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Formation of leukocyte inclusions in normal polymorphonuclear cells incubated with synovial fluid.

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Formation of Leukocyte Inclusions in Normal
Polymorphonuclear Cells Incubated with Synovial Fluid
Eric R. Hurd, Joseph LoSpalluto and Morris Ziff
Normal polymorphonuclear cells phagocytosed IgG, IgM and plC (CB)-containing inclusions from the majority of rheumatoid factor-positive synovial f.uids, but
not from factor-negative fluids, as determined by fluorescent antibody staining.
Addition of rheumatoid factor (but not Waldenstrom IgM) to factor-negative
rheumatoid fluids produced phagocytosable complexes of similar composition in
these fluids, indicating the presence of altered IgG in factor-negative as well as
factor-positive rheumatoid effusions. Inclusions were infrequently phagocytosed
from nonrheumatoid effusions.
~ l ' h esignificance of CytoplasiiiiL inclusions in synovial fluid leukocytes has received much attention. Parker and Schmid
(1) demonstrated that complexes containing heat-aggregated IgG and rheumatoid
factor are phagocytosed and appear as cytoplasmic inclusions. Hollander and coworkers (2,3) have shown that such inclusions,
observed in the polymorphonuclear cells of
rheumatoid synovial fluid, contain IgG and
IgM. They suggested that the phagocytosis
of complexes made u p of these immunoglobulins results in the release of lysosomal
enzymes which, in turn, lead to inflammation in the joint. With regard to the formation of such complexes, Hannestad (4) has
demonstrated a precipitin reaction between
high ,titer rheumatoid sera and certain rheumatoid synovial fluids. T h e precipitating
factor of the synovial effusions, which
reacted with IgM rheumatoid factor in the
sera, appeared to be structurally altered
IgG. Winchester and Kunkel ( 5 ) have
shown that 10s to 19s complexes, which
contain mainly IgG and form precipitates
with IgM rheumatoid factor, are present in
rheumatoid synovial fluid.
I n the present study, synovial fluids were
examined for (a) the presence of immunoglobulin complexes capable of forming cytoplasmic inclusions upon incubation with
normal polymorphonuclear cells and (b)
the presence of altered 7-globulin capable
of forming such complexes by reaction
with IgM rheumatoid factor.
Supported by Project Grant AM-09989 US Public
Health Service; Grant 1-155 The Robert A. Welch
Foundation and an Arthritis Foundation Clinical
Study Center grant.
ERICR. HURD,MD: Postdoctoral Fellow, The
Arthritis Foundation; Assistant Professor, Department of Internal Medicine, The University of Texas
Southwestern Medical School, Dallas, Texas.
PhD: Associate Professor of Biochemistry, The University of Texas Southwestern
Medical School, Dallas, Texas. MORRISZIFF, PhD.
MD: Recipient, Research Career Award, US Public
Health Service; Professor of Internal Medicine, The
University of Texas Southwestern Medical School,
Dallas, Texas.
Address for reprint requests: Eric R. Hurd, MD,
Department of Internal Medicine, The University
of Texas Southwestern Medical School, 5323 Harry
Hines Blvd., Dallas, Texas 75235.
Preparation of white blood cell suspensions.
Submitted for publication June 5 , 1950; accepted
Fresh, heparinized blood was obtained from either
August 7, 1970.
Arthritis and Rheumatism, Vol. 13, No. 6 (November-December 1970)
normal persons or trauma patients. Three millilitcrs of a 6y0 solution of dextran were added to each
lOml of blood and gently mixed. The sample was
then allowed to stand for 30 min. The supernatant buffy coat cells were removed, centriluged at
lOOOrpm and washed twice in 0.9% normal saline.
Synovlal fluld. Synovial fluids were obtained
from both outpatients and hospitalized patients.
Following heparinization, a leucocyte count was
performed on each fluid, which was then clarified
by centrifugation at 17,300 x g for 20 minutes. The
supernatant cell-free fluid was then stored in
aliquots at -70° C.
Incubation of white blood cells In synovlal fluid.
Four million washed peripheral white blood cells
This mixture was incubated for one hour at 37" C
were suspended in 1.0 ml of cell-free synovial fluid.
with gentle shaking. After incubation, the cells were
centrifuged, washed twice, and resuspended in
saline. Slides of this suspension were then prepared
for staining in a Shandon cyt0centrifuge.l
rected against human Igbl, IgC, pl. and fibrin, and
then washed with 0.01 M phosphate-buffered
saline, p H 7.0, with gentle shaking for 30 min.
Sections were then covered with a drop of 90%
coverglass, and read in a Leitz-Orthomat microscope
glycerine in phosphate buffer, covered with a
fitted with appropriate darkfield condensers, an
Osram mercury lamp, HBO 200 W. BG 12, UGI
primary filters, and K 430 and K 460 secondary
filters. Cells were examined for the presence of
cytoplasmic inclusions and recorded as positive only
when the majoiity of cells contained distinct cytoplasmic granules. Rare tiny inclusions, seen in only
a few cells, were read as negative. In all preparutions where inclusions were present, sequential
staining with fluoresceinated and rhodaminated
antibodies was carried out to determine the
presence of more than one component. Control
preparations were made using cells not previously
incubated in synovial fluid. No significant staining
of leukocytes exposed to fluorescein isothiocyanate
(FITC) conjugated and rhodamine isothiocyanate
(RITC) -conjugated antisera was observed in the
absence of prior incubation with synovial fluid.
Preparatlon and purification of immunoglobulins.
IgM was prepared from sera of patients with
Waldenstrom's macroglobulinemia and IgM rheumatoid factor from rheumatoid sera by gel filtration
first through Sephadex G-200 and then through
Biogel A-5 M. Immunodiffusion tests, using specific
goat antisera, indicated the presence only of IgM in
these preparations.
Tests for rheumatoid factor. These tests were
performed on synovial fluid by the latex slide$ test
and by the sensitized sheep cell agglutination
(SSCA) test, sensitizing with one-fourth the basic
agglutinating titer of amboceptor.
Statistleal analysis. Fisher's Exact Probability
Test was used for analysis of the data.
Addition of rheumatoid factor and Waldenstrom
IgM to synovial flutd. Synovial fluids which gave
negative tests for rheumatoid factor and which
produced no cellular inclusions when incubated
with normal white blood cells were used in these
experiments. Such fluids were divided into two
portions: to one portion, the purified IgM rheumatoid factor was added and to the other, the
Waldenstrom IgM, with no rheumatoid factor activity, was added, each to a final concentration of 1
mg/ml. Normal buffy coat leukocytes were then
added to each fluid, as described, and the mixture
incubated for 1 hr a t 37O C. The cells were then
centrifuged, washed, and slides prepared in the
Slides were stained for 30
min with fluorescein-conjugated goat antisera? di'Shandon Scientific Co, Ltd, London, England.
tHyland Laboratories, Los Angeles, Calif.
Rheumatoid factor positive synovial
fluids. Figure 1 illustrates the type of
large inclusion phagocytosed from rheumatoid factor-positive synovial fluid. This type
of inclusion, which stained positively for
IgG, JgM and the plo (CS) component of
complement, was phagocytosed from the
synovial fluid of patients with seropositive
rheumatoid arthritis. I n the case illustrated, the patient had severe seropositive rheumatoid arthritis; the synovial fluid SSCA
titer was 1:448 and the slide latex fixation
test was 3+ reactive.
Nine of 13 synovial fluids with SSCA
fHyland Laboratories, Los Angeles, Calif.
Arthritis and Rheumatism, Vol. 13, No. 6 (November-December 1970)
titers of 1:14 or greater, obtained from
patients with adult rheumatoid arthritis,
yielded IgG staining inclusions after incubation with normal buffy coat cells (Table
1). I n contrast, no such inclusions were
seen after incubation with rheumatoid factor-negative fluids. Similarly, eight oE 13
factor-positive fluids yielded IgM-staining
inclusions while no IgM-staining inclusions
were obtained from the factor-nega'ive
fluids. Both of these differences were statistically significant (IgG p<O.Ol, IgM
p<0.025). Although a greater percentage of
factor-positive fluids had p,,-staining inclusions than did factor-negative fluids, this
difference was not significant. I t is of interest that all eight of the Igh4-positive inclusions also stained for IgG and that five of
these eight also stained for p,,, suggesting
that the IgM-staining inclusions were
present as IgG-IgM-plc complexes. Fluorescent antibody staining for fibrin revealed
only rare inclusions.
tis, produced inclusions which stained for
IgG, IgM and plc. From two other fluids,
derived from patients with gonococcal arthritis and psoriatic arthropathy, only IgG
and plC staining inclusions were obtained
and in the case of one fluid from a patient
with palindromic rheumatism, only PIC
was stained. Since ,the three IgGpositive
fluid's, which were obtained from 2 patients
with gonococcal arthritis and on- with psoriatic arthropathy, respectiveIy, were also
positive for plc, both by single and combined staining techniques, these data suggest that IgG-plc complexes are present in
the synovial effusions of some patients with
non-rheumatoid inflammatory arthritides.
Addition of rheumatoid factor to factor-negative fluids. Results of experiments in which preparations of IgMrheumatoid factor and IgM purified from
the serum of a patient with Waldenstrom's
macroglobulinemia were added to rheumatoid factor-negative synovial fluids are
Juvenile rheumatoid arthritis synovial shown in Tables 3, 4 and 5. All of thes:
fluids. In a small group of 5 patients fluids were previously screen-d for the
with juvenile rheumatoid arthritis (Table presence of inclusion-yielding complexes
l), none had IgM-staining inclusions. One and all were found to be negative. After
of 2 patients with rheumatoid facto-- the addition of rheumatoid factor (Table
positive effusions had IgG-containing inclu- S), five of nine adult factor-negative fluids
sions and one of three factor-negative fluids developed IgG and IgM-staining inclusions
had both p,, and IgG-staining inclusions. and four of these nine developed PICstaining inclusions. After the addition of
Synovial fluids from miscellaneous Waldenstrom IgM, tiny inclusions were ocarthritides. T h e results obtained with casionally seen, but were not considered to
fluids from 22 patients with miscel'aneous be significant, when compared with those
arthritides are shown in Table 2. None of produced with rheumatoid factor, becausthese synovial fluids gave positive rheuma- of their very small size. T h e differences
toid factor tests. I n contrast to the results observed after the addition of IgM rheumaobtained with rheumatoid factor-positive toid factor and Waldenstrom IgM were
effusions, it can be seen that only one of statistically significant.
,the 22 fluids produced IgM-containing inI n the juvenile rheumatoid group, one
clusions, four yielded p,,-containing inclu- of five fluids developed I g E and IgMsions and 3 IgG-containing inclusions. One staining inclusions after the addition of
fluid, from a patient with gonococcal arthri- IgM rheumatoid factor, while none de726
Arth~itisand Rheumatism, Vol. 13, No. 6 (November-December 1970)
Fig 1. lmmunofluorescent staining of normal leukocytes after incubation with rheumatoid factor
positive synovial fluid: A. Large intracellular inclusions staining for IgG; B. Inclusions staining for IgM;
C. Inclusions staining for pic; and D. Control preparation stained with anti-IgG, showing absence of inclusions. Control preparations stained with anti-lgM and anti-pIc also failed to demonstrate inclusions.
Table 1. Relation of SSCA Titer of
Synovial Fluid To Inclusions Phagocytosed
From Rheumatoid Effusions
Reciprocal SSCA titer
Adult RA
antibody ___-__
Juvenile RA
veloped Plc-staining inclusions. In this
group, as in the adult group, no fluid
yielded significant inclusions with the addition of WaIdenstrom IgM.
Table 4 gives the results obtained when
either IgM rheumatoid factor or Waldenstrom IgM was added to nonrheumatoid
synovial fluids. I n these experiments, only
three of 16 fluids developed significant I g G
and IgM-staining inclusions after the addition of rheumatoid factor. T w o oE these
fluids were from patients with acute Reiter’s syndrome and one was from a patient
Table 2. Inclusions Phagocytosed From
Nonrheumatoid Synovial Fluids*
FI uorescent
antibody staining
(No. positive/No. tested)
* Degenerative joint disease, 6; gout, 4; gonococcal arthritis, 3; Reiter’s syndrome, 3; systemic
lupus erythematosus, psoriatic arthropathy. palindromic rheumatism, pneumococcal arthritis,
post-transplantation arthritis, and sickle cell
disease. one each.
with degenerative arthriiis who had a n
effusion oE the knee of three weeks’ duration. One of the Reiter’s fluids also developed &.staining inclusions. After the
addition of Waldenstrom IgM to these
fluids, no significant inclusions were seen
on staining with any of the fluorescent
Table 5 presents a statistical comparison
of the results previously shown in Tables 3
and 4 in which rheumatoid factor was
added to factor-negative adult rheumatoid
and nonrheumatoid synovial fluids. T h e
differences observed between the rheumatoid and nonrheumatoid fluids were statistically significant.
Hollander and coworkers (2, 3) described
the presence of leukocytes with cytoplasmic granules in the synovial fluid of most
patients with rheumatoid arthritis and ok
some patients with other types oE inflammatory arthritis. They hypothesized that
rheumatoid factor-IgG complexes in the
synovial fluid stimulate leukocytosis and
that this stimulation is followed by the
subsequent phagocytosis of these complexes
and the release oE lysosomal enzymes which
produce inflammation. Rawson et al (3)
showed that leukocyte cytoplasmic inclusions had positive fluorescent staiaing for
IgG and IgM.
I n studies with suspensions of synovial
lining cells obtained by trypsinization of
synovial tissue, Kinsella et a1 (6) demonstrated cytoplasmic inclusions that stained
for IgG and plC and for IgG, IgM and PIC
in rheumatoid synovial lining cells. Inclusions containing IgM were observed only in
seropositive patients. Fluorescent staining
of immunoglobulin and complement components in undigested rheumatoid synovium had been previously demonstrate:l by
several groups (7-9).
Arthritis and Rheumatism, Vol. 13, No. 6 (November-December 1970)
Table 3.
Production of Inclusions Upon Addition of Rheumatoid Factor To
Factor-Negative Rheumatoid Synovial Fluids
antibody staining
Immunoglobulin added
Immunoglobulin added
factor IgM
factor IgM
p< 0.025
T h e present study demonstrates that normal polymorphonuclear celis ingest aggregates from the majority of rheumatoid factor-containing synovial fluids. I n mojt instances, the resulting inclusions stained
positively for IgG, IgM and the p,, cornponent of complemmt. Since the IgG- and
IgM-staining inclusions were obiaineg primarily from rheumatoid factor-positive
fluids, it is likely that the-e inclu,'crlons consisted of a complex of altered IgG with
IgM rheumatoid factor. Indeed, in no instance was IgM staining observed wit1io:it
the concomitant staining of IgG.
Combined staining with fluorescein- and
r h o d a m i ne-labeled anti-immunoglobulin
sera showed that these inclusions contained
complexes of IgG with plc, IgM and plC
and IgG with IgM. Although it is not
possible to conclude from thes:: observations whether these represented individual
binary complexes or the single ternary
complex, IgG-IgM-p,,, i t appears likely
that the PIC was combined with the IgGIgM complex to form an IgGIgM-p,,
complex, since Zvaifler and Schur (10)
demonstrated that complexes of rheumatoid factor with mercaptoethanol reduced,
heat-aggregated IgG do fix human complement.
Vaughan and coworkers (11) identified
C3 in joint fluid leukocyte inclusions by
fluorescent staining in approximately 50%
of bo'h rheumatoid and nonrheumatoirl
patients, although all joint fluids which
contained abnormally low hemolytic complement activity also showed y-globulin inclusions in the synovial fluid leukocytes.
Ruddy et n l (12) demonstrstel th3t thinclusions in r1ieumatoi:I fluids also contain Clq and C4. I n the pre-ent experiments, inclusions were not obtained from
most rheumatoid factor-negative synovial
fluids. However, one of 22 coytrol fluids,
obtained from a patient with go iocoxal
arthritis, produced inclusions that stained
for IgG, IgM and PIC.I n two other fluids
(from patients with gonococcal arthritis
and psoriatic arthropathy), IgG- and Picstaining inclusions were present and in one
fluid from a patient with palindromic rheumatism only PIC was stained. In the single instance in which IgM was present in
inclusions obtained from a nonrheumatoid
fluid, it is possible that small amounts of
rheumatoid factor were present in the
kthritis and Rheumatism, Vol. 13, No. 6 (November-December 1970)
Table 4. Effect of Added Rheumatoid Factor IgM
or Waldenstrom IgM on Phagocytosis of
Inclusions From Nonrheumatoid Synovial Fluids
Immunoglobulin added
Anti body
factor IgM
effusion. although not detected by the
SSCA test. T h e fluid coxerned did in fact
show a If slide latex-fixation test.
Immunoglobulin-containing inclusions
have been observed in leukocytes of
nonrheumatoid synovial effusions. Brandt
et al (9) found IgG- and IgM-staining
inclusions in the synovial fluid leukocytes
from a patient with Reiter’s syndrome.
Nevertheless, from the present study, it is
apparent that leukocyte inclusions are
much more frequently phagocytoserl from
factor-positive rheumatoid fluids than from
factor-negative fluids and nonrheumatoid
Table 5. Production of Inclusions on
Addition of Rheumatoid Factor: Comparison of
Factor-Negative Rheumatoid and
Nonrheumatoid Fluids
Factor-negative synovial fluid
( 6%)
p< 0.025
fluids. Moreover, although IgG-plc aggregates may be obtained from a small minority of nonrheumatoid fluids, IgM-containing
inclusions are rare in such fluids, suggesting that rheumatoid factor plays a n important role in the formation of the inclusions
obtained from factor-positive fluids.
When IgM rheumatoid factor and Waldenstrom Ighl were added to factornegative synovial fluids from which inclusions had not been phagocytosed, the majority of these fluids yielded inclusions after
the addition of IgM rheumatoid factor but
not after the addition of the Waldenstrom
IgM. I n every instance, the inclusions that
formed stained positively for IgG and IgM,
and in most cases also for PIC.I n contrast
to this experience with factor-negative rheumatoid effusions, only 3 of 16 nonrheumatoid fluids developed IgG and JgM inclusions after the addition of rheumatoid factor. As with the factor-negative rheumatoid
fluids, addition of Waldenstrom IgM to the
nonrheumatoid fluids did not yield significant inclusions.
These observations on the development
of inclusions after the addition of rheumatoid factor to factor-negative rheumatoid
fluids, as opposed to the results obtained
with most fluids from other arthritides,
suggest that factor-negative rheumatoid
fluids contain an altered form of IgG which
is not often present in effusions of patients
with other arthritide:. I n factor-positive
rheumatoid fluids, the altered IgG is presumably already combined with rheumatoid factor, leading to the phagocytosis of
the complexes which show combbed staining for IgG, IgM and PIC. I n factornegative fluids, the presence of altered IgG
becomes evident by virtue of its reaction
with the rheumatoid factor added in vitro.
T h e above findings are consistent with
the observations of Hannestad (4) who
Arthritis a d Rheumatism, Vol. 13, No. 6 (November-December 1970)
demonstrated a precipitin reaction between Barbara Smith for their excellent technical
high titer rheumatoid sera and certain rheu- assistance.
matoid synovial fluids. T h e precipitating
factor in the sera was shown to be a n IgM
Schmid FR: Phagocytosis of
rheumatoid factor. T h e precipitating comparticulate
of 7-globulin and
ponent of the synovial effusions was, prerheumatoid factor. J Immunol 88:519-525,
sumably, structurally altered IgG. T h e
presence of 10s to 19s IgG complexes,
9 Hollander JL, McCarty DJ, Astorga G,
which form precipitates with added IgM
et al: Studies on the pathogenesis of rheu
rheumatoid factor, has been demonstrated
matoid joint inflammation. I. The “R.A.
in rheumatoid effusions by Winchester and
Cell” and a working hypothesis. Ann InKunkel (5). It seems probable that these
tern Med 62:271-280, 1965
precipitates would be phagocytosed and
3. Rawson AJ, Abelson NM, Hollander JL:
Studies on the pathogenesis of rheumatoid
visualized as IgG-IgM staining inclusions.
joint inflammation. 11. Intracytoplasmic
More recently, Winchester et a1 showed
particulate complexes in rheumatoid s y n
(IS) that depression in the levels of Clq in
ovial fluids. Ann Intern Med 62281-284,
rheumatoid effusions correlated with their
content of IgG complexes containing IgG
4. Hannestad K: Presence of aggregated -,-Grheumatoid factor. They concluded that
globulin in certain rheumatoid synovial
the decrease in the level of complement
effusions. Clin Exp Immunol 2:5 11-529,
was due to binding by these complexes and
suggested that the resulting activation of
5. Winchester R, Kunkel HG: High molecuthe complement system might initiate local
lar weight complexes involving y-globulin
in joint fluids from rheumatoid arthritis
injury. Kinsella et al (6) have, in fact,
(RA) patients. Arthritis Rheum 11:849,
observed the presence of a n IgGp,, com1968, abstr
plex in the phagocytic lining cells of the
6. Kinsella TD, Baum J, Ziff M: Immunosynovial membrane of rheumatoid patients
fluorescent demonstration of an IgG-p,,
with factor-negative effusions. It seems likecomplex in synovial lining cells of rheuly, however, that the combination of IgM
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7. Fish AJ, Michael AF, Gewurz H, et al:
sulting in the formation of phagocytosable
Immunopathologic changes in rheumatoid
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arthritis synovium. Arthritis Rheum 9:
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tant mechanism for initiating and perpetu8. Rodman WS, Williams RC Jr, Bilka PJ,
ating synovial inflammation through the
et al: Immunofluorescent localization of
release of lysosomal enzymes and other
the third and fourth component of comcellular constituents, as has been suggested
plement in synovial tissue from patients
previously (11, 14-16).
with rheumatoid arthritis. J Lab Clin Med
We thank Dr. Chester Fink for supplying
synovial fluids from patients with juvenile
rheumatoid arthritis. We also thank Mrs.
Gwendolyn Davis, Mrs. Joan Sims and Miss
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J Lab Clin Med 72631-647, 1968
10. Zvaifler NJ, Schur P: Reactions of aggregated Mercaptoethanol treated gamma
Arthritis and Rheumatism, Vol. 13, No. 6 (November-December 1970)
globulin with rheumatoid factor-precipitiii
and complement fixation studies. Arthritis
Rheum 11:523-536, 1968
11. Vaughan JH, Barnett EV, Sobel MV, et al:
Intracytoplasmic inclusions of immunoglobulins in rheumatoid arthritis and other diseases. Arthritis Rheum 11:125-134, 1968
12. Ruddy S , Britton MC, Schur PH, et al:
Complement components in synovial fluid:
activation and fixation in seropositive rheumatoid arthritis. Ann NY Acad Sci 168:
161-172, 1969
IS. Winchester RJ, Agnello V, Kunkel HG:
The joint Auid yG-globulin complexes and
their relationship to intraarticular comple-
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Vol. 13, No. 6 (November-December 1970)
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