS. Similarly, there exists a broad spectrum of severity within each
disorder. T h e families of 6 patients with B27-associated arthritis were
studied clinically and radiographically. H L A genotypes were determined independently and then correlated with clinical data.
Two of the 6 probands (families I and 2) are homozygous for
827 and heterozygous a t the A, C , and D loci. Both have extraordinarily severe disease. The homozygous female proband of family 1 has
a I 5-year history of J C P with severe peripheral and axial disease and
recurrent uveitis. The mother and a sister with the maternal B27
haplotype have only mild, nondeforming, seronegative peripheral arthritis, whereas the father has only sacroiliitis (SI). The homozygous
male proband of family 2 is crippled by a progressive, deforming
peripheral and axial arthritis beginning at age 18 (JCP-AS), aortic
regurgitation, and uveitis. A heterozygous brother has RS without
crippling despite similar duration of disease (30 years). One heteroeygous child has mild JCP. Six B27-positive relatives are well. The
male proband of family 3 is possibly homozygous and has RS with
severe. deforming arthropathy and mucocutaneous disease. Two HLA
identical brothers are well.
Family 4 contains a mother, daughter, and son with RS. all
heterozygous for 827, and none has axial disease. Family 5 consists of
a mother, 2 daughters, and 1 son, all with SI, and 1 son with AS: all are
B27 heterozygotes and none has peripheral disease. Family 6 contains
4 B27 heterozygous siblings, I female with severe peripheral and axial
arthritis (JCP-AS), I male with mild AS, and 1 male with SI; 1 female
is well.
These data suggest that a ) homozygosity for B27 may result in
more severe rheumatic disease; b ) there may be exclusivity to the type
and pattern of rheumatic disorder associated with a given B27 haplotype within families; and c ) the presence of normal B27 positive
individuals within these families implies that other environmental
and/or genetic factors a r e necessary for disease expression.
CRP in SLE Patients: An Aid in Diagnosing Superimposed Infections
Stephen Honig, Peter Gorevic, and Gerald Weissmann, New York. New York
The presence of C-reactive protein ( C R P ) in the sera of
patients with SLE is not well documented. C R P positivity is, however,
well established in sera from patients with various other acute and
chronic inflammatory processes. In order t o determine the incidence of
C R P in sera of patients with SLE, 70 patients with SLE were studied.
Results of C R P positivity were correlated with disease activity and
superimposed infections. Seventeen hospitalized patients with documented infections were also studied.
Strong C R P positivity (2 2 + ) was found in the sera of 13 of
61 patients with active SLE (21%). Of these 13 patients, 11 had
superimposed infections at the time that C R P was found in their sera.
Only 2 patients with active SLE had strong C R P positivity in their sera
in the absence of a superimposed infection. Excluding the SLE
patients with infections, the incidence of CRP-positive sera in patients
with active SLE was 4%. Only 1 patient continued t o have C R P in her
sera after treatment of the superimposed infection. In the absence of
infection, there is a lack of association between C R P positivity and
active SLE ( P < 0.002). The activity of the uninfected SLE group is
shown by the negative correlation between ESR and C, ( r = -0.16)
and C, ( r = -0.41), results reflecting patients with elevated ESR and
hypocomplementemia. The finding of C R P positivity in sera of
patients with SLE is therefore strongly associated with the presence of
infection (P < 0.002). There were n o instances of infection documented in the active SLE patients without strong C R P positivity in
their sera.
The 17 patients with infections, including 1 patient with a
viral infection, all had C R P in their sera at the time of initial diagnosis.
Appropriate treatment resulted in the subsequent loss of C R P from
their sera and reaffirmed its utility as an indicator of acute infections.
The data suggest that patients with active SLE rarely have
strongly positive C R P in their sera and, when positive, this acute phase
reactant indicates a superimposed infection and not disease activity.
Early Diagnosis and Treatment of Ischemic Necrosis of the Femoral Head
David S. Hungerford, Baltimore, Maryland
The hemodynamic status of the proximal femur has been
evaluated in 90 hips with ischemic necrosis of the femoral head
( I N F H ) in 60 patients and 18 normal controls. The methods constitute
a simple, rapid system for establishing the diagnosis of I N F H in the
patient with suspicious clinical findings. Treatment of 70 hips in 44
patients has consisted of core decompression and removal of an 11
mm diameter plug of bone from the central axis of the femoral head
and neck.
Baseline intraosseous pressure (IOP) and the pressure response t o a 5 cc saline load injected intraosseously were measured by
an electromanometer via a rigid, percutaneously inserted 3 mm cannula. In selected patients an intraosseous phlebogram, using Renografin, was carried out. All patients with I N F H had their diagnosis
histologically confirmed.
Thirty-eight of 90 hips with I N F H showed an IOP of greater
than 30 mmHg and/or a sustained hypertensive response of more than
10 mmHg to the injected saline load. All controls demonstrated IOP
less than 28 mmHg and none had a hypertensive response to the saline.
lntraosseous venograms in hips with I N F H showed diaphyseal reflux.
poor filling of extraosseous veins, and delayed intraosseous evacuation
of dye, in marked contrast to the normal controls.
Hips having had core decompression have been followed
from 6 t o 42 months. All hips were painful preoperatively. Of 18 hips
in the preradiologic stage, 17 are asymptomatic and show no radiologic changes at last follow-up. Successful relief of symptoms and
prevention of progression decrease with advancing stages.
Forty-four hips in this series were in patients with connective
tissue disease who were on or had been on large doses of steroids. Ten
of these patients and 18 in the whole series had painful hips but normal
or near normal x-rays. The methods presented provide the opportunity of establishing the diagnosis of I N F H in the preradiologic stage of
the disease, at a time when core decompression appears to be an
effective means of interrupting the natural history of the disease.
ARA ABSTRACTS
122
Influence of Interstitial Infiltration on Tubule Cells in Kidneys of NZB X NZW FI (B/W) Hybrid Mice and the EfFect of
Cyclophosphamide (CY)
E. R . Hurd and M . Zif, Dallas, Texas
We have reported that kidney glomerular cell proliferation
can be quantitated by 3HTdr injection, autoradiographic examination
of kidney sections, and enumeration of labeled glomerular cells. In the
present studies we have examined renal tubule cells (TC) by the same
technique to assess the effect of interstitial infiltration on renal T C
proliferation.
Three, 5, 7, 9, and Il-month B/W mice were injected with
SHTdr daily for 8 days prior to sacrifice. Autoradiographically labeled
sections of kidney were prepared and the number of labeled TC per
lo00 TC was counted. At the same time interstitial mononuclear cell
infiltration, which is commonly present in these mice, was scored on a
0-4+ basis. Percentages of labeled T C adjacent to interstitial infiltrates and distant from them were tabulated. In other mice the effect of
CY (15 mg/kg/day), injected over a 2-month period, o n both the
interstitial infiltrate and amount of T C proliferation, was examined.
NZB, CBA/J, and C57B1/6J mice were used as controls.
In B/W mice interstitial infiltration began at 5 months and
reached a peak at 9 months. Proliferation of T C began somewhat
later, i.e. at 7 months, but also reached a peak (45 labeled cells per
lo00 T C ) at 9 months. Control mice (15 labeled T C ) showed no
increase with age. In the areas adjacent to infiltrates at 7 and 9 months,
an average of 72 TC/1000 were labeled, as opposed to 19 cells at a
distance, an increase of about 4-fold. CY administered 2 months
before sacrifice virtually obliterated interstitial infiltration and significantly decreased TC proliferation at 3, 5, 7, and 9 months of age.
These studies suggest that mononuclear cells in the interstitial
infiltrate may produce a lymphokine or other factor that may be
injurious to kidney T C and lead to regeneration after injury or death.
CY reverses or markedly inhibits these phenomena. The present findings suggest that in diseases in which interstitial nephritis is present,
such as Sjogren’s syndrome, the interstitial infiltrate may contribute to
the observed T C dysfunction by immunologically induced injury.
Characterization of the Proteins Synthesized by Sclerodermatous Skin in Organ Cultures
Sergio A . Jimenez. Philadelphia, Pennsylvania
Full-thickness skin biopsies from the leading edge of actively
involved sclerodermatous lesions were incubated in an organ culture
system in the presence of “C-proline. At the end of the incubation, the
labeled proteins from the media and the tissues were separated and
characterized. The organ cultures studied showed marked increase in
the incorporation of “C-proline and in the synthesis of “C-hydroxyproline when compared to normal skin. The distribution of radioactivity between media and tissue proteins showed that 77-93% of the
total 14C-prolineincorporated and 6 1-9 I % of the IT-hydroxyproline
synthesized, were present in the media. Determination of “C-hydroxyproline showed that only 1-3’70 of the total “C-proline incorporated
was hydroxylated to “C-hydroxyproline, indicating that collagen represented only a minor fraction of the labeled proteins synthesized
during the incubation. When the media proteins were examined by gel
filtration under denaturing conditons, it was found that most of the
labeled proteins were of small molecular weight, and that only 10-12%
of the radioactivity eluted in a region corresponding t o collagenous
molecules. The elution of “C-hydroxyproline-containing proteins in-
dicated that essentially all the labeled collagen in the media was
present as procollagen. Determination of the hydroxyproline content
of the purified procollagen indicated that the protein was markedly
under-hydroxylated, because it contained only one-third of the
hydroxyproline expected for a fully hydroxylated molecule. The small
molecular weight proteins did not contain hydroxyproline and therefore they were noncollagenous. N o molecules with molecular weights
greater than that of procollagen were present in the media. The tissues
were sequentially extracted with 0.15 M NaCI, 0.5 M acetic acid, 100
jtg/ml pepsin, and I % sodium dodecyl sulfate. About 40% of the
labeled collagen was extracted in the neutral salt and acid buffers. An
additional 35% was solubilized after pepsin digestion of the tissues,
and the remaining was solubilized under denaturing conditions. In
contrast, approximately 75% of the total “C-proline incorporated was
extracted in the neutral salt and acid buffers, indicating that these
buffers extracted preferentially noncollagenous molecules or underhydroxylated collagen.
Detection of Circulating Immune Complexes in Patients with Rheumatic Diseases and Vasculitis
Gary M.Kammer, David N . Glass, Nicholas A . Soter, and Peter H . Schur, Boston, Massachuserts
The sera of patients with rheumatoid arthritis or Sjogren’s
syndrome complicated by vasculitis, and of patients with SLE were
studied for circulating immune complexes. The IC assays performed
were Clq-1z51 binding and inhibition of antibody-dependent lymphocyte-mediated cytotoxicity (ADLMC). Both Clq binding and
A D L M C inhibition are expressed as the equivalent of micrograms of
aggregated IgG (AgglgG) per ml. Mean Clq binding in 34 patients
with vasculitis, histopathologically defined a s cutaneous necrotizing
venulitis (CNV), was 315.5 pg AggIgG/ml (range: 10-1840), and in 34
normals it was 44 pg AgglgG/ml (range: 10-200) ( r = 4.16, P <
0.001). Of the 34 patients with CNV, hypocomplementemia and Clq
binding were detected in 1 I , normocomplementemia and Clq binding
in 8, and both normocomplementemia and n o detectable Clq binding
in 15. The 19 CNV patients with Clq binding had significantly higher
binding [572.6 pg AggIgG/ml f 474.1 (1 SD)] when compared with
patients with active SLE [155.0 jtg AgglgG/ml f 125.7 ( I SD)] (f =
3.31, P < 0.001). Cryoproteins were detected in 5 of the CNV patients
with Clq binding. Mean ADLMC inhibition in 13 of the 34 CNV
patients studied was 1.97 pg AgglgG/ml (range: 0.1-9.0). and in 17
normals, 0.17 pg AgglgG/ml (range: 0.1-0.82) ( I = 2.40, P < 0.01). In
the 13 patients assayed by ADLMC inhibition, there were 4 with
hypocomplementemia and inhibition, 4 with normocomplementemia
and inhibition, and 5 with normocomplementemia and without inhibition. There was n o significant difference between CNV and SLE
patients with the A D L M C inhibition method.
These data suggest a ) that there are circulating immune complexes in sera of some patients with CNV complicating rheumatic
diseases; b ) that there is a relationship between hypocomplementemia.
Clq binding, and ADLMC inhibition; c ) that sera from CNV patients
contain appreciably more Clq binding material than do sera from
patients with active SLE; and d ) that both the Clq binding and
ADLMC inhibition assays are more sensitive than hypocomplementemia as criteria for the detection of immune complexes.
ARA ABSTRACTS
123
Increased Incidence of Malignancy in Sjogren’s Syndrome
Stuart S. Kassan. Robert Hoover, Robert P . Kimberly, Daniel R . Budman, John L. Decker, and Thomas M.Chused. Bethesda.
Maryland
The case records of 128 women and 6 men with Sjiigren’s
syndrome (SS) treated at the National Institutes of Health (NIH) from
1955 through 1975 were analyzed to estimate the risk of malignancy.
Only those cases of malignancy developing after the first admission
were included as cases in calculating risk of cancer in this population.
The number of cancer cases in the study group was compared to the
expected incidence of various forms of cancer in the population derived from the Connecticut Cancer Registry (for the incidence of
lymphoma) and the cancer registries of Dallas-Fort Worth and Iowa
(for the incidence of skin cancer). The relative risk ( R R ) for each type
of cancer observed, observed casedexpected cases, was then determined. Five patients developed non-Hodgkin’s lymphoma (RR =
100, P < 0.001) from I t o 9 years following their first NIH admission
for SS. The R R for lymphoma in patients with sicca complex alone
was similar to that for SS patients with rheumatoid arthritis. Six cases
of Waldenstrom’s macroglobulinemia occurred in this study group,
but control incidences for this entity are not available to allow an
estimation of the RR. Four patients developed nonmelanotic skin
cancer, a figure in excess of that expected from rates in either low(Iowa) o r high-risk (Dallas-Fort Worth) areas in the US. Other tumors occurred at their expected frequency.
Low-dose therapeutic parotid irradiation may have predisposed to lymphoid malignancy in SS patients so treated. SS patients
who received low-dose parotid irradiation had an incidence of lymphoma 33 times that of nonirradiated SS patients. This particular
mode of therapy may, however, have selected a subpopulation of SS
patients at greater risk of developing malignancy. Cytotoxic drug
therapy had no evident effect on tumor development in this SS population. However the number of patients treated with cytotoxic drugs
was small (13) and the duration of follow-up for these patients was
short (longest = 6 years).
This is the only quantitative consideration of this matter
available. T h e nature or severity of SS upon referral to N I H may of
course introduce selective biases, but none was apparent.
HLA-D Locus Typing in Ankylosing Spondylitis
Kip L . Kemple. Richard A . Gatti, Rodney Bluestone, and James Klinenberg. Los Angeles. CaliJornia
The high association of the HLA antigen B27 with ankylosing
spondylitis (AS) and other spondyloarthropathies has led to the speculation that a disease susceptibility gene within the major histocompatibility complex is responsible for disease production. The incriminated
gene is presumed to be in marked linkage disquilibrium with the 827
antigen and possibly with other histocompatibility determinants.
Analogous Ir genes in the mouse H-2 complex are known to be located
near or at the main MLR locus. We have therefore evaluated the
patterns of D locus typing in humans suffering from AS to determine
whether there is a n altered distribution of L D alleles comparable to
that seen with the 827 antigen.
T o date 44 patients with definite or probable AS have been
evaluated. Both B27-positive (35) and 927-negative (9) patients a r e
included. Typing was carried out with a panel of 24-40 stimulator
lymphoblastoid cell lines which represent the Sixth International
Workshop specificities DW 1-6 plus 107-108. These lines a r e derived
from individuals known to be homozygous at the D locus. They are
mitomycin-treated and frozen in typing plates for storage at -80”.
When they are thawed for use in standard one-way M L R testing, they
give patterns of stimulation comparable to typing with fresh cells.
Results have been compiled by separating weak (typing) responses
from strong MLR responses pending more sophisticated computer
analysis of the data.
These studies indicate that there are no significant differences
between AS patients (either 927-positive or negative) and controls in
the distribution of DW alleles or in responses to 40 individual cell
lines. Specific gene frequencies will be discussed. In addition, there is
no significant difference in the overall level of M L R responsiveness
between AS patients and controls.
It appears on the basis of these results that disease susceptibility in AS is not determined by factors associated with the D locus, as
appears t o be the case with multiple sclerosis and possibly other
diseases.
Effects of Aspirin on Renal Blood Flow, Glomerular Filtration Rate, and Renal Prostaglandins in SLE
Robert P . Kimberly, John R . Gill Jr., Robert E. Bowden and Paul H . Plotz, Bethesda. Maryland
Antiinflammatory doses of aspirin have been shown to affect
serum creatinine (SCr), blood urea nitrogen (BUN), and creatinine
clearance (CCr) in some patients, but the nature of the changes in
renal function remains controversial. Five patients with systemic lupus
erythematosus (SLE) have been studied under controlled metabolic
conditions in order to examine the effects of approximately 7 days of
oral aspirin (3.3-6.0 g/day) on SCr, BUN, CCr, and urinary excretion
of PGE-like material determined by radioimmunoassay. Inulin and
PAH clearances were performed before, during, and after aspirin
therapy. The BUN rose an average of 5.8 f 3.1 mg/dl (mean f SEM);
SCr increased 0.3 f 0.06 mg/dl (P < 0.01); and CCr fell 15.6 f 1.9
ml/min ( P < 0.01). PAH clearance (CPAH) dropped 29% or 128 f
22.5 ml/min (P < 0.01). while inulin clearance (CIn) fell 15% o r 12.2
f 2.4 ml/min (P < 0.01). With the cessation of aspirin, all parameters
showed a significant return toward baseline (P < 0.05) within 5 days,
except for CIn which demonstrated some variability. Two additional
patients without complete CIn and CPAH studies also showed increases in SCr and BUN with decreases in CCr. However in I the
change in CCr had regained the control level by the end of the
treatment period. Measurement of urinary PGE-like material showed
a mean decrease of 47% (P < 0.001) during aspirin therapy.
These data demonstrate that aspirin given in antiinflammatory doses to patients with SLE for several days causes a significant
decrease in renal blood Row and glomerular filtration rate, which may
be reflected in serum parameters. These changes are reversible with the
cessation of aspirin therapy. The decrease in renal blood Row concomitant with the measured decrease in renal prostaglandins suggests
that the mechanism for the aspirin-induced change in renal physiology
may be the inhibition of renal prostaglandin synthesis. This hypothesis
predicts that this effect will not be limited t o aspirin in the class of
nonsteroidal antiinflammatory drugs.
ARA ABSTRACTS
I24
Efficacy of Antiviral Agents in the Treatment of Lupus Nephritis in NZB/NZW F, Mice
L. W . Klassen. D . R. Budman, G. W . Williams,A . D . Steinberg. and N . L. Gerber, Bethesda. Maryland
Four antiviral agents were evaluated in the treatment of lupus
nephritis in NZB/W mice. One hundred fifty-two NZB/W female
mice aged 20 weeks were randomly selected to receive I P injections of
ribavirin (250 mg/kg) twice weekly; phosphonoacetic acid (750
mg/kg) five times weekly; levamisole (10 mg/kg) twice weekly; ara-A
(600 mg/kg) twice weekly or saline twice weekly. All mice were followed for development of proteinuria, anti-DNA antibodies, and survival.
Control mice had a median survival of 10 months with a 15%
survival at 14 months. Ribavirin therapy was associated with 76%
survival at 14 months. Levamisole and phosphonoacetic acid prolonged median survival to I I .4 and I 1.6 months respectively, and were
associated with a 2% survival at 14 months for both groups. Ara-A
treated mice had a median survival of 9 months (Table).
This study was designed t o determine the efficacy of selected
antiviral agents in prolonging life in NZB/W female mice. The results
clearly demonstrate the efficacy of ribavirin in prolonging survival,
reducing proteinuria, and decreasing the titer of antibodies against
native DNA.
Elfects of Antiviral Agents on Proteinuria and DNA Antibodies in NZB/W Mice
Therapy Group
Evaluation
Proteinuria*
Age (mo)
Ara-A
Levamisol
Phos Acid
7
0/25t
0/7t
64 f 3
62 f 7
4/22
11/17
55 f 3
65 f 74
13/28
11/17
52 f 2
69 f 6f
II
Anti-DNA$
7
II
* Animals with urinary protein concentration greater than I00 mg/day/number
t P < 0.05 vs saline control by x 2 analysis.
Ribavirin
10/29t
0/26t
44 f 24
42 f 2
Saline
21/31
8/13
59 f 2
47 f 4
surviving.
$ Expressed as percent binding f SEM.
< 0.05 vs saline control by student's I test.
tj P
Effects of Soluble Immune Response Suppressor (SIRS) on the Course of Autoimmune Disease in NZB/W Mice
Randall S . Krakauer, Warren Strober, and Thomas A . Waldmann,Boston. Massachusetts, and Bethesda, Maryland
Female NZB/W mice develop autoimmune abnormalities
similar to those seen in systemic lupus erythematosus (SLE). As they
age, these mice lose precursors of suppressor T cells, and their ability
to make soluble immune response suppressor (SIRS) in response to
concanavallin A exposure for 36 hours. Yet even the adult mice
respond to the suppressor signals of SIRS produced from other
sources. These observations suggested the possibility that such mice
could be treated with the suppressor T-cell product, SIRS. Therefore
we felt SIRS may provide a means of adoptive immunotherapy for
these mice, and by extension, for SLE.
We treated female NZB/W mice, beginning at age 4 weeks,
with thrice weekly injections of SIRS, the supernatant of splenocytes
incubated with concanavallin A (2 rg/cc) for 36 hours, from BALB/c
and young (4-week-old) NZBiW mice spleen cells. Treated mice over
the 28-week observation period had decreased immunoglobulin levels
(75% reduction in IgG and IgA, 50% reduction in IgM) and strikingly
less antinuclear antibody (absent or present in very low titer in all
treated animals, present in higher titer in all untreated animals) compared to animals receiving a control preparation. They also had less
proteinuria (22 f 24 mg/100 ml urine in treated animals. 105 f 28
mg/100 ml in untreated animals) and renal pathology (by light microscopy, severe glomerulonephritis in untreated animals, near normal
glomeruli in treated animals) compared to NZB/W mice receiving a
control preparation or no treatment at all. At the end of a full year of
study, 93% of the treated animals and 7% of the control animals
survived.
Thus SIRS administration appears to be an effective therapy
of the autoimmune disease found in this animal model of SLE and
holds promise for the successful treatment of human SLE.
Liver Involvement in Essential Mixed Cryoglobulinemia (EMC)
Yoram Levo. Peter Gorevic, Hannah Kassab. Hillel Tobias, and Edward C. Franklin, New York, New York
Essential mixed cryoglobulinemia is characterized by purpura
arthralgia and weakness. Although an immune complex glomerulonephritis is the major cause of death. the frequency of clinically
apparent liver involvement has not been emphasized.
Between 1960 and 1976, 104 patients with cryoglobulins were
seen, 30 of whom were considered to have EMC. Purpura was present
in 90%. arthralgia in 70%. and renal disease in 43%. Twenty-six cryoglobulins were composed of IgG and IgM; 4 also contained IgA. The
IgM was polyclonal in 22 and monoclonal (IgMk') in 8. Evidence of
hepatic involvement was present in 25 (83%). Hepatomegaly was
noted by physical examination in 18 and by liver scan in only 3
additional ones. Seventeen patients had abnormal liver function tests,
with elevation of alkaline phosphatase the most sensitive parameter.
Four patients had severe clinical manifestations of liver involvement
such as ascites or hepatic encephalopathy. Liver tissue was obtained
from I I patients and showed pathologic changes in 10. Two patterns
of liver pathology were observed-one similar to that in chronic active
hepatitis characterized by periportal infiltration with lymphocytes,
histiocytes, and plasmacytes, piecemeal necrosis, and fibrosis; the
other resembling portal cirrhosis. All 4 patients with IgA in their
cryoprecipitate had liver involvement documented by biopsy. Because
of the high incidence of hepatic dysfunction, sera and in several
instances cryoprecipitates from 21 subjects were tested for H&Ag only
in their cryoprecipitate and one had it in his serum (14%). Twelve
ARA ABSTRACTS
125
patients (57%) had significant titers of anti-H$, antibodies.
Thus 62% of the patients had evidence of exposure to the
HBV. These findings emphasize the frequency of hepatic involvement
in EMC and raise the possibility that this syndrome may be another
example of extrahepatic manifestations as a secondary immunologic
response to HBV.
Presence of y-Carboxyglutamic Acid in the Proteins Associated with Ectopic Calcification
Jane B. Lian. Martha Skinner, Melvin J. Climcher, and Paul M . Gallop, Boston, Massachusetts
A specific calcium-binding amino acid, y-carboxyglutamic
acid (Gla), discovered in the blood clotting factor prothrombin, is
synthesized in a vitamin K-dependent and bicarbonate ion requiring
post-translational enzymatic reaction. Recent studies have demonstrated the presence of Gla in an EDTA-soluble, noncollagenous
protein in both chicken and bovine bone. Because a Gla-containing
protein is normally present in bone and absent in unmineralized
tissues, it was of interest to examine several kinds of ectopic calcifications.
Studies were done on a 21-year-old female with dermatomyositis and a 52-year-old woman with a 30-year history of scleroderma.
In the case of dermatomyositis, the patient had many large nodules of
soft tissue calcification throughout her body. Due to the acid lability
of Gla, hydrolysis in 2 N KOH for 22 hours is required to detect it by
amino acid analysis. Alkaline hydrolysis of biopsy material from both
the normal and calcified skin and subcutaneous tissue from the patient
with dermatomyositis revealed the presence of Gla only in the plaque
specimens. The chalky paste-like exudate from subcutaneous nodules
and from an infrapatellar knee bursa of the patient with scleroderma
showed a higher concentration of Gla (6 res/IOOO amino acids) than
normal whole bone (0.67 res/1000). The EDTA-soluble and nondialyzable proteins extracted from this exudate were enriched in Gla
(20 res/IOOO amino acids).
Because X-ray diffraction analysis showed hydroxyapatite to
be the major mineral component of the plaque in these two cases, the
question arises as to whether the Gla-containing protein(s) in the
plaque is similar to the Gla-containing protein in normal bone. I t is
also relevant to inquire if the Gla in the plaque is derived from the
vitamin K-dependent circulating blood clotting factors or if perhaps it
is synthesized de novo by cells at the site of calcification. In view of the
known calcium and phospholipid binding properties of y-carboxyglutamic acid, the presence of Gla in pathologically mineralized tissues is
a potentially significant finding.
Effect of Prostaglandins on Metabolism of Isolated Chondroycytes
Louis Lippiello, Dragoslav Mitrouic. and Henry J . Mankin, Boston, Massachusetts
Prostaglandins (PG)are found at elevated levels in synovial
fluid from patients with inflammatory arthritis and have been implicated as a possible agent in erosion of the articular cartilage of such
joints. Experiments were designed to examine the role of PGs in
articular cartilage metabolism and to define the nature and specificity
of their effect on proteoglycan metabolism in vitro.
Chondrocytes isolated from calf metatarsal cartilage were
counted and assayed for viability with the tryphan blue dye exclusion
test. Cells (1V) were aliquoted to tubes containing I ml media, and the
cells “rested” for 24 hours. The media was changed and PGs at
varying dosages were added for an additional 24 hours. At this time
labeled substrate (ssSO,+ SH-glucosamine) was added for 30 minutes,
and incorporation of isotope was terminated by heating cells media
at 100” for 5 minutes. Radioactivity of the TCA insoluble (lo%), I N
NaOH solubilized material was assayed by liquid scintillation counting.
The results showed that there was considerable variation in
the response of the cells to different members of the PGs. With the
exception of PGA,, only PG of the 2 series (PGA,, PGE,, PGF-)
+
inhibited uptake of substrate. The observed inhibitions were statistically significant and the most potent inhibitor was PGA, (>SO% at a
dose of0.5 pg/ml; <50% at a dose between 0.06pg/ml and 0.5 pg/ml).
Concentrations of PGA, of less than 0.03 pg/ml had little effect.
Inhibition by PGA, at 25 pg/ml was irreversible up to 60 hours after
exposure, and treatment of cells with indomethacin (5 pg/ml) for 6
hours before exposure of the cells to PGA, at 25 pg/ml did not alter
the inhibitory pattern.
The results indicate that calf chondrocytes in suspension culture are very sensitive to and selectively inhibited by PG PGA, and all
members of the 2 series (PGA, > PGA, > PGE, > PGF,). The
absence of a reduction of the inhibition by indomethacin confines the
action of the drug at the PG synthesis level, rather than after the active
material is formed. Previous demonstrations of an inhibitory effect of
PG on cartilage in vivo and in vitro in organ culture, and now using
isolated cells, supports the thesis that these fatty acids may play a
major role in the development of cartilage destruction in inflammatory
arthritis and conceivably in osteoarthritis.
Joint Allotransplantation: A Metabolic and Biomaterial Study
Mitchel Lipton, Joseph M . Lane, Carl T. Brighton, John Irwin, and Chester Shadle, Philadelphia, Pennsylvania
and New York. New York
Joint replacement with allotransplants has received renewed
clinical interest. A successful transplant necessitates the continued
viability of the articular chondrocytes and structural stability of the
cartilage matrix. Utilizing a mature rabbit osteochondral joint allotransplant model, we studied the metabolic, biochemical, and biomateria\ properties of the transplanted cartilage for up to 1.5 years, and
the cartilage remained metabolically and materially intact in the majority of long-term rabbit transplants.
Forty mature New Zealand rabbits underwent osteochondral
shell joint allotransplantation of the left femoral condyle. The major-
ity of long-term transplants remained grossly and histologically intact
for as long as I .5 years. The subchondral bone of the shell graft was
replaced by creeping substitution by 2 months. Twenty-eight percent
of the transplants failed because of the surgical defects (pseudarthrosis, malalignment, incongruous fit) and manifest secondary osteoarthritic morphologic changes with no evidence of rejection. At 1.5
years the transplanted cartilage was comparable statistically to control
cartilage in proteoglycan hexosamine, collagen hydroxyproline, and
protein content.
The transplanted cartilage was indistinguishable biomate-
ARA ABSTRACTS
126
rially from control in terms of relaxed sheer modulus (0.9 vs 0.9) and
instantaneous sheer modulus (5.1 vs 5.0) (dynes/cmP X 1V).
Successful long-term joint allotransplantation with viable
chondrocytes and intact cartilage matrix is possible. The limited failures appear to be directly related to surgical malalignment and incongruities resulting in secondary osteoarthritis. N o rejection was
evident morphologically. These metabolic and biomaterial studies give
strong support for continued clinical trials ofjoint allotransplantation.
Hexosarnine
Control
Transplant
(ag/mg dry wt)
Hydroxyproline
Olg/mg dry w t )
52.6 f 0.5
43.7f 11.3
75.6 f 8.5
85.2f 10.6
Protein
dry w t )
(mg/mg
0.48 f 0.09
0.58f 0.07
In Vivo Gold Kinetics-Lymphocyte Binding and Effect on Lymphocyte Responsiveness
Arthur Lorber, Timothy Simon, and Stuart Wilcox. Long Beach, California
tionation. The lymphocyte fraction, >95% lymphocytes by Geirnsa
stain, was removed and washed until the effluent was devoid of detectable gold. Aliquots of 0.25-0.5 ml containing 3 X l(P cells of pre- and
post-gold specimens were sonicated, and then 5-pl volumes were quantitated for gold content. This procedure included the application of an
internal standard. Aliquots taken before sonication were used to assess
the effect of superficial (membrane) bound gold on lymphocyte PHA
and concanavallin A response and t o compare these values with the
differences in lymphocyte gold content of the I-hour post-gold administration. We observed in all patients a decreased lymphocyte responsiveness and increased gold binding 1 hour post-gold administration,
when compared to responsiveness and binding of lymphocytes obtained immediately before gold administration. A consistent suppression of the post-gold injection specimen is a remarkable finding, not
attributable to usual sources of variability in the mitogen procedures.
Carbon rod atomic absorption analysis (CRA) enables the
quantification of gold in soluble and cellular materials of small sample
volume ( 1 5 p l ) containing <I0 pg of gold at a precision of 6% RSD.
The application of this technique was developed to quantitate the gold
content of circulating lymphocytes in RA subjects receiving chrysotherapy. Hence we are able t o monitor blood gold kinetics and binding
to lymphocytes and their subpopulations under in vivo conditions
during intervals following gold administration. Prior methods for in
vivo cellular quantification of gold, i.e., atomic absorption flame analysis, neutron activation analysis. or isotopic applications, have not
been practical or feasible for long-term gold administration, because
of limited sample size, detection limits, or amount of isotope required.
Circulating lymphocyte gold content was quantitated by C R A analysis
after timed periods of gold administration. Blood specimens were
collected in heparinized tubes and subjected to Ficoll-Hypaque frac-
Patient
Serum gold
fig%
Pg Au/ lo*
lymphocytes
Pre
Post
Pre
Post
1
2
3
4
5
I32
441
1310
1510
99
381
360
490
93
313
210
320
238
495
380
720
205
644
650
3470
PHA
c p m X lo3
6
7
n
9
10
94
214
360
234
306
144
45
34
229
666
295
I57
209
745
887
764
214
61 I
339
I32
-
Osteonecrosis of the Knee Without Bone Collapse
Paul A . Lotke and Malcolm L. Ecker. Philadelphia. Pennsylvania
Osteonecrosis of the knee presents with severe pain in the
knee, x-ray evidence of subchondral radiolucent lesion in the femoral
condyle, and a positive radionuclide bone scan. This report reviews
osteonecrosis of the knee and broadens its spectrum to demonstrate
that radiographic changes d o not have to be present.
Eight patients, aged 58 to 75 years old, presented with the
sudden onset of pain over the medial (7) or lateral ( I ) aspect of the
knee. The pain was worse at night and unrelieved with antiinflammatory medication. Examination revealed trace synovitis, trace effusion, and marked tenderness just above the joint line. Synovial fluid
analysis did not reveal any abnormalities. X-rays were normal in 5
patients, and 3 patients showed minimal degenerative changes. Laboratory studies did not reveal apparent cause for knee pain. In all
patients the technetium diphosphonate radionuclide bone scans were
markedly positive with definite localization of radionuclide uptake
over the affected condyle. Technetium sulfur colloid scans of 2 patients
did not reveal abnormalities in the marrow. Craig needle bone biopsies
in 2 patients revealed small spicules of dead bone without new bone
reaction. An autopsy performed on 1 patient showed an osteonecrotic
focus within the subchondral bone plate, localized within 3 mm of the
subchondral plate. Histologic section showed a well-localized necrotic
area within the subchondral plate. There was a fibrocartilage surface
and fibrous tissue reaction within the adjacent marrow space.
In summary, we have shown that osteonecrosis of the femoral
condyle can present without x-ray changes and it must be considered
in patients without other obvious causes for knee pain. Because this
problem may resemble diseases treated surgically, i.t. torn meniscus,
recognition of this diagnosis can help avoid unnecessary surgery.
Association of Autoantibodies to Certain Cytoplasmic Antigens with Systemic Lupus Erythematosus and Other Rheumatic Diseases
Peter J . Maddison, Morris Reichlin. and Thomas T. Provost, Buflalo, New York
Antibodies t o a soluble cytoplasmic antigen (Ro) have been
found in approximately 25% of patients with systemic lupus erythema-
tosus. In order t o investigate their clinical significance we have studied
60 patients in whom these antibodies have been identified by double
ARA ABSTRACTS
immunodiffusion. In 35% of cases they were accompanied by precipitating antibodies to another soluble cytoplasmic antigen, a ribonucleoprotein (La). Complement-fixing antibodies to ssDNA were present in
40%. and in 7 instances anti-Ro was associated with antibodies to
nuclear-RNP. Sixty percent of the patients fulfilled the preliminary
criteria of the ARA for SLE. The clinical course of these patients
resembled that of other patients with lupus, but noteworthy were the
presence of arthritis in almost 100%.and a higher incidence of photosensitive skin rashes. Sjogren’s syndrome occurred in 20%. All the
patients were remarkable for a high degree of hyperganimaglobulinemia and for positive tests for rheumatoid factor, which occurred in
60% of those with antibodies to Ro alone and in 100% of those who
also had antibodies to La. A further 20% had features typical of lupus
but failed to fulfill the ARA criteria. A group of 10 patients (17%) had
127
a connective tissue syndrome consistent with lupus, although 5 did not
meet the criteria, but their sera lacked demonstrable antinuclear antibodies. Of the remaining 20%. half had evidence of other connective
tissue diseases, while the other half had apparently unrelated disorders.
Although antibodies to Ro are not exclusively found in
patients with SLE, they are most commonly found in this context, with
a significant overlap with Sjogren’s syndrome. It is interesting that
certain patients respond predominantly to cytoplasmic antigens, with
a low incidence of antibodies to the soluble nuclear antigens, nuclearR N P and Sm. Furthermore, we have identified a population of
patients with lupus in whom no antinuclear antibodies can be detected
at all.
ProliferativeSynovitis in Hemophilia: Morphologic and Biochemical Observations
Carlo L. Mainardi, Zena Werb, and Edward D. Harris, Jr., Hanooer, New Hampshire
Recurrent hemarthroses in hemophiliac patients lead to synovial proliferation, deposition of iron pigment in articular structures,
and accelerated joint destruction. Most previous studies have examined the effects of hemarthrosis on articular cartilage. We report here
light and electron microscopic studies of the proliferative synovitis
along with measurements of neutral proteinase and collagenase release
by primary cultures of the synoviuni and by monolayer cultures of
enzymatically dissociated synovial cells.
Fresh tissue was obtained from synovectomy of the knee of a
10-year-old boy with hemophilia A. The synovium was deep brown in
color, quite distinct from the color of rheumatoid synovium. Microscopic studies showed synovial lining cell proliferation without inflammatory cell infiltration and iron pigment in the superficial cells.
This pigment was also seen in the dissociated cells and was found t o be
membrane-bound by electron microscopy. Many of these cells had Fc
receptors on surface membranes. After passage, Fc receptors were
found rarely, intracellular pigment decreased, and fibroblast-like cells
began to predominate the culture.
Medium from primary cultures of synovial fragments was
activated with 10 pg TPCK trypsin at 22°C for 30 minutes followed by
a 4-fold excess of soybean trypsin inhibitor, and was subsequently
assayed for enzyme activity. Large amounts of latent collagenase (73.5
14.2 p g collagen degraded at 37’C/min/mg DNA/48 h) and neutral proteinase (gelatin and azocasein substrates) were found. Lysozyme
was detectable in the medium from primary cultures but not from the
monolayer cultures. Collagenase secretion (60.25 & 13.5 pg collagen
degraded/min/mg cell protein) and neutral proteinase secretion by the
monolayers were significant until they were passaged, then the enzyme
activity fell precipitously.
We conclude that recurrent hemarthrosis in hemophilia stimulates synovial proliferation and an increase in collagenase and neutral
proteinase release by synovial cells. The uniform, dense iron deposits
are greater than those seen in rheumatoid arthritis, but the proteolytic
enzyme release is similar in quality and quantity t o that released by
rheumatoid cells in previously reported studies. This proliferative
lesion has the potential to destroy articular structures. These observations support the rationale for early synovectomy in selected joints of
certain patients with hemophilia.
+
Abnormal Collagen Synthesis by Cultured Dermal Fibroblasts in Osteogenesis lmperfecta
Ralph E. Marcus and Peter G. Bullough. New York. New York
Osteogenesis imperfecta (01)is a primary connective tissue
disorder that is broadly divided into two clinical types-a severe,
spontaneously occurring “congenita” type, and a milder, inherited
“tarda” type. Both are characterized by a diminished amount of
matrix in tissues normally rich in type I collagen, and to a lesser extent
type I l l collagen, its fetal analog.
To determine if the normal amounts of types I and 111 collagen are produced by 01 dermal fibroblasts, cells were obtained from
the skin of 6 0 1 (4 congenita, 2 tarda) and 4 normal patients and
grown in secondary monolayer culture in the presence of ascorbic acid
and 6-APN. Procollagen in the medium, labeled with C-14 glycine and
proline, was converted to collagen by pepsin digestion, purified on
DEAE-cellulose. and finally separated into component a chains o n a
CM-cellulose column.
In control cultures the ratio of type 111 to total (types I + 111)
collagen in the medium was 0.085-0.143 (av: 0.1 16). inversely dependent on patient age. In 3 of the 4 01 congenita cultures, however, the
ratios were 0.160-0.259 (av: 0.220). The ratios for the 01 tarda specimens were only slightly elevated above controls. Electron micrographs
of the cultured 01 fibroblasts and fibroblasts from intact 0 1 skin
revealed the cytoplasm to be packed with increased amounts of microtibrillar material as compared to normal controls.
Previous investigators have found that the total amount of
skin collagen is reduced in 01 patients and that urinary excretion of
cysteine, found in type 111 but not type I collagen. is increased in 01
patients. With this new data we postulate that there is a quantitative
defect in the synthesis (or transport) of type 1 collagen in 0 1 congenita,
and that in many instances there is a relative increase in the synthesis
of fetal type 111 collagen, altering the normal ratio of the two collagen
types.
Responses of Whole and T-Lymphocyte Populations to Mitogens in Patients with SLE, Rheumatoid Arthritis, and in Normal
Volunteers
Joseph A . Markenson, John W . Morgan, Catherine L. Joachim, and Michael D. Lockshin, New York, New York
To define more precisely the reason for poor lymphocyte
response to mitogens in systemic lupus erythematosus (SLE) and
rheumatoid arthritis (RA), mononuclear cells from SLE and RA
patients and from normal volunteers were fractionated and tested for
ARA ABSTRACTS
128
their responses to phytohemagglutinin (PHA), concanavalin A (Con
A), and pokeweed mitogen (PWM).
Mononuclear cells were isolated by Ficoll-Hypaque gradient
centrifugation and passed over rabbit anti-human Fab columns. In
some experiments half of the initial sample was incubated at 37OC
overnight in a glass petri dish t o remove the adherent cell population.
Whole mononuclear cell preparations or the separated or reconstituted fractions were cultured in RPMI with 10% AB serum and
varying concentrations of mitogen, and were assayed for response at 3
days by H, thymidine uptake.
In normals and RA patients the response of the columnpurified T-cell population was less than that of the unfractionated
population for PHA, Con A, and PWM. SLE patients demonstrated
an increase in the response of isolated T cells compared to unfractionated cells for PHA and Con A, but not for PWM. Removal of the
adherent cell population and subsequent fractionation also improved
the low response of unfractionated SLE cells but did not alter the
response of normal fractionated or unfractionated cells.
The data suggest a suppressor cell population among the
adherent cells in patients with SLE, which may account for poor
lymphocyte response to mitogen. The poor response of lymphocytes
from RA patients is not explained by this suppressor cell population
alone.
Mechanism of Probenecid-Induced Uricosuria: Inhibition of Reabsorption of Secreted Urate
Allen D. Meisel and Herbert S.Diamond, Brooklyn, New York
,
Probenecid-induced uricosuria may be attributed t o diminished reabsorption of either filtered or secreted urate. To study the
mechanisms of probenecid uricosuria, 72 clearance studies were performed in 18 normal subjects. Probenecid was adminstered as a I-g
intravenous dose.
PAH administration as a constant intravenous infusion resulted in a decrease in probenecid excretion (UPbV) (control UPbV.
20.1 f 5.0 pglmin; UPbV during PAH infusion, 9.2 f 3.2 pg/min), and
probenecid administration resulted in a decrease in tubular secretion
of PAH (TmPAH) (control TmPAH, 103 f I2 mg/min; TmPAH
post-probenecid, 55 f 1 1 mg/min, P < 0.01). This result suggests that
probenecid and PAH compete for secretion a t a common secretory
site, and that urinary probenecid is derived largely from secretion.
PAH administration attenuated the uricosuric response to probenecid
13 f 1.7 ml/min, P < 0.005
(dCur) (dCur during PAH infusion,
compared t o dCur with probenecid alone), a fact suggesting that
probenecid must be secreted in order t o exert its uricosuric effect. Thus
the intraluminal concentration of probenecid is likely to be low in the
early proximal tubule, where reabsorption of filtered urate occurs, and
+
higher in a more distal segment of the tubule, where secreted urate is
r eabsorbed.
Probenecid and uric acid d o not appear to compete for secretion, because urate loading by feeding yeast RNA (16 g/day) did not
alter probenecid excretion or affect the uricosuric response to probenecid (control dCur, 19.8 f 2.5 ml/min; dCur after RNA, 24.1 f 7.8
ml/min).
Pyrazinamide, an inhibitor of urate secretion, did not alter
probenecid excretion (UPbV, 14.3 f 3.6 pg/min; UPbV post-PZA,
16.6 & 3.6 pg/min). However pyrazinamide resulted in a marked
decrease in the uricosuric response to probenecid (dCur after probenecid and PZA, -0.5 ml/min). Thus intact urate secretion was required
for probenecid t o be uricosuric.
It is concluded that a ) probenecid secretion is required for its
uricosuric effect; b) probenecid and PAH are secreted a t a common
site independent of the urate secretory site; and c) probenecid uricosuria results predominantly from inhibition of reabsorption of secreted urate.
Spontaneous Activation of B Lymphocytes and Macrophages in Young New Zealand Black Mice
H. M . Moutsopoulos, M . S . Meltzer, J . J . Oppenheim and T.M . Chused, Bethesda, Maryland
New Zealand black (NZB) mice spontaneously develop autoimmune disease at about 6 months of age. Because the pathogenesis
of their disorder is not understood, we have investigated the status of
their immune system very early in life.
The activity of B lymphocytes was assessed by measuring the
IgM in spleen cell extracts, in supernatants of 4-hour cultures of spleen
cells, and in serum with an ultrasensitive two-site immunoradiometric
assay. From 2 to 5 weeks of age, 10' NZB spleen cells produced
between 21 and 32 ng IgM in 4 hours of culture, compared to I to 2 ng
by the same number of NZW or C57BI /6J spleen cells. This increased
to 340 ng at.6 weeks and 2100 ng at 10 weeks. Spleen cells from NZW
or C57B1/6J mice produced only 5-7 ng a t 8-10 weeks of age. NZB
spleen contained 52 ng IgM/IO' cells at 2 weeks, 76 ng at 4 weeks, and
250 ng at 8 weeks. NZW and C57B1/6J spleen contained 5. 6, and 7
ng/lO' cells at 2, 4, and 6 weeks, respectively. Because IgM release
requires active metabolism, because the amount released into the
medium during4 hours culture is a large fraction of that present in the
unincubated cells, and because there is no difference in viability of
NZB and control spleen cells, the elevated level of IgM produced by
the NZB lymphocytes is not due to cell lysis. NZB serum contains
0.017 mg IgM/ml at 2 weeks, 0.17 at 4 weeks, and 1.8 at 6 weeks.
Normal mice have 10-fold less at each of these ages.
The level of activation of peritoneal macrophages was assessed by the degree of monocyte chemotactic responsiveness and by
the amount of lymphocyte activating factor (LAF, assayed by the
stimulation of thymidine incorporation by mouse thymocytes) produced by supernatants of 72-hour cultures of adherent peritoneal cells.
Both chemotactic responsiveness and L A F production of peritoneal
macrophages from 6-week old NZB mice were elevated 6- t o 8-fold
above controls.
These findings indicate that both splenic B lymphocytes and
peritoneal macrophages are spontaneously activated in NZB mice by 6
weeks of age, long before they develop symptoms of autoimmune
disease. Whether this phenomenon is part of an immune response
(perhaps to an unusually presented self-antigen) or a primary abnormality that may initiate the autoimmune sequence remains to be
determined.
ARA ABSTRACTS
129
Neutral Protease from Bovine Nasal Cartilage that Digests Proteoglycan
Hideaki Nagase and J . Frederick Woessner, Jr.. Miami, Florida
The observation that highly-purified cathepsin D is unable to
digest proteoglycan at pHs greater than 5.5 prompted a search for
proteases in cartilage that could act at physiologic pH. Such a protease
has been detected and purified from bovine nasal cartilage, based o n
an assay employing proteoglycan subunit (PGS) of bovine nasal cartilage as substrate. Following a suggestion of A. J . Barrett, the PGS is
incorporated in beads of cross-linked polyacrylamide gel. Uronicacid-containing fragments are released from the beads following protease action on the protein backbone of PGS. The bovine enzyme was
purified about 4000-fold from crude cartilage extracts by cetylpyridinium chloride precipitation of polysaccharide material, 40-7570 ammonium sulfate fractionation of the supernatant, chromatography on
Bio-Gel P- 100, DEAE BioGel-A Sephadex G-50, and C M Bio-Gel-A.
The protease has an apparent molecular weight of approximately
15,000daltons based on Sephadex G-50 filtration. The p H optimum is
7.5-8.0, and split products of PGS are shown t o arise by proteolysis,
not by splitting of carbohydrate chains. T h e protease is inhibited by
EDTA and o-phenanthroline, and activity is restored by several divalent cations.
It is concluded that the enzyme is a metalloprotease. N o
effect is produced by soybean trypsin inhibitor, TPCK, TLCK,
AcAla,CK, Trasylol, iodoacetate, cysteine, EACA, or pepstatin.
These properties distinguish the cartilage protease from proteases
found in leukocytes and synovial membrane. Such an enzyme may
play a role in normal matrix turnover and in pathologic destruction of
the matrix.
(Supported by NIH Grant AM-16940.)
Impaired in Vitro Immunoglobulin Synthesis by Peripheral Blood Lymphocytes in SLE
Kenneth M . Nies and James S . Louie, Torrance, California
Despite increased in vivo synthesis of immunoglobulin (Ig) in
systemic lupus erythematosus (SLE), lymphocytes stimulated in vitro
with pokeweed mitogen (PWM) show a depressed Ig synthetic response.
Peripheral blood lymphocytes (PBL) (2 X IP) isolated from
20 patients with SLE and 20 normal individuals were incubated at
37°C for 7 days in RPMl 1640 with 10% A B serum with and without
PWM. The cultures were washed and pulsed with 'H-leucine for 6
hours in Medium 199 without leucine. Secreted Ig was isolated by
immune precipitation and compared to total TCA precipitable radioactivity. Classes of Ig secreted were determined by polyarcylamide gel
electrophoresis (PAGE).
The total amount of Ig secreted, expressed in cpm, was decreased in SLE compared to normal lymphocytes (6.6*6.4 X I @ vs
24.2f I I .6X IDS), P<O.001. A smaller difference resulted when secreted
Ig was expressed as a percentage of secreted protein (15.5f13.7% vs
24.7f I I % ) P=0.05. Unstimulated PBL cultures in both groups secreted little or no Ig. On PAGE, the major Ig class synthesized by PBL
in SLE was IgG>lgA>lgM, whereas normal lymphocytes synthesized
IgM>lgA>IgG. Co-culture of SLE lymphocytes with normal PBL
and PWM produced a slight but not significant increase in Ig synthesis. Co-culture of SLE PBL with nylon wool separated normal
lymphocytes (<4% membrane Ig bearing cells), and PWM led to a
further depression of Ig synthesis. All abnormalities were most
marked in patients with clinically active disease and those taking
> lOmg/d prednisone.
These findings suggest that nonspecific stimulation of PBL is
impaired in SLE and cannot be normalized by co-culture with normal
PBL. IgG rather than IgM synthesis suggests a fundamental functional difference between SLE and normal PBL.
Modulation of Human Lymphocyte Response by Cartilage Proteoglycans
Masaru Nozoe, Marie V . Dennis, and Jerome H . Herman, Cincinnati, Ohio
Highly negatively charged proteoglycans (PG), in part sharing antigenicity with those contained in cartilage matrix, have been
detected in pathologic synovial fluids and a cell-mediated immune
response (CMI) to disruptive P G preparations identified in patients
having diverse cartilagenous destructive disorders. Because immunologic events potentially play a significant role in the pathogenesis of
articular inflammation, we determined the effect of a broad range of
doses of more highly purified PG upon modulation of spontaneous
DNA synthesis and phytomitogen (PHA-M) responsiveness of normal
and rheumatoid (RA) peripheral blood lymphocytes (PBL) derived by
Hypaque-Ficoll gradient sedimentation.
PG were isolated by dissociative measures from normal human cartilage in conjunction with cesium gradient ultracentrifugation
under aggregative and dissociative conditions to provide aggregate,
link protein, and subunit fractions. PBL were cultured for 3, 5, and 7
day periods in microtiter wells containing cells alone, cells + P H A
+ PG fractions ranging from 200 t o 0.001y,and cells
+ P H A + P G in varying doses. D N A synthesis was assessed following
(25-1OOy), cells
3H-thymidine pulsing.
Without affecting PBL viability, PG significantly modified
spontaneous D N A synthesis in both a stirnulatory and/or suppressive
manner, depending upon the fraction employed, its concentration, and
the duration of cell exposure. PG were able to'modulate mitogeninduced D N A synthesis in a similar manner, and total CPM was
potentially suppressed and/or enhanced, depending on the quality and
quantity of fraction employed. A dichotomy in mitogen response was
observed wherein P G suppression of spontaneous D N A synthesis was
associated with enhanced PHA responsiveness, spontaneous activation of synthesis by PG, and a suppressed PHA effect. RA response
could be clearly differentiated from that of normal subjects. It thus
appears that PG in synovial fluid, o n a concentration-dependent basis,
may potentially modulate CMI responses.
ARA ABSTRACTS
130
Cytotoxic Activity of Rheumatoid Arthritis Sera on Rheumatoid Synovial Cell Cultures
Stephen A . Paget. Karen Anderson, and Paul E. Phillips, New York, New York
The immunopathology of rheumatoid arthritis (RA) is consistent with a response to a chronic antigenic stimulus. Cell cultures
from RA synovial membranes have persistent biochemical and immunologic abnormalities that suggest the in vivo abnormalities may
persist in vitro. We have tried t o determine whether R A sera contain
antibodies specific for persisting antigens in RA synovial fibroblast
cultures by using a complement-mediated cytotoxicity assay.
Synovial membranes from 16 patients with definite RA for
0.1-30 years and 10 non-RA control patients were trypsinized and
cultured in medium with 20% heat-inactivated fetal calf serum for 1-30
months and 1-9 subcultures before testing as confluent monolayers in
Linbro 24-well multiplates. Sera obtained aseptically from 2 I patients
with definite RA for 0.3-30 years ( 8 of whom had synovial cultures as
well) and non-RA controls were stored at 4'C and heat-inactivated
before testing. The RA and control synovial cultures were 61 Crlabeled (Na Chromate, 100pCi/ml in medium at 37OC, 90 min) and
washed 6 times. RA and control sera were diluted at 1:2 and 1:8 in
medium containing 1:32 freshly thawed rabbit serum (stored at
-70°C) as a source of complement and added to the cultures. After
incubation (37°C. 60 min), serum dilutions were removed and Cr
cpm counted; the cells were then removed by trypsinization and 61 Cr
cpm counted. Percent cytotoxicity was calculated as serum cpm X
100/cpm in cells cpm in serum. The positive control was rabbit antinon-RA synovial fibroblast antiserum; negative controls were included. R A sera were tested on both R A and non-RA cells. Preliminary control experiments with herpes simplex virus (HSV)-infected
non-RA synovial fibroblasts and HSV-antibody containing sera from
both normal and RA patients showed that their HSV cytotoxic antibody titers were similar to their virus-neutralizing antibody titers,
and that other constituents of RA serum did not interfere with this
known antigen-antibody system. One hundred and twenty experiments using both autologous and heterologous RA culture-serum
combinations were then done. No increased cytotoxicity over controls
was found. Some RA sera showed anticomplementary activity, which
was overcome by using rabbit complement at 1:8; repeat testing of 12
RA sera against 7 RA cultures showed similar negative results. Thus,
in this system, R A sera did not contain detectable antibody specific for
a persisting antigen in cultured R A synovial fibroblasts.
+
Circulating Complement-Fixing Immune Complexes in Mixed Connective Tissue Disease
Michael D . Parker and Tony Marion, Winston-Salem, North Carolina
The presence of high titers of antinuclear antibodies in mixed
connective tissue disease (MCTD) reactive with nuclear ribonucleoprotein (RNP) suggests that immunologic factors might be important
in the pathogenesis of this illness. T o determine whether circulating
immune complexes (IC) might contribute to tissue injury, we have
assayed to date sera from 8 patients with MCTD for presence of IC.
Results were compared to those from 63 patients with other rheumatic
diseases, including 8 with SLE, 46 with rheumatoid arthritis (RA), 3
with scleroderma, 1 with eosinophilic fasciitis, 2 with R A vasculitis, 2
with Reiter's disease, and 1 with psoriatic arthritis. The recently described Raji cell assay was employed to detect IC. Complement-fixing
IC bind t o these lymphoblasts via surface complement receptors.
Surface-bound IC are then detected by immunofluorescence. The sensitivity of the assay was 15 pg IC/ml Serum (Table).
Results demonstrate that circulating immune complexes are
found consistently in M C T D and thus establish MCTD as an illness in
which vascular deposition of IC may play an important pathologic
role. The frequency with which IC were detected was comparable t o
that in SLE, wherein IC have been previously found in a similarly high
percentage of patients. IC were infrequently found in uncomplicated
RA. Preliminary studies indicate that titers of IC were equivalent in
SLE and MCTD. Despite the presence in M C T D of circulating IC
with the capacity to fix complement, serum concentrations of C3 were
normal by radial immunodiffusion. In SLE, IC-especially those involving DNA-have been implicated in the pathogenesis of nephritis.
The absence of nephritis among patients with M C T D despite the
presence of circulating IC suggests that there is a fundamental difference in the pathologic potential of 1C involving D N A and those
involving RNP.
Disease
No. of
patients
Percent
positive
MCTD
SLE
RA
RA-Vasculitis Other
8
8
46
2
7
100
88
4
loo
30
.
Characterization of an IgG, Rheumatoid Factor
David N. Podell, Martin Isenman, John J . Condemi, and George N. Abraham, Rochester, New York
Understanding of the unique properties of rheumatoid factors has been clarified by structural studies of these anti-IgG autoantibodies. These investigations have been primarily confined t o monoclonal IgG-K or IgM-K rheumatoid factors, because automated amino
acid sequence analysis of most antibodies with lambda chains is difficult due to blocked amino termini. Protein LON is an IgG,-X cryogel
rheumatoid factor isolated from a patient with Atrophie Blanche, a
vasculitis that affects only lower extremities and occurs primarily in
middle-aged women. It is the first reported human monoclonal IgG-X
rheumatoid factor with a defined antigenic specificity and an unblocked amino terminus. Upon analytical ultracentrifugation of se-
rum, this protein sediments as a very prominent 7.5 spike, which
decreases t o normal concentration after serum is depleted of protein
by cryogelation. Immunodiffusion, immunoelectrophoresis, and liquid
and polyacrylamide gel electrofocussing were employed to identify
and elucidate the nature of the isolated cryogel. LON consisted of at
least two serum components: an IgG rheumatoid factor of restricted
heterogeneity, and the third component of complement, with isoelectric points at pH 6.75 and 4.83 respectively. Reduction and alkylation
of the intact immunoglobulin molecule yielded heavy and light chains
which were monoclonal by isoelectric focussing. Automated amino
acid sequence analysis revealed the light chains to belong to the V A-iii
ARA ABSTRACTS
variable region subgroup. The antigenic specificity was determined by
hemagglutination-inhibition assay and was nearly equivalent for intact
IgG and isolated heavy chains of subclasses 1,2, and 3, but not IgG,.
Reactivity was with an antigenic determinant present on the Fc region
of IgG. The specificity pattern noted has been reported for some
131
monoclonal IgM-K rheumatoid factors. This protein will be useful in
determining the structural similarities or differences between autoantibodies with the same antigenic specificity, but with different light chain
variable region subgroups.
Chemotactic Attraction of Fibroblasts to a Fragment from the Fifth Component of Complement
Arnold E. Postlethwaite. Ralph Snyderman. and Andrew H . Kang. Memphis, Tennessee and Durham, North Carolina
Fibroblasts accumulate at sites of tissue damage and inflammation and synthesize new connective tissue elments that constitute the scar. The mechanism(s) by which these effector cells are
attracted to such areas is poorly understood. The complement system
participates in inflammatory reactions resulting from immune or nonimmune etiologies. We have examined the complement system for a
possible source of a chemotactic stimulus for fibroblasts, and have
found that activation of the complement sequence by either the classic
or alternate pathways generates a factor that is chemotactic for human
dermal fibroblasts.
Aliquots of normal human serum were incubated with heataggregated y-globulin (HAGG), zymosan (Z), lipopolysaccharide
(LPS), or saline (S) fractionated by gel filtration, and effluent column
fractions were assayed for fibroblast and monocyte chemotactic activity (CTX). Fibroblast CTX was measured by a sensitive in vitro assay
that employs gelatin-treated polycarbonate filters and modified Boyden chambers. Separate peaks of fibroblast CTX (MW: 48,000daltons) and monocyte CTX ( M W 14,000 daltons) were found when
HAGG, Z, or LPS activated serum samples were fractionated, but
such activity was not found when S- treated serum was fractionated.
When fractions from the 48,000and 14,000 dalton peaks weyreacted
with monospecific antibody to C5, fibroblast and monocyte CTX in the
respective samples were destroyed. The 14,000dalton factor has been
previously described as being C5a, a potent anaphylatoxin.
Limited proteolysis of the 48,000 dalton factor by trypsin
conjugated to polyacrylamide beads was performed, and the reaction
products were fractionated by gel filtration. A factor with a MW of
14,000daltons with chemotactic activity for monocytes was recovered,
suggesting that the fibroblast chemotactic factor contains a subunit
similar to or identical with C5a.
Activation of the complement cascade generates a fragment
with a MW of 48,000daltons, presumably from the fifth component of
complement, that is chemotactic for fibroblasts but not for monocytes.
This factor might act as a chemoattractant for fibroblasts in inflammatory reactions mediated by the complement system.
Substantial Purification of the S m Antigen and Association of High Titer Antibody to Sm with a Clinical Subset of Systemic Lupus
Erythematosus (SLE)
Runas Powers, Masashi Akizuki, Marilyn J . Boehm-Tmitt, Virginia Daly, and Halsted R. Holman, Stanford, California and Bethesda,
Maryland
Specific autoantibodies are associated with certain subsets of
SLE, i.e., antibody to DNA associated with nephritis and antibody to
nuclear ribonucleoprotein (RNP) associated with mixed connective
tissue disease (MCTD). We describe another subset associated with
high titer antibody to the Sm antigen and present data on the purification of the Sm antigen.
The usual source of the Sm antigen is in the soluble nuclear
extract from mammalian cells, where the antigen is thought to be
inseparably associated with RNP. However RNP-free Sm antigen can
be recovered by a digestion of isolated, insoluble chromatin with
DNAse. Ammonium sulfate (3040%) fractionation of the supernatant from DNAse-digested chromatin yields the RNP-free Sm antigen, as determined by the appropriate pattern of enzyme sensitivity
using agar diffusion, passage hemagglutination and complement fixation. Chromatin Sm antigen is further purified by affinity chromatography, using Sepharose 4B activated by cyanogen bromide coupled
with IgG that has been separated from selected sera of patients with
SLE and higher titer antibody to the Sm antigen. The resultant preparation migrates as an apparent homogenous and immunologically
reactive band on polyacrylamide gel electrophoresis.
Chromatin Sm is stable between pH 5 and 10, but its antigenic activity is lost after heating at 60°C for 45 minutes. Sm antigen is
stable in lyophilized form, has certain characteristics of an acidic
glycoprotein, is resistant to enzymatic digestion with RNAse and
DNAse, but is gradually digested by tryspin.
Sixteen consecutive patients with high titer antibody to the
Sm antigen without demonstrable RNP antibody were characterized
by major skin lesions, clinically mild and apparently nonprogressive
nephritis, substantial hypocomplementemia, significant antibody to
DNA (demonstrated by binding assay but not by passive hemagglutination), and positive LE cell tests. On the basis of these observations,
these patients can be distinguished by at least some characteristics
from classic SLE with antibody to DNA, from MCTD, and from
patients with antibody to the Ha antigen.
Factors Affecting the Wear of Articular Cartilage in Vitro
Eric L. Radin, Nicholas J . Myttas, David A . Swann, and Igor L. Paul, Boston, Massachusetts
I t is usually assumed that the degradation of articular cartilage by enzymes is enhanced by mechanical factors. The relative
contribution of these two mechanisms and the relationship between
the wear rate and the chemical structure of the cartilage, however, are
not known.
In an attempt to answer some of these questions, bovine
metatarsal-phalangeal joints were worn under various conditions in a
mechanical system and the degree of wear of articular cartilage was
determined histologically. The treatments used to alter the structure of
the articular cartilage included extraction with 4 M guanidinium chloride, fixation with glutaraldehyde, scarification (A la Meachim) to
disrupt the surface, and replacement of subchondral bone under the
contact area with methacrylate.
The results showed that wear was enhanced when sub-
I32
ARA ABSTRACTS
chondral area was stiffened with methacrylate, when the cartilage was
extracted with 4 M guanidinium chloride and then the wear test run
with veronal buffer as the lubricant, and also when the wear test was
performed with 4 M guanidinium chloride as a lubricant without prior
extraction of the cartilage. Scarification alone had n o effect, whereas
glutaraldehyde fixation of the cartilage retarded wear.
These data suggest that the proteoglycans contribute significantly to the wear characteristics of articular cartilage, in that under
conditions in which these constituents were removed from the cartilage. the wear was increased, whereas following crosslinking reactions
that prevent loss of proteoglycan, wear .was decreased. These conclusions, based on the depth of cartilage remaining after the wear test,
were supported by histochemical analyses. More detailed chemical
studies are now in progress to confirm these observations.
(Supported by N I H Grant No. A M 15216.)
Sex Differences in the Formation of Anti-T-cell Antibodies
E. S. Ravecht, L . Kfassen. and A . D. Steinberg, Bethesda, Maryland
Hybrids of NZB and DBA mice were studied at I year of age
for the presence of naturally occurring thymocytotoxic antibodies
(NTA). All NZB parents had NTA, whereas none of the DBAs did.
Half of the F, mice of both crosses were castrated at 5 weeks of age in
order to determine the contribution of sex hormones to the mice. The
titer was the last dilution giving greater than 50% I’ Cr release. A
positive animal had a titer of a t least 1:4 (Table).
Castration of the females did not affect the development of
NTA, whereas manipulation of the males did. Castrated males
reached the level of their normal female littermates. The data suggest
that the lack of NTA in intact male NZB hybrids may be due t o the
presence of male sex hormones that suppress the development of
autoimmunity.
Naturally Occurring Thymoeytotoxic Antibodies ( N T A )
Positive NTA Test
F, Hybrid
NZBQ X D B A d
NZBQ X D B A d
NZBQ X D B A d
NZBQ X D B A d
DBAO X N Z B d
DBAQ X N Z B d
DBAQ X N Z B d
DBAQ X N Z B d
Sex
Q
9 castrated
6
castrated
No./Total
Percent
11/20
9/16
0/15
10/18
55
53
0
55
2/8
2/7
0/6
2/7
25
29
0
29
0
9 castrated
d
6 castrated
Synovitis and Periostitis in Secondary Syphilis
A.J. Reginato. H.R. Schumacher, F. Valenzuela. S. Jimenez, and K . Maurer. Philadelphia, Pennsylvania and Santiago, Chile
Arthritis is frequently described in secondary syphilis, and
there have been n o previous studies t o support an infectious basis for
the arthritis or periostitis. This report describes clinical, dark-field,
serologic, and pathologic studies in 7 cases presenting synovitis or
periostitis. There were 6 women and 1 man. Ages were 18-44. The
diagnosis of a secondary syphilis was documented by maculopapular
generalized rash in 7, oral or perigenital mucus plaques in 5 , positive
STS and FTA absorption in 7, and positive dark-field examination of
the perigenital mucus plaques in 5 . N o patients had antinuclear antibodies.
Two patients presented with elastic, round, mildly painful,
localized swelling of the scalp. X rays showed localized bone resorption of the outer table of the skull. Bone biopsy in I case revealed
subperiosteal inflammation with gelatinous exudate. There were abundant spirochetes on dark-field study. Four patients had symmetric
nonmigratory polyarthritis involving MCPs, PIPS, wrists, knees, and
ankles. One of these and one additional patient without arthritis had
symmetric tenosynovitis at wrists and ankles. Musculoskeletal manifestations appeared at the same time as rash and other systemic aspects
of secondary lues. Synovial fluid in the 4 patients with arthritis was
5-20 cc, cloudy, and yellow with 4,100-13,OOO WBC. PMN predominated in 2 effusions, while PMN and mononuclear cell numbers
were similar in 2. Dark-field, silver stain, EM, and fluorescent antibody study on I fluid were negative for treponemes; routine cultures
were negative. Synovial needle biopsy in 3 cases showed synovitis with
infiltration by lymphocytes, plasma cells and PMN, vascular congestion, and increased synovial lining cells. Electron microscopy in
these 3 synovial membranes in addition showed many necrotic PMN,
much interstitial debris and fibrin, and in I patient bodies characteristic of treponema pallidum in the interstitium and vessel lumens.
There were no electron-dense deposits in vessel walls. In all cases the
periostitis and tenosynovitis rapidly subsided with penicillin. Two
patients had Herxheimer reactions.
Periostitis of the skull, tenosynovitis, and symmetric polyarthritis can occur in secondary syphilis along with the mucocutaneous manifestations. Spirochetes can occasionally be identified in
subperiosteal and synoival lesions.
Recurrent Myoglobinuria Due to Muscle Carnitine Palmityl Transferase Deficiency
Michael J . Reza. Nirmal C . Kar, Pieter Kark. and Carl M . Pearson, Los Angeles, California
Recurrent myoglobinuria ( R M ) is a dramatic syndrome commonly thought to be “idiopathic.” The identification of specific defects
in muscle carbohydrate metabolism, e.g. myophosphorylase deficiency
(McArdle’s syndrome), has led to improved diagnostic ability. Yet
there remain patients with RM in whom no defect in carbohydrate
metabolism is apparent.
Bank et a / have recently described 2 brothers in whom defec-
tive muscle lipid metabolism due to absence of muscle carnitine palmityl transferase (CPT) was associated with RM. CPT is an enzyme that
facilitates the transfer of cytoplasmic long-chain fatty acids into mitochondria, where oxidation takes place. We have since assayed CPT
activity in frozen muscle tissue in all available cases of “idiopathic”
R M a t the UCLA Medical Center during the past 10 years. Of I I
patients studied, 3 were found to have absent or markedly reduced
ARA ABSTRACTS
levels of skeletal muscle CPT, compared to 9 normal controls and 5
patients with intermittent muscle cramps without myoglobinuria.
Three patients had myophosphorylase deficiency. The 3 CPT-deficient
patients had distinctive histories of life-long episodes of muscle pain,
weakness, and cramps associated with myoglobinuria. The epidodes
were usually exercise- induced, especially with prolonged exercise after
fasting, but sometimes occurred spontaneously and could be induced
by fasting alone. Patients were entirely normal between attacks. As
opposed to patients with McArdle’s syndrome, these patients could
sometimes engage in vigorous muscle activity without ill effects. In the
I patient so studied, fasting-induced ketosis was absent, and excess
133
lipid was demonstrated on muscle biopsy. Administration of the ketone body, beta-hydroxybutyrate, to this patient resulted in a prompt
increase in muscle strength. Our studies show that CPT is probably
absent in the liver as well, and emphasize the importance of lipids as a
fuel for muscle energy metabolism.
In summary, of I I patients studied with RM, we have uncovered 3 additional cases of CPT deficiency, hitherto described in only 2
patients in the world’s literature. In the future, many of the cases of
what we now label as “idiopathic” RM may well be traced to specific
metabolic abnormalities.
Eosinophilia and Serologic Abnormalities in Linear Localized Scleroderma
Gerald P. Rodnan. Esther Lipinski. Bruce S . Rabin, and Morris Reichlin, Pittsburgh, Pennsylvania and Bufalo, New York
The presence of both rheumatoid factor and antinuclear antibodies, including anti-DNA antibody, has been described in patients
with localized (or focal) scleroderma (Hanson et al: Pediat Res
8:806-809, 1974). In an effort to obtain more information concerning
the nature of the antinuclear antibodies present in these individuals,
we have studied 17 patients with linear localized scleroderma (1 3 F, 4
M), I 1 patients with morphea (single or multiple plaques) (6 F, 5 M),
and 2 patients with scleroderma en coup de sabre (2 F).
Linear localized scleroderma: Rheumatoid factor (RF) was
present in titers of 1:80-1:2560 in 4/16 patients. Antinuclear antibody
(ANA) was detected (immunoflourescent technique utilizing rat liver
slices) in 7/15 individuals in titers of 1:64-1:256, with a speckled
pattern in all. As determined by a radioimmunoassay-double antibody
technique, high titers of antibody to single- stranded deoxyribonucleic
acid (denatured E coli and calf thymus DNA) were found in the sera of
6/6 patients with strong ANA reactions and in 1 young girl in whom
this latter test had been only weakly positive. Four of these seven sera
fixed complement with heat-denatured calf thymus DNA, while only
one yielded a weakly positive complement fixation reaction with native
calf thymus DNA. Levels of C3 and C4 components of serum complement were normal in 14/14 cases. Anti-ribonucleoprotein antibody
(anti- RNP) was not found (I4 cases). Peripheral eosinophilia. ranging
from 7 to 14% of the total white blood cell count, was noted in 9/14
patients and exceeded 600 eosinophiles per mms in 6. Both the eosinophilia and the serologic abnormalities were limited to patients with
evidence of clinically active disease. These observations, together with
the presence of heavy infiltrations of lymphocytes in the affected skin,
subcutaneous tissue, and muscle suggest that immunologic mechanisms may play an important role in the pathogenesis of this form of
localized scleroder ma.
Morphea and scleroderma en coup de sabre: Serum immunoglobulin and complement levels (C3, C4) were normal in all patients,
and serum RF, ANA, anti-I)NA, and anti-RNP reactions were.
uniformly negative, with the exception of a low titer of anti-RNP
(l:2560) in 1 case.
Augmentation of Immunoglobulin (Ig) Production in Connective Tissue Disorders (CTD)
I . Jon Russell, Doyt L . Conn, Charles H . McKenna. and John D. Stobo. Rochester, Minnesota
Compelling evidence indicates the importance of soluble
products of lymphocyte metabolism in the regulation of Ig synthesis
by B lymphocytes. We have investigated the possible relationship of
such factors to polyclonal elevations of Ig noted in 26 patients with
systemic lupus erythematosus (mean total serum Ig = 20.1 mg/ml),
and 6 patients with mixed connective tissue disease (mean total serum
Ig = 18.3 mg/ml, normal = 14.2 mg/ml).
Peripheral blood mononuclear cells (PBMC) from these
patients and 16 normal individuals were suspended in 5% fetal calf
serum (FCS) and incubated in a humidified atmosphere of 95% air, 5%
CO,, at 37OC. At 2, 4, and 7 days, aliquots of culture fluid were
removed and added, in a final concentration of 25%. to allogeneic
normal PBMC in 5% FCS. These admixtures were then cultured for 7
days, without any allogeneic normal PBMC in 5% FCS. These admixtures were then cultured for 7 days, without any mitogens. The net
total Ig synthesized and released during this time was measured by a
competitive binding radioimmunoassay.
After 48 hours of culture, PBMC from 18 of the patients
(mean total serum Ig = 20.7 mg/ml) released a soluble material (AF)
capable of augmenting Ig production (mean 6.0-fold). In contrast,
culture fluids from 14 patients (mean total serum lg = 19.6 mg/ml)
and the 16 normals failed to do so (mean augmentation = 1.1-fold).
Comparable augmentation of lg synthesis by A F occurred despite
variable histocompatibility differences between patients and the target
PBMC. Culture fluids from patients’ T-cell enriched (92 4% T ) or Bcell and monocyte enriched (50 f 4% lg bearing, 40 f 8% esterase
positive cells) preparations were relatively depleted of AF. This result
indicates that interactions between these two populations may be
required for its production. The ability of A F to augment Ig synthesis
among target populations depleted of T cells was markedly reduced, a
fact suggesting that augmentation of Ig synthesis by A F was T dependent. However AF was not blastogenic for T cells. Fractionation of
culture fluids by column chromatography and density gradient centrifugation indicated that A F has a molecular weight of approximately
190,000 daltons. Most importantly, AF could be specifically depleted
by solid support media to which anti-lg or monoclonal rheumatoid
factor was covalently linked.
These studies suggest that in some patients with CTD and
hypergammaglobulinemia, an immune complex may augment Ig synthesis by B cells, perhaps through its effect on regulatory T-cells.
+
134
ARA ABSTRACTS
Multiple Forms of Neutral Metalloprotease Activity from Human Cartilage
Asher I . Sapolsky, Harold D . Keiser. David S. Howell, and J . Frederick Woessner, Jr, Miami. Florida and Bronx, New York
Human articular cartilage contains a novel metal-dependent
neutral protease that can digest the proteoglycan of the cartilage
matrix. Because this protease also penetrates cartilage slices and depletes them of proteoglycan, it is postulated that it may play a role in
the pathogenesis of osteoarthritis and in normal matrix turnover.
The neutral protease can be resolved into four distinct electrophoretic forms distributed between two weight groups of I1,OOO
and 22,000 daltons. The forms are cationic, with isoelectric points up
to pH 8.4. The protease forms are all inhibited by o-phenanthroline
and D-penicillamine; this inhibition is reversed by zinc, cobalt, and
ferrous ions. Three of the four protease forms are inactive against
casein and histone, a fact indicating a certain degree of specificity for
the proteoglycan substrate. This protease is believed to belong to a
previously undescribed group of mammalian metalloproteases.
A 400-fold purified preparation of this neutral activity that
includes all the forms digests proteoglycan subunit optimally at pH
7.25, and produces fragments containing 5-12 chondroitin sulfate
chains. However it does not digest the link proteins of proteoglycan
complex nor destroy the antigenic determinants of the complex or link
proteins. No single or fragmented chondroitin sulfate chains are produced. Thus the human cartilage neutral protease degrades proteoglycan by a limited attack on its protein core.
Clq Metabolism in a Clq-Deficient Lupus Syndrome
Frank R . Schmid, Adoracion F. Suarer, Chester R . Zeiss, Adrian M . Jaffer,and Henry Gewurz, Chicago, Illinois
An unusual lupus-like syndrome (urticaria, erythema multiforme, vasculitis) has been described, in which Clq levels are disproportionately reduced compared to other complement proteins. We
studied the metabolism and the assembly of radiolabeled Clq from
normal donors in 2 such patients. The Clq tracers gave a single sharp
1 IS peak on ultracentrifugation and a single preciptin line by autoradiography in gel diffusion against whole human antiserum. They
were >80% precipitable by immune complexes, and <0.67 pg restored
hemolytic activity to 0.1 ml RClq reagent. In contrast to normals and
classic lupus subjects, both patients showed an accelerated catabolism
Patient
Clq1-D
Clql-B
SLE-L
SLE-R
Normal-S
Normal-Z
of Clq without marked change in synthesis (Table ).
Addition of the Clq preparation to one Clq-deficient serum
doubled the number of functional CI molecules. In both patients 1 IS
Clq rapidly became incorporated into a series of heavier (> 11-19s)
units after intravenous injection or after in vitro addition to serum.
Such units were dissociable by EDTA into lighter (<15-llS) units.
Thus Clq appears to be formed and assembled normally into larger
(CI?) units in these patients, but it is more rapidly catabolized by
mechanism(s) still to be determined.
Clq level
Olg/ml)
TM
(hrs)
Hourly Fractional Plasma
Pool Catabolism
Hourly Plasma Pool
Turnover Rate (mg/kg)
32
14
.065
.064
45
8
28
28
32-48
39
.02
,015
.015
,096
.092
.072
160
180
180
250
-
-
Decreased Cytotoxic Potential in Synovial Fluid in Rheumatoid Arthritis
Stuart L. Silverman, Donna M . Yonkosky, and Edgar S. Cathcart, Boston, Massachusetts
Cytotoxicity in synovial fluid of patients with RA was found
to be markedly decreased (7%) as opposed to synovial fluids of
patients with gout (73%) and septic arthritis (50%). In contrast, cytotoxicity in peripheral blood of patients with RA (31 f 12%) was
found not to be significantly different (31 & 12%) from that ofcontrols
(24 f 12%). The amount of slCr released depended dosage of PHA
and was directly related to the number of effector cells in suspension.
There is evidence that both polymorphonuclear leukocytes
and monocytes are effector cells in this assay (J Clin Invest 57:826,
1976). However, despite high percentages of both these cell lines in
synovial fluid of patients with RA, the cytotoxicity was found to be
markedly decreased. This abnormality in synovial fluid as opposed to
normal findings in peripheral blood suggests a local phenomenon in
synovial fluid. These observations have relevance in the clearer under-
standing of the total cytotoxic potential of synovial fluid in RA and
other disease states.
Cellular cytotoxicity of synovial fluid and peripheral blood
cells using the mitogen-induced cellular cytotoxicity (MICC) assay
was studied in patients with erosive, seropositive rheumatoid arthritis
(RA), and in controls.
Hyaluronidase-pretreated synovial fluid cells and Ficoll-Hypaque-purified peripheral blood cells were cultured with chromated
Y r chicken red blood cells in the presence of phytohemagglutinin
(PHA). Percentage of cytotoxicity was determined by the release of
W r from chicken red blood cell targets. Monocytes were quantified by
the nonspecific serine esterase stain (NSE). Polymorphonuclear
(PMN) and mononuclear (MN) cells were determined by a Wright
Giemsa stain.
Synovial Fluid Cytotoxicity (Mean f Standard Deviation)
Diagnosis
RA
Gout
Septic arthritis
WBC/m ma
% PMN
% MN
% NSE
% MICC
13,000f6.000
9,700f700
37.000f 17,000
6 0 f I4
86f13
91f 6
40f I4
15f10
IOf 6
17f3
7f 2
73fll
50f20
10
2fI
ARA ABSTRACTS
135
Total Hip Replacement (THR) in Children with Arthritis
Bernhard H . Singsen. Alvin S . Isaacson, Michael J . Patzakis, Bram H . Bernstein, Helen K. Kornreich, Karen K . King,
and Virgil Hanson, Los Angeles and Downey. California
THR for rheumatoid hip involvement in children under 18
years old has received little attention in the literature. Between 1972
and 1975, 8 girls and 6 boys with severe, polyarticular juvenile rheumatoid arthritis, and 2 boys with ankylosing spondylitis, received 29
THRs. The median age at disease onset was 6.8 years (range: 1-13),
the median duration of hip involvement was 7 years (range: 2-12), and
the median age at THR was 15.2 years (range:12-18). All 29 hips had
radiographic evidence of severe destructive changes, and none showed
evidence of further growth potential. Clinically, 5 of 29 THRs were for
pain alone, 16 for pain and severe flexion contractures (FC) and 8
because of severe FC alone. Among the 16 children, 7 were complete
nonambulators, 6 household ambulators with support, and 3
limited community ambulators without support. At the time of THR
12 children had severe, bilateral knee involvement; 4 had normal
knees. Previous hip surgery, done in 5 patients, included 8 synovectomies, 6 soft tissue releases, and 1 cup arthroplasty. The median
preoperative hip FC was 35" (range: 10-85'). median hip flexion range
of motion (ROM) was 25" (range: 0-80"), and the median abduction
was 8' (range: - 15-35"). All prostheses were the Charnley type, and
many were custom made because of small patient size. The greater
trochanter was transplanted in 26 of 29 cases. Postoperative complications were limited to low-grade fever (<38.5") in 20 cases All 29 cases
had 1 day of preoperative and 3 days of postoperative intravenous
antibiotics.
The median follow-up duration was 2.2 years (range:l-4), at
which time hip ranges were: median FC 20" (range: 0-45'), median
hip flexion ROM 61" (range: 35-105'), median abduction 20" (range:
-5-35"). All children had complete pain relief after the initial postoperative period. The ambulatory status included 4 full community
ambulators, 7 limited community ambulators, 4 household ambulators with support, and I non-ambulator. Late postoperative complications were 2 femoral shaft stress fractures, 1 acetabular component
subluxation, 1 acetabular cement stress fracture, and 1 painful trochanteric wire. At follow-up, 15 of 16 children felt they were improved. THR can be successful in the severely afflicted rheumatoid
child. Motivation and cooperation are limiting factors, but the late
results are encouraging.
Ferric Hydroxide Macroaggregates as Particles for Radiation Synovectomy
Clement B. Sledge, Jonathan Noble, Sonya ShortkroJ, and Donald J . Hnatowich, Boston and Cambridge, Massachirsetrs
Radiation synovectomy has been used in Europe, but possible side effects have precluded its acceptance in the United States.
These include lymphocyte aberrations (10% of patients), and leakage
of OOYfrom treated knees (52-91%). If leakage could be prevented, the
advantages of this simple procedure would be enormous, especially
since the results of radiation synovectomy compare favorably with
those of surgical synovectomy.
Our approach is based on the principle of having a short-halflife, beta-emitting particle, which could be readily produced and
whose tissue penetration was effectively confined to the rheumatoid
synovium. Retention of such an isotope in the joint is made possible
and leakage minimized by incorporating it with biodegradable particles of a given size. The pure beta emitter "'Dysprosium, whose halflife is 137 minutes and which has a Mev of I .3, fulfilled these criteria.
Ferric hydroxide macroaggregate and laDy can be associated with
99% efficiency. The 137 minute half-life of Dy prevents its use for
tracer experiments. Therefore we used another rare earth, I%d,
whose half-life is 240 days, but whose chemistry is similar to Dysprosium.
The ferric hydroxide-lS3Gd macroaggregate was injected into
the knees of rabbits. Eleven normal rabbits were injected and sacrificed at 5 hours, and a further 10 at 24 hours. The amount ofactivity in
tissue specimens was expressed as a percentage of the dose injected
into the knee. Greatest leakage was <0.33% in the liver. Identical
experiments were then repeated in 7 rabbits with antigen-induced
arthritis in which leakage rates were nearly identical to those found in
normal knees. Finally, a further 7 normal rabbits were studied by
means of a preparation of Fe(OH), and lsaDy. Leakage in this group
given a therapeutic dose of lesDy was extremely low, a result suggesting improvement in particle preparation.
Our conclusions are a) In all cases the leakage was <0.3390
and was greatest in the liver. b) Leakage in the arthritis model is
similar to that in normals. c) A rabbit model has been produced in
which the leakage of a short half-life, beta emitting-isotope, bound to
ferric hydroxide macroaggregates never exceeded 0.33%, 5 and 24
hours after injection. This rate of leakage would be clinically acceptable.
Xanthine and Uric Acid as Xanthine Oxidase Inhibitors
Hugh A . Smythe, Toronto, Ontario, Canada
A dramatic feature of primate evolution has been the rise in
plasma urate concentrations, so that human values are about 10 times
higher than those of monkeys or nonprimates. This elevation has been
achieved in steps, by the deletion of uricase and by complex alterations
in renal transport mechanisms. No biologic function served by this
change has been identified. Uric acid (UA) is not re-utilized, and its
immediate precursor, xanthine (X), is re-utilized only slightly. Hypoxanthine (HX) is however, extensively and rapidly re-utilized, and
absence of its re-entry in the Lesch-Nyhan syndrome has destructive
effects on human brain function.
Flexible inhibition of xanthine oxidase (XO) by X and UA
might protect HX levels at critical periods. The kinetics (KI) of XO
inhibition by X and UA were therefore studied, and the results are
shown in the table on the next page.
The KI of X is close to its normal plasma concentration, so
that it would be a very effective inhibitor of HX oxidation, especially
in the postabsorptive period when purine supply must switch from
exogenous to endogenous sources. The relatively high KIs for UA
make a biologic role for hyperuricemia less obvious. However, at 7
mg%, inhibition of xanthine oxidation is sufficient to raise by 64% the
concentration at which xanthine formation and oxidation are in equilibrium.
For technical reasons, little is known about the diurnal variations for metabolism of Hx and X, but the results of these in vitro
studies indicate the need for in vivo studies of the inhibitory effects of
X and UA on xanthine oxidase functions.
ARA ABSTRACTS
136
Source of XO
Substrate
KI ( r M )
Inhibitor
KI ( r M )
KI (mg%)
Human liver
HX
HX
X
16
16
II
X
UA
UA
9.3
4600
I600
0. I4
77.0
27.0
Induction of Cell-Derived Chemotactic Factor (CF) and of Arthritis by Amorphous Diamond Crystals
Isaias Spilberg, David Rosenberg, and Jagdish Mehta. St. Louis, Missouri
The phagocytosis of Na urate or Ca pyrophosphate crystals
by neutrophils leads to the generation of a chemotactic factor (MW
8400). Current evidence suggests that the above CFs are identical.
Therefore experiments were designed to test the ability of diamond
dust to induce generation of chemotactic activity from neutrophils and
to produce arthritis in rabbits.
Peripheral blood neutrophils (6 X 10’) were incubated with 6
mg of diamond dust (2-12 microns) for 45 minutes at 37OC. Cells were
disrupted and the lysosomal fraction was obtained by differential
centrifugation. The fraction was passed over a calibrated Sephadex
G-50 column, and the fractions were tested for chemotactic activity by
radioassay. Chemotactic activity was found localized at 0.1 I of eluate
(MW 8400). When the chemotactic eluate was placed on polyacrylamide gel electrophoresis, a line representing the chemotactic
material appeared at the same distance from the cathod as the chemo-
tactic factor induced by Na urate or Ca pyrophosphate crystals. Control fractions showed no activity.
In a separate set of experiments, 10 mg of diamond dust in 0.3
ml saline was injected into the left knee joints of 6 rabbits. Contralateral joints were injected with saline. Animals were killed at 4
hours, synovial membrane was fixed, and cell counts were made in
synovial fluid. Marked leukocytosis was observed in the joints injected
with diamond dust, and only minimal leukocytosis in control joints.
In conclusion, amorphous diamond dust is capable of inducing
generation of chemotactic factor from neutrophils following phagocytosis and also of producing a violent arthritis in vivo. Although
evidence is incomplete, it is conceivable that the generation of CF may
represent a uniform response of the neutrophils to phagocytosis of
different crystals and perhaps other materials.
Lyme Arthritis: Serum Cryoprecipitates Associated with Skin and Joint Lesions
Allen C. Steere. John A . Hardin. and Stephen E. Malawista. New Haven, Connecticut
We have reported a cluster of 51 patients who developed a
similar type of inflammatory, oligoarticular arthritis, “Lyme arthritis,” the epidemiology of which suggests transmission of a causative
agent by an arthropod vector (Arthritis Rheum 19:824, 1976). Onequarter of the 51 patients described a preceding skin lesion thought to
be an arthropod bite and consistent with the entity “erythema chronicum migrans.” Because circulating immune complexes might occur in
this disease, we tested sera for cryoprecipitates in 14 new patients who
developed the skin lesion, with or without subsequent arthritis, in June
or July 1976.
The initial lesion, usually on a proximal extremity or the
trunk, was a red papule that developed into an expanding, red circle,
as large as 50 cm in diameter, often with central clearing. The patients
had a median of 5 such lesions (range: 1-20) that lasted from 0.5 to 8
weeks. All 14 patients had profound fatigue, 13 fever, 9 stiff neck and
headache, 7 backache, 5 myalgia, 3 nausea and vomiting, 2 malar rash
and periorbital edema, and 1 Bell’s palsy and sensory neuropathy of
the TI0 dermatome. In the table below the patients have been grouped
according to whether the skin lesion was acute (group A ) or fading
(group B), or gone recently but followed by a first attack of arthritis
(group C). Three additional patients had recurrent attacks of arthritis
during this period (group D).
As the table shows, there was a strong tendency for cryoprecipitates to be associated with active skin lesions-in all of 6 patientsand with attacks of arthritis-in 5 of 7, plus the 2 in group A, who also
had active skin lesions. We are currently examining the presence and
composition of cryoprecipitates in individual patients serially.
Correlation Between Clinical Activity of Skin or Joints and Presence of Cryoprecipitates
Skin
Arthritis
Total
Patients
Total
Group A: Active
Group B: Fading
Group C: Gone, 2-4 wks
Group D: Gone, 1 year
(2 with 1st attack)
None
1st attack
Recurrent attack
6
4
4
3
6
0
3
2
Delinition of GrouDs
Patients with Cryoprecipitates
IBM
IgG
Both
0
0
2
0
1
0
2
1
4
0
I
0
Circulating DNA in SLE Patients with Active Vasculitis and/or CNS Disease
Charles R. Steinman, New York. New York
DNA, in association with antibody, has long been thought to
play a central role in the pathogenesis of SLE. Until recently, however,
only conflicting information was available regarding the prevalence of
circulating DNA in normals or in patients with SLE. With improve-
ARA ABSTRACTS
137
ments in technique and the recognition by Davis and Davis that most
serum samples contain D N A released in vitro, it has been recently
shown that normal plasma contains n o detectable DNA. Surprisingly,
the prevalence of plasma D N A in SLE, determined by a technique
capable of detecting as little as 1.5 pg/ml of plasma, was reported not
t o differ significantly from that in normals.
In the present study, a modified counterimmunoelectrophoresis technique capable of detecting as little as 20 t o 50
ng/ml of DNA in plasma (and which shows no DNA to be present in
normals) was used to examine, blindly, plasma from patients with
active SLE. Persistent circulating D N A was defined as detection of
DNA in plasma samples collected o n at least 2 different days. Patients
with a clinical condition other than SLE that had been previously
known or suspected of being associated with circulating DNA were
excluded. Because preliminary studies suggested possible association
between circulating D N A and active vasculitis and/or C N S involvement, the presence of persistent D N A in patients with these
manifestations was specifically examined. A total of 43 patients with
suitable specimens were studied. Eighteen were found t o be positive
for persistent circulating DNA. In patients with C N S involvement, 8
of 11 (73%) were positive. Of those with vasculitis with or without
concomitant C N S disease, 8 of 13 (62%) were positive for circulating
D N A . Of the combined group with C N S disease and/or vasculitis, 16
of 24 patients (67%) were positive. Of patients with active SLE but
lacking vasculitis or C N S disease, 2 of 19 (1 1%) had circulating D N A .
N o correlation with active nephritis was noted.
Neither inflammatory disease per se, as judged either clinically or by elevation of the ESR, nor high dose corticosteroid therapy
could account for these findings. It is therefore concluded that the
presence of circulating DNA may be clinically useful as a diagnostic
tool in some SLE patients. Further elucidation of the source, mode of
release, and consequences of circulating D N A may shed light on
pathogenetic mechanisms currently not understood.
Reversible Renal Insufficiency in Systemic Lupus Erythematosus
M . Sugarman. A . Kamdar, B . H . Barbour. F. P. Quisrnorio, S . Massry, and E. L. Dubois. Los Angeles, California
Advanced renal failure in SLE has been associated with a
universally poor prognosis. In the past 5 years we have seen 7 of 43
SLE patients with renal insufficiency (serum creatinine 4 mg/dL)
whose renal function improved; 6 survived 2.5 to 4 years.
Six presented with rapid renal deterioration extending over
weeks, associated with SLE activity both clinically and immunologically. None had significant hypertension with the renal failure.
Treatment included dialysis in 4. Three of these patients
improved without recent treatment with corticosteroids o r other immunosuppressive drugs. Two of these 3 had been declared drug failures. The first underwent renal transplantation, graft rejection, and
removal of the transplanted kidney 4 months prior t o improvement in
her natural kidney’s function. Renal function of the other drug failure
improved after 11 months of chronic hemodialysis. Of the 3 not
requiring dialysis, all received high-dose corticosteroids, and in addition 1 was given intravenous nitrogen mustard.
Renal histopathology in 5 showed active diffuse proliferative
glomerulonephritis. Significant interglomerular variability in SLE was
noted; some glomeruli were obviously lost, whereas others showed
marked inflammatory reaction, which may be reversible. Inflammation involved the interstitium, which may have adversely affected renal function. Vascular changes were minimal and no fibrin
thrombi were noted.
These patients demonstrate that this disease is too variable to
indicate withholding therapy because of poorly functioning kidneys.
Even patients with apparent end stage kidney disease may improve.
Renal biopsy evidence of intact glomeruli, profound interstitial and
glomerular inflammation, and normal arteries in a patient with rapid
renal deterioration and multisystem disease may be helpful in determining which patients have a more favorable prognosis. The role of
hemodialysis in these patients is uncertain, but it has been shown to
activate the alternative pathway of complement, which may dissociate
immune complexes.
Augmentation of Leukocyte Motility by a Neutrophil Cytoplasmic Factor
Akiteru Takeuchi and Robert H . Persellin, San Antonio, Texas
The chemotactic attraction of polymorphonuclear (PMN)
leukocytes is one requisite of the inflammatory reaction. We have
presented evidence that directional cell motility is influenced not only
by the concentration of the chemotactic attractant, but also by cell
density. The number of P M N leukocytes required for the augmentative response must be adequate to permit cell-to-cell contact. Further
studies of this cytoplasmic factor were performed.
Directional cell motility was studied in a modified Boyden
chamber. Normal human neutrophils were layered on a 3-p Millipore
filters by means of a cytocentrifuge. E coli lipopolysaccharide-activated serum served as the chemotactic stimulus. Granulocyte cytoplasm was found t o activate cellular response. Fractionation of
isolated neutrophils in hypertonic sucrose was performed by differential centrifugation. Neither the nuclear (400 X g, 10 min), the lysosoma\ (8,200 X g, 15 min), nor the microsomal fractions (1 hour at
100,00 X g), would activate cell migration in response t o the chemotactic stimulus. However the post-microsomal supernatant fraction,
when placed in the top chamber in direct contact with viable cells,
increased chemotaxis, and this action was dose-dependent. Activation
was not due to protein content alone, because equivalent amounts of
protein from either other subcellular fractions or human serum albumin failed to cause a comparable migration. Heating at 56°C for 30
minutes did not alter the activity, but dithiothreitol at 5 mM inhibited
its effect. Studies performed with the cytoplasmic fraction either alone
or in combination with the chemotactic factor in the top and/or
bottom chambers indicated that the cytoplasm did not function as a
chemotaxin but acted t o potentiate the cellular response to C5a. To
further investigate this phenomenon, cell electrophoresis was performed. Using a Rank Mark I1 cytophorometer, we measured the
surface charge of cells suspended in 5% sorbitol, 0.13 mM phosphate
buffer. Chemotactic factors decrease the negative surface charge and
increase cellular motility. The lysosomal fraction, previously shown to
contain a chemotaxin, prolonged cell motility 10.35 f 0.32 seconds
(SEM) being required for travel of 48 p . However, the cytoplasmic
fraction, at an identical protein concentration, did not alter motility,
7.45 f 0.26 seconds being the same as control. Thus the cytoplasmic
activator of neutrophil migration does not induce directional migration but renders a cell more responsive to a chemotactic stimulus.
ARA ABSTRACTS
138
Specific Extraction of DNA Antibodies by Extracorporeal Circulation over DNA Immobilized in Collodion Charcoal
David S. Terman, Ronald I . Harbeck, and Ronald Carr, Houston, Texas and Denver, Colorado
It is now well recognized that tissue inflammation in some
cases of systemic lupus erythematosus (SLE) is mediated by antibodies
to DNA which, when combined with circulating DNA, give rise to
pathogenic immune complexes. The selective withdrawal of circulating
DNA antibodies would therefore be a valuable therapeutic measure.
A novel immunoadsorbent was prepared in which significant
quantities of DNA were entrapped in collodion membranes adherent
t o small particles of activated charcoal. These D N A collodion charcoal (DCC) particles were placed in an extracorporeal circulation
system, and arterial blood was circulated through a continuous flow
celltrifuge in which plasma was separated from formed elements.
Rabbit anti-BSA and human anti-DNA antibodies were passively
infused into mongrel dogs, and only plasma was pumped through the
DCC at a flow rate of 40 ml/min. Complete removal of DNA binding
activity in serum was observed over the ensuing 15 to 30 minutes, with
only a slight change in BSA binding during the same time period.
There were no significant changes in lZsI DNA on the charcoal before
and after the perfusion experiments and no recovery of lZsl DNA in
the serum, urine, or vital organs of dogs at the conclusion of the
studies. Further, there were no demonstrable hematologic or biochemical alterations.
These findings suggest that this extracorporeal immunoadsorbent may rapidly and specifically withdraw circulating DNA antibodies with minimal host toxicity and may offer a specific approach to
therapy of SLE when it is desirable to remove D N A antibodies.
The Arthritogenicity of Type I1 Collagen: A New Animal Model
David E. Trentham, Alexander S. Townes, and Andrew H . Kang, Memphis, Tennessee
Immunity t o collagen could play a role in inciting or perpetuating the inflammation that occurs in the connective tissue diseases.
We have found that about 40% of Wistar rats injected with Type I I
collagen from human or chick cartilage in incomplete Freund's adjuvent (ICFA) develop an inflammatory arthritis and both humoral and
cell-mediated immune responses to the Type I1 immunogen.
Outbred female Wistar rats were injected intradermally on
the back with 0.5 mg of soluble native collagens extracted from human
skin (Types I and HI), or human or chick cartilage (Type 11) with
complete or ICFA. Booster doses consisting of the immunogen without adjuvant were given intraperitoneally on day 21. Forty-three of
106 rats injected with four separate Type I1 collagen preparations
developed a chronic, erosive polyarthritis 15 to 60 days following
primary immunization. Peak incidence occurred at 20 days. Type I or
Ill collagen, Type II without adjuvant, MgCI, extractable cartilage
proteoglycan, denatured a l ( I I ) chains, and complete or ICFA were
not arthritogenic in a total of 206 rats. Booster doses of Type II
collagen as long as 6 months after the initial immunization resulted in
recurrence of inflammation only in previously arthritic animals. Pre-
liminary results also indicate induction of arthritis following injection
of homologous rat Type I I collagen.
Passive hemagglutination using antigen fixed to glutaraldehyde-treated human 0 red cells indicates a significant (P < 0.001)
correlation of mean antibody titers to Type I1 with arthritis (mean in
27 arthritic rats, 1 :5120; mean in 39 nonarthritic rats, I : 160). Peripheral blood lymphocyte cultures show type-specific transformation
to Type I1 [mean stimulation index (SI) in 15 arthritic rats 10.8; in 14
nonarthritic rats 3.31 that also correlates significantly with arthritis (P
< 0.05). Rats injected with Types I and 111 collagen yielded lower
antibody titers (mean 1:40 with I 1:80 with 111) and lymphocyte
transformation (mean SI 2.8 with I, 2.7 with Ill). N o antibody or
transformation to collagen was observed in normals or rats immunized with cartilage proteoglycan.
The demonstration of immunity to collagen in this new animal model of arthritis suggests a potential role for cartilage type of
collagen in provoking or perpetuating joint inflammation in the rheumatic diseases.
+
Immunologic Study of Arthritis Associated with Adenovirus Infection
Peter D. Utsinger, Chapel Hill, North Carolina
Three unrelated patients who developed an acute arthritis
associated with a febrile upper respiratory illness (URI) were studied.
All were females and the age range was 18-35 years. The arthritis
began 3 to 8 days after the onset of the URI and was symmetric
involving large and small joints of the upper and lower extremities. On
joint examination the involved joints were red, hot, and tender. Two
patients had a macular erythematous rash. N o lymphadenopathy or
hepatosplenomegaly was found. Synovitis decreased gradually and
after 4 weeks the joint examination was normal.
Lymphocytotoxic antibodies (LCTA) were determined by a
microlymphocyte cytotoxicity assay. T lymphocytes were determined
by spontaneous rosetting with sheep blood cells, B lymphocytes by
surface immunoglobulin using F(ab'), antisera; the Raji cell test for
immune complexes (IC) was done by the method of Dixon. C 3 and C4
were determined by a nephelometric technique.
The presence of adenovirus infection was proved by throat
culture and complement fixation. None of the patients had cold agglutinins, rheumatoid factor, Australia antigen, DNA antibodies, or
antinuclear antibodies. Gonococcal and mycoplasma cultures were
negative. All patients had serum and synovial fluid (SF)cryoglobulins
and IC as determined by the Raji cell test. When Raji cells were
incubated with these serum cryoglobulins and subsequently stained
with a fluorescein-conjugated adenovirus antiserum (FCAA), immunofluorescent staining was found. Cryoprecipitable protein (CP)
found in the S F of these patients also fixed to Raji cells and were also
stained with FCAA. IgG and C 3 were also found in the cryoprecipitates. LCTA were found in 2 of the 3 patients. These antibodies had
specificities for membrane antigen determinants on both T and B
lymphocytes. Selective concentration of the LCTA in the cryoprecipitates was not found. The peripheral blood lymphocyte populations of
these patients were decreased with proportionate reductions of both 7
and B lymphocytes. Serum C 3 and C 4 were decreased in 2 patients and
normal in the third. Synovial fluid white blood cell counts were less
than 4000/mma in 2 patients with predominance of mononuclear cells,
and 8500/mma in the third with predominant PMNs. C D and IC were
not detectable 3 to 4 weeks after the onset of the illness. These data
suggest that serum and intraarticular IC are involved in the pathogenesis of adenovirus-associated arthritis.
ARA ABSTRACTS
139
Immunologic and Therapeutic Study of Erosive Osteoarthritis
Peter D. Utsinger, Donald Resnick, and Robert Shapiro, Chapel Hill, North Carolina, San Diego, and Sacramento, California
The purpose of this study was to define immunologic abnormalities and the efficacy of intraarticular steroid treatment in erosive
osteoarthritis (EOA). In 12 females and 3 males, originally seen because of a dip or pip arthritis, the diagnosis of erosive osteoarthritis
was made on blind reading of hand radiographs. The patients, whose
mean age was 62 (range: 48-70) were followed for 18-22 months.
Constitutional symptoms consisted of morning stiffness less than 1
hour and rare afternoon fatigue. The initial joint examination was
notable for erythema, warmth, and swelling of either pip or dip joints
in all patients, with symmetry in 1 1. Four patients had knee effusions.
Lymphocytotoxic antibodies (LCTA) and histocompatibility
typing were performed by microlymphocyte cytotoxicity assays. T
lymphocytes (L) were determined by spontaneous rosetting with sheep
blood cells, and B-L by surface immunoglobulin using F(ab’), f r a g
ment antisera. C 3 and C4 were determined by a nephelometric technique. Ficoll-Hypaque separated L were transformed by different
PHA concentrations in 2-4 cultures. M L C were performed by the
miniaturized microtechnique of Hartzman.
Serum rheumatoid factor and A N A were absent. Serum C3
and C4 were normal. ESR ranged from 18 t o 43 with a mean 30. The
percentage of T- and B-L was normal. L PHA and M L C responses
were normal. There was no significant deviation of HL-A antigen
frequencies as compared t o normals. Analysis of dip or pip synovial
fluid revealed a WBC less than 2,000/mma in 10 patients and a WBC
from 2,000 to 16,000/mm8 in 5 patients. LCTA were absent.
Each patient had individual joints injected with 10 mg triamcinolone hexacetonide. Prompt decrease in objective synovitis was
noted after 26/33 injections. Radiographs taken from 6 to 18 months
after the injections revealed a n increase in erosions in 7/33 injected
joints, no change in 18, and a decrease in 4. Similar changes were
noted in noninjected joints. Marked pip subluxation developed in 2
injected joints and in none of the noninjected joints.
These data d o not support the concept of significant immunologic abnormalities in EOA and suggest that little permanent benefit
accrues from intraarticular steroid injection in EOA.
Structural and Immunologic Changes of Synovium of Hypertrophic Osteoarthropathy (HPO)
Angel F. Vidal. Roy D. AItman, Victoriano Pardo, and Duane Schultz, Miami, Florida
Recent interest in immunology of malignancy and Schumacher’s findings of electron-dense deposits (Arthritis Rheum 19:629,
1976) in synovial blood vessels of patients with H P O have raised the
question of whether the synovitis of H P O is due to an immunemediated mechanism. We attempted to confirm the presence of electron-dense particles in H P O synovitis and to investigate the possible
immune origin of this disease.
A study was made of 2 subjects with bronchogenic carcinoma
and HPO-related synovitis. Synovial biopsies were examined by light
microscopy (LM) and electron microscopy (EM), and cryostat sections were reacted with fluorescein-conjugated antisera t o the following human immunoglobulins: polyvalent IgG, IgA, IgM, C3, and C4.
Functional hemolytic complement assays were assessed in synovial
fluid utilizing control fluids from degenerative arthritis.
The H P O synovial fluids were noninflammatory, and synovial LM demonstrated minimal infiltration with inflammatory cells.
EM revealed lesions involving segments of the microcirculation underlying the synovial lining cells. Some capillaries and venules were
dilated and contained masses of platelets, necrotic desquamated cells,
occasional lipid droplets, and dense granular material in their lumens.
Many endothelial cells exhibited swollen cytoplasm-poor in organelles-with endothelial cell gaps. Rare electron-dense deposits were
observed in the subendothelial areas of thickened capillary walls. In
this, the first investigation of its kind to our knowledge, there was no
localization of fluorescein-conjugated immunoglobulins or complement components in the synovium, and functional synovial fluid
complement levels were within the range of the matched controls.
The etiology of HPO synovitis remains unknown. The electron-dense deposits of the inner portion of the vascular walls with the
cytoplasmic abnormalities may represent a permeation of the vascular
wall by plasma components as a result of increased vascular permeability. The preliminary results of our study, with normal complement assays and absence of immunoglobulin deposits in the synovia, d o not support an antigen-antibody immune deposit
mechanism.
Autoantibodies in Palmerston North (PN) Mice, a New Animal Model of SLE
Sara E. Walker, Maureen Fulton. Robert H. Gray, and Richard D. Wigley,Ann Arbor, Michigan and Palmerston North, New Zealand
In 1966 Wigley described polyarteritis-like lesions in outbred
P N mice (Nature 21 1 :319). The current study investigated the natural
history of spontaneous autoimmune abnormalities, vasculitis, and
glomerulonephritis in these animals.
Colonies of PN mice were established, consisting of mice
selected for antinuclear antibodies (ANA) through 3 generations and
inbred for 9 generations. Ninety mice were followed t o death to
determine life spans, and 19 of these animals were bled a t 6-month
intervals until death and autopsied. Ninety additional mice aged 1-19
months were bled to study age-dependent autoantibody formation.
Serum was tested by indirect immunofluorescence o n guinea pig stomach, kidney, aorta, and liver. Anti-double-stranded DNA was assayed
by modified Farr technique; binding greater than 20% was abnormal.
Ten kidneys from mice aged 4-21 months were tested by direct immunofluorescence. Tissue from mice aged 14 and 18 months was exam-
ined by electron microscopy. Results were compared with data from
autoimmune B/W mice, recognized a s animal models of SLE.
Mean life spans in PN mice were 1 I .4 months in females and
14.1 months in males (P < 0.01). Vasculitis was found in 10/19 old
autopsied mice, and 11/19 mice had glomerulonephritis. Lymphomas
arose in 10% of these animals. Sera from 35% of mice aged 6 months
produced specific fluorescence of stomach smooth muscle and renal
arteries. These positive tests persisted throughout life. ANA were
positive in 55% of mice aged 6 months and 92% of mice I2 months of
age. Anti-DNA were first detected in 6-week-old mice. Mean DNA
binding increased from 22% in mice aged 4-5 months to 30% in mice
aged 12 months. Granular deposits of IgG and C 3 were found in
glomeruli of all kidneys tested. Irregular thickening of glomerular
basement membranes and dense subendothelial deposits were identified in renal tissue examined by electron microscopy. Thymic tissue
ARA ABSTRACTS
I40
contained cytoplasmic viral particles 700 A in diameter that were not
identical to C-type viruses.
Like B/W mice, female PN mice died earlier than male mice.
Although anti-DNA appeared earlier in PN mice, PN renal disease
was less severe than B/W glomerulonephritis. Thymic ultrastructure
was different in both strains of mice. PN mice are an anti-DNA
producing model simulating human SLE more than polyarteritis.
Endogenous Activation of Collagenase Secreted by Rheumatoid Synovial Cells: Evidence for the Role of Plasminogen Activator
Zena Werb, Carlo L. Mainardi, Carol A . Vater. and Edward D. Harris, Jr., Hanover. New Hampshire
Adherent cells obtained by dissociation of human rheumatoid synovium secrete collagenase in a latent form (E,,)into culture
medium. We report that conversion of EL t o active collagenase (E')
may be mediated by another enzyme produced by these cells, a plasminogen activator.
Previous studies have shown that exposure of conditioned
culture medium to trypsin resulted in activation of EL t o E'. Plasmin, a
proteinase that could be found in rheumatoid joints, also activated EL;
100 pM of plasmin/ml (2OoC, 30 min) completely activated EL to E'.
Both E' and E,, bound to reconstituted guinea pig collagen fibrils.
Substrate-bound EL was resistant to inhibition by a,-macroglobulin
but could be activated by adding plasmin to the system. Addition of
plasminogen (Pg) alone did not activate EL.
The capacity of synovial cells secreting EL t o activate Pg to
plasmin was examined next. Pg activator was assayed by growing cells
on radioactive fibrin layers in the presence of 5% acid-treated dog
serum, a source of Pg. Time-dependent release of fibrin peptides
occurred when Pg was present, but not when the serum used first had
been depleted of Pg by passage through lysyl-Sepharose. Addition of
purified Pg to the cultures reconstituted the system. The Pg-dependent
fibrinolysis also could be measured in serum-free culture medium from
these cells. In the presence of an excess of plasmin inhibitors such as
a,-macroglobulin of a,-proteinase inhibitor in culture medium, no
active plasmin was formed and collagenase was found only in latent
form. In the absence of such inhibitors, addition of 10 pM of Pg/ml to
the cultures for 48 hours resulted in conversion of the Pg t o plasmin
and activation of El, to EE' (collagenolytic activity capable of degrading 8 pg of fibrillar collagen/min/ml a t 37°C).
These data suggest that in rheumatoid arthritis, collagenase is
secreted in a latent form, EL. that is trapped locally by binding to
collagen fibrils, its natural substrate. The endogenous activation of EL
to E' may be accomplished by plasminogen activator released from the
synovial cells. Plasmin is generated which, after saturating proteinease
inhibitors present locally, can activate EL to E', and collagen degradation can begin.
Marrow-Dependent (M) Cell Function in Casein-Induced Experimental Amyloidosis
Donna Yonkosky and Edgar S . Cathcart, Boston, Massachusetts
We have previously reported that mitogen-induced cellular
cytotoxicity is completely depleted in the amyloidotic CBA mouse.
Recent results from our laboratory indicate that an effector cell in the
MlCC assay is a marrow-dependent ( M ) cell. M cells are responsible
for rejection of selected marrow allografts in lethally irradicated recipients, and there is evidence that the same cell population may control
resistance to Friend virus and Listeria monocytogenes infections. In
the present study normal WB and CBA. strontium 89, and caseintreated CBA mice were lethally irradiated and injected with 2-4 X 16'
WB marrow cells. Four days later cellular growth was detected by
uptake of iodinated deoxyuridine in spleen tissue. Results are reported
with respect to 100%growth in syngeneic animals (Table).
Marrow allograft rejection was completely abolished following multiple casein injections. Similar results were obtained in CBA
mice receiving spa, a bone-seeking isotope that destroys marrow and
selectively eliminates M cells. Increased rejection of marrow allografts
was noted during the preamyloid phase, at a time when MlCC is also
markedly enhanced. Thus MICC and marrow allograft rejection, two
functions that detect M cell activity, are increased in the preamyloidotic phase and depleted in the amyloidotic phase. These new
findings are additional evidence that M cells play a key role in pathogenesis of amyloid.
Percent Growth
WRCBA
CBA/preamyloid
CBA/amyloid
CBA/S~~
2 X I@ cells
4 X l(P cells
100
100
46
0
0
100
100
0
100
100
Prostaglandin E, (PGE,) Treatment of NZB/NZW Mice
Robert B. Zurier, Donna M . Sayadoff, Ivan Damjanov, and Naomi F. Rothfield, Farmington. Connecticut
Because experimental evidence suggests that prostaglandins
might modulate immune/inflammatory responses, NZB/NZW F,
mice were treated with PGE,. After 1 year of treatment (with 200 pg
PGE, sc once or twice daily from 6 through 58 weeks of age), 18 of 19
female mice survived, whereas only 2 of 19 control (saline sc once or
twice daily) female mice were alive. Male mice treated with 200 pg
daily PGE, were also protected: 10 of 1 I alive vs 2 of 9 untreated
control mice alive at 58 weeks.
The 9 female mice treated with PGE, twice daily were sacrificed at 58 weeks. Immunofluorescence and light microscopy studies
done on kidney tissue were compared to kidneys from 9 untreated
female control mice that had died of nephritis (proteinuria > 2+,
BUN > 25) between 6 and 10 months of age. All of the untreated
NZB/NZW mice exhibited heavy (4+ fluorescence) diffuse and globular glomerular deposits of IgG, whereas there were only minimal
(*-2+) mesangial or focal deposits of IgG in PGE,-treated mice.
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1976, scientific, december, section, proceedings, florida, 911, 41st, associations, annual, rheumatic, beach, miami, arthritis, american, foundations, session
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