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Platelet-activating activity in synovial fluids of patients with rheumatoid arthritis juvenile rheumatoid arthritis gout and noninflammatory arthropathies.

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PLATELET-ACTIVATING ACTIVITY IN SYNOVIAL
FLUIDS OF PATIENTS WITH RHEUMATOID
ARTHRITIS, JUVENILE RHEUMATOID ARTHRITIS,
GOUT, AND NONINFLAMMATORY ARTHROPATHIES
CHRISTINE SHAPLEIGH, FRANK H. VALONE, PETER H. SCHUR, EDWARD J. GOETZL,
and K. FRANK AUSTEN
The predominant activating activity in rheumatoid synovial fluid for human platelets, as assessed by
the release of I4C serotonin, was previously isolated and
shown to be a complex of IgG of molecular weight
450,000. Thus, a pooled 5 ml concentrate of the 450,000
molecular weight fraction obtained by Sepharose 6B gel
filtration of 1 ml of synovial fluid was used to compare
the platelet-activating activity (PAA) of fluids from 28
patients with inflammatory and noninflammatory arthropathies. One unit of PAA was arbitrarily defined as
that volume of the 5 ml concentrate required to obtain
release equal to that obtained with a standard dose of
thrombin, and total units per milliliter of synovial fluid
were calculated as the reciprocal of the volume of concentrate in milliliters containing one unit multiplied by
five. A platelet-activating activity of >766 units/ml of
synovial fluid was arbitrarily defined as elevated, since
this value represents two standard deviations above the
mean for the noninflammatory synovial fluids. Each of 7
noninflammatory fluids (range <50 to 695) and 3 of 4
gouty fluids (range <50 to 1,250) fell within the normal
From the Departments of Medicine, Harvard Medical.
School, and the Robert B. Brigham Hospital Division of the Affiliated
Hospitals Center Inc., Boston, Massachusetts 021 15.
Supported in part by Grants AI-07167, AI-07722, AM-I 1414,
AM-05577, and RR-05669 from the National Institutes of Health, and
by a grant from The New England Peabody Home for Crippled Children.
Christine Shapleigh, MD: Postdoctoral Trainee, National Institutes of Health; Frank H. Valone, MD: Research Associate of the
Howard Hughes Medical Institute; Peter H. Schur, MD, Edward J.
Goetzl, MD: Director of the Howard Hughes Institute Laboratories
for the Study of Immunological Diseases; K. Frank Austen, MD.
Address reprint requests to Dr. K. Frank Austen, Room 604,
Seeley G. Mudd Building, 250 Longwood Avenue, Boston, Massachusetts 021 15.
Submitted for publication January 11, 1980 accepted in revised form March 20, 1980.
Arthritis and Rheumatism, Vol. 23, No. 7 (July 1980)
range, whereas 9 of 13 adult rheumatoid fluids (range
<50 to 11,880), and 4 of 4 JRA fluids (range 1,299 to
5,100) had elevated levels of PAA per ml of synovial
fluid. The finding that the purified synovial fluid 450,000
molecular weight complex of IgG was inhibited in its
platelet-activating capacity by purified monoclonal rheumatoid factor suggests that a role for IgM rheumatoid
factor could be to block IgG complex Fc receptor-dependent proinflammatory effects on platelets, and perhaps other cells.
Intermediate molecular weight complexes of IgG
present in the serum of patients with rheumatoid arthritis (1) have been shown to contain IgG rheumatoid factors (2) which preferentially associate with each other to
the exclusion of IgG molecules of other specificities (3).
Certain rheumatoid synovial fluids contain IgG complexes which were initially considered to resemble IgG
aggregated by physicochemical procedures (4) but were
subsequently shown to be immune complexes composed
of IgG rheumatoid factors (5). Both experimentally
formed IgG-containing immune complexes and aggregated IgG induce platelet aggregation and secretion of
serotonin by interaction with the Fc receptor (6,7). Although platelet aggregation has been used as an assay of
immune complexes in serum (8), it has not been applied
to synovial fluid. Platelet-activating activity (PAA), as
assessed by the release of I4Cserotonin in the absence of
lactic acid dehydrogenase from human platelets, has
been identified in rheumatoid synovial fluid and purified by cation exchange chromatography, gel filtration,
and preparative isoelectric focusing to yield a 450,000
molecular weight principle termed purified synovial
fluid platelet-activating activity (SF-PAA); SF-PAA
PLATELET-ACTIVATING ACTIVITY
contained only IgG by immunochemical analysis (9).
T h e level of platelet-activating activity in a 450,000 molecular weight fraction of synovial fluid has been found
to be elevated in adult and juvenile rheumatoid arthritis
but not in gout or noninflammatory arthropathies.
MATERIALS AND METHODS
5-[2-’4C]hydr~xytryptamine
binoxalate (14C serotonin,
specific activity 47 mCi/mmol), Aquasol Universal LSC cocktail (New England Nuclear Corp., Boston, Massachusetts),
human thrombin (Fibrinidex, Ortho Diagnostics Inc., Raritan, New Jersey), five times recrystallized human serum albumin (Miles Laboratories, Inc., Kankakee, Illinois), and Sepharose 6B (Pharmacia Fine Chemicals, Uppsala, Sweden) were
obtained as noted.
Platelet suspensions were prepared with siliconized
glassware (Pro-Sil-28, Fisher Scientific Co., Pittsburgh, Pennsylvania) or with plasticware (Walter Sarstedt Inc., Princeton,
New Jersey). Protein concentration was determined by optical
absorbency at 280 nm on a Gilford spectrophotometer (Oberlin, Ohio). Protein concentration of synovial fluid was assessed by the method of Lowry et a1 (lo), synovial fluid rheumatoid factor titers were determined by nephelometry (1 1),
and serum rheumatoid factor titers by latex fixation (12) or
nephelometry. Clq binding (13), inhibition of antibody-dependent cellular cytotoxicity (ADCC) (14,15), and the complement components Clq, C4, C3 (16), and B (17) were measured as described.
Purified monoclonal IgM rheumatoid factor (mRF)
containing 3.26 mg/ml IgM and less than 0.031 mg/ml IgG
was obtained from Dr. Vincent Agnello (18). SF-PAA was
isolated as described and stored at a concentration of IgG of
1.4 mg/ml (9).
Patient population. Synovial fluids were obtained
from 28 patients (Table 1). The thirteen patients with adult
rheumatoid arthritis satisfied the American Rheumatism Association criteria for definite or classic rheumatoid arthritis
(RA) (19). Eleven were seropositive with rheumatoid factor titers greater than 1:64, and 2 were seronegative. The diagnosis
of juvenile rheumatoid arthritis (JRA) was based on the criteria established by the JRA subcommittee of the American
Rheumatism Association (20), and subtypes were classified by
observations made in the first 6 months of disease. Three of
the JRA patients were females with oligoarticular disease
without iridocyclitis and with negative serum titers for rheumatoid factor and antinuclear antibodies (ANA). The male
JRA patient had oligoarticular inflammatory arthritis, psoriatic skin lesions present from birth, intermittent diarrhea, a
negative serum rheumatoid factor, a positive ANA titer of
1 :40, and an HLA-B27 tissue type. The 4 patients with gout
had negative birefringent crystals in the synovial fluid; 1 patient had tophaceous disease. Of 7 patients with noninflammatory arthropathies, 3 had osteoarthritis, diagnosed by the radiologic criteria of Kellegren (21), 3 had mechanical
derangements, and one had a traumatic arthropathy.
Processing of synovial fluid. Synovial fluid was obtained during diagnostic or therapeutic arthrocentesis and was
immediately placed on ice. The fluid was transferred to 14 ml
plastic tubes, and the cells and other debris were sedimented
80 1
Table 1. Patient characteristics
Diagnosis
Adult rheumatoid
arthritis
Juvenile rheumatoid
arthritis
Gout
Noninflammatory
arthropathies
Number
of patients
M
F
Range
Mean
13
6
I
26-81
56
4
4
1
3
0
9-28
16
4
35-60
49
7
5
2
30-80
57
Sex
Age (years)
by centrifugation at lOOOg at room temperature for 10 minutes. Samples of each supernatant were analyzed for PAA immediately or after storage at 4°C for 1 to 5 days; additional
samples were frozen immediately at -70°C and assessed later
for C l q binding, inhibition of ADCC, rheumatoid factor titer,
and total protein concentration. Two milliliters of each unfrozen synovial fluid supernatant were dialyzed against one
change of 1 liter of 0.01M Tris buffer, pH 7.4, containing 0.1M
NaCl at 4°C overnight, and any residual debris was removed
by centrifugation at 7000g for 2 minutes. One milliliter of supernatant was then applied at room temperature to an 80 x
1.8 cm column containing Sepharose 6B equilibrated in the
same buffer. Fractions of 3.8 ml(2% of bed volume) were collected at a flow rate of 15 ml per hour, and 1.0 ml portions of
every other fraction were dialyzed in separate bags against
one change of 4 liters of 0.01M phosphate bbffer, pH 7.0, containing 0.1M NaCl at 4°C overnight; replicate 250 p1 samples
of each portion were assayed immediately or after 1 to 14 days
of storage at 4°C for their capacity to release I4C serotonin
from platelets.
Fractions from Sepharose 6B filtering with an approximate molecular weight of 450,000 and yielding more than
10% net I4C serotonin release were pooled along with the adjacent fractions and concentrated to 5 ml at 4°C with an Amicon ultrafiltration apparatus (Amicon Corp., Lexington, Massachusetts). If less than 10% net 14C serotonin release was
detected, all fractions from the 350,000-550,000 molecular
weight region were pooled and concentrated. The 5 ml concentrate was dialyzed overnight at 4°C against one change of
I liter of 0.01M phosphate buffer, pH 7.0, containing 0.1M
NaC1, stored at 4°C for 1 to 14 days, and assayed in a doseresponse fashion for I4Cserotonin release.
Platelet serotonin release assay. Fifty to 100 milliliters of blood were obtained by venipuncture with sterile plastic syringes from normal adult volunteers who had not taken
aspirin-containing compounds for the previous 7 days. The
samples were aliquoted by 5 ml portions into 14 ml plastic
tubes containing 1 ml of citrate anticoagulant (0.076M citric
acid, 0. ISM sodium citrate, pH 5.2). The erythrocytes and leukocytes were sedimented by centrifugation at 170g for 15 minutes at room temperature. The platelet-rich plasma supernatants from each tube were aspirated with siliconized Pasteur
pipettes, pooled into a 50 ml plastic tube, and mixed with a final concentration of 0.4 x 10-6M I4C serotonin. After incubation at 37°C for 30 minutes, the labeled platelets were
sedimented at lOOOg for 10 minutes, and the cell pellet was
washed 3 times with 0.01M phosphate buffer, pH 6.5, containing 0.1M NaCl, 0.1 1M glucose, and 20% (v/v) citrate anti-
SHAPLEIGH ET AL
802
coagulant. The platelets were suspended in 0.01M phosphate
buffer, pH 7.0, containing 0.1M NaC1, 0.1 1M glucose, 1.8
mM CaCl,, and 0.1% (w/v) human serum albumin to yield a
final cell concentration of 2-3 x lox platelets/ml. Two hundred fifty microliters of radiolabeled platelets were added to 3
ml plastic tubes containing 250 p1 of either buffer or test
sample in buffer, and the mixture was incubated at 37°C for
30 minutes with gentle agitation. The platelets were sedimented at lOOOg for 10 minutes at room temperature; the supernatant was mixed with 10 ml of Aquasol in 20 ml liquid
scintillation vials, and radioactivity was determined in a
Searle Mark IV Liquid Scintillation Counter. The total 14C
serotonin in 250 pl of washed radiolabeled platelets was determined by adding the sample directly to counting vials containing Aquasol. Results were expressed as the net percent release of 14Cserotonin according to the following formula:
Net % serotonin released =
(“C serotonin released) - (14Cserotonin leak)
x 100
(Total I4C serotonin) - (I4Cserotonin leak)
In 74 experiments the spontaneous leak of 14C serotonin by
platelet suspensions incubated with buffer ranged from 0.5%
to 16.6%, with a mean of 6.6%.
Statistical analysis was performed with a one-sided
Wilcoxon signed rank test and Spearman’s rank correlation
coefficient.
RESULTS
In preliminary studies crude rheumatoid synovial fluid stimulated release of platelet 14Cserotonin, but
a dose-response relationship was observed only after sequential chromatographic purification of the 450,000
molecular weight principle containing IgG, termed SFPAA (9). Thus, a clinical study was initiated with a
450,000 molecular weight fraction of synovial fluid obtained by Sepharose 6B gel filtration, as depicted in Figure 1 for 1 ml of synovial fluid from a patient with rheumatoid arthritis. The fractions containing the peak of
platelet-activating activity (PAA) of approximately
450,000 molecular weight, as defined by the position of
the ferritin marker, were pooled, concentrated to 5 ml
and assayed in a dose-response fashion for platelet I4C
serotonin release. Volumes of 100, 10, 2, 1, 0.5, and 0.1
p1 gave a net percent I4Cserotonin release of 52%, 63%,
54%, 42%, 9%, and 0%, respectively. Release was doserelated, reaching a plateau for most synovial fluids, the
exceptions generally being synovial fluid with insignificant concentrations of PAA. Maximal 14C serotonin releases (mean -C SD) for fluids from patients with
adult rheumatoid arthritis, juvenile rheumatoid arthritis, gout, and noninflammatory arthropathies were 53 k
12%, 59 k 12%, 45 f lo%, and 47 f lo%, respectively.
The additional peak of PAA (Figure 1) in the limit vol-
Peak of Approx,
m
MW 450 OOO
Figure 1. Platelet-activating activity in eluant fractions from gel filtration on Sepharose 6B after application of l ml of synovial fluid
from a patient with rheumatoid arthritis. The column was calibrated
with blue dextran (exclusion volume), ferritin (molecular weight
450,000),and B12 (molecular weight 1355).
ume was variably present in all patient groups, whereas
that at the void volume was present in 8 adult rheumatoid synovial fluids only. These two additional peaks
consistently contained less than one-tenth the PAA of
the 450,000 molecular weight peak as measured by the
dose-response release of I4Cserotonin and are not considered further.
To minimize day-to-day variations in platelet
sensitivity, a thrombin standard was analyzed in parallel with test samples in a final concentration from 0.01
to 0.40 units/ml which gave a dose-related net percent
14Cserotonin release from 5% to 95%. In 10 consecutive
experiments, net percent serotonin release by 0.08
units/ml thrombin was 44 11% (mean k SD), and one
unit of synovial fluid PAA was defined as that volume
required to obtain a net percent I4C serotonin release
equal to that generated by 0.08 units/ml thrombin in
that assay. Total units of PAA for 1 ml of synovial fluid
were expressed as the total number of units present in
the 5 ml concentrate of the 450,000 molecular weight
peak and were calculated as the reciprocal of the sample
volume containing one unit, expressed in milliliters,
multiplied by 5. If all volumes of a sample (up to 100 pl)
generated less serotonin release than 0.08 units/ml
throdin, units of PAA were expressed as 4 0 . The stability of PAA in crude synovial fluid was assessed by gel
filtration before and after storage for 4-6 months at
-7OOC. Units of PAA before and after freezing were
11,880 and 10,000, respectively, for one rheumatoid synovial fluid, 65 and 4 0 for a gouty fluid, and 695 and
4 0 for a noninflammatory fluid. Assessment of the reproducibility of PAA was determined for 4 fluids in 3
PLATELET-ACTIVATING ACTIVITY
803
80
60
40
20
0
VOLUM€ O f 5 m l CONCENW'ATE ON
Figure 2. Dose response of I4C serotonin release by incremental volumes of the 5 ml concentrate of the
450,000 molecular weight fraction of 1 ml of synovial fluid from patients with seropositive rheumatoid
arthritis, gout, and noninflammatory (traumatic) arthritis, each assayed on 3 separate occasions (U,
M,
A-A). The corresponding release by 0.08 units/ml thrombin on each of these occasions was 30%
(O), 34% (0),
and 58% (A).
experiments over a 6-week period using samples of the 5
ml concentrate which had been stored at 4°C. Units of
PAA ranged from 10,640 to 12,500 with a mean of
11,880 and from 5,555 to 10,000 with a mean of 7,350
for 2 rheumatoid fluids, from t50 to 103 with a mean of
67 for one gouty fluid, and from 4 0 to 565 with a mean
of 255 for one noninflammatory fluid (Figure 2).
Platelet-activating activity, expressed as units of
PAA per ml of synovial fluid, is depicted in Figure 3.
The mean PAA for the 7 noninflammatory fluids was
264 +. 251 units (mean k SD) per ml and, therefore,
fluids were designated as containing elevated PAA if
they contained greater than 766 units, which equaled 2
standard deviations above the mean for the noninflammatory fluids. Each of the 7 noninflammatory and 3 of 4
gouty synovial fluids contained less than 766 units of
PAA. Nine of the 13 adult rheumatoid synovial fluids
had elevated levels of PAA with units ranging from
2,630-1 1,880, while 4 had less than 766 units of PAA. In
patients with adult rheumatoid arthritis, units of PAA
correlated inversely with synovial fluid C3 protein (rs =
-0.74, n = 12, P < 0.01) (Table 2) but not with C l q (rs
= -0.13, n = 12, P > O.lO), C4 (r$= -0.39, n = 12, P >
0.10), or B (r. = -0.18, n = 12, P > 0.10) proteins, and
there was no apparent relationship between units of
PAA and synovial fluid leukocyte count, synovial fluid
protein concentration, sedimentation rate, hematocrit,
disease duration, or extraarticular manifestations. Levels of PAA in rheumatoid synovial fluid were significantly higher than in noninflammatory fluids (P <
0.03), and tended to be higher than in gouty fluids, al-
-=
though statistical significance was not achieved (P
0.08). High levels of PAA were detected in each juvenile
rheumatoid synovial fluid, ranging from 1299 to 5100
units; these levels were significantly higher than those in
noninflammatory (P< 0.005) or gouty (P < 0.02) fluids.
1
I
0
11,200-
9600 -
'3
P
8
80000
6400-
i?
4800-
0
0
8
3200 16001
0
RA
JRA
lnflommatory
Figure 3. Units of platelet-activating activity (PAA) for 1.0 ml of
partially purified synovial fluid from patients with adult rheumatoid
arthritis (RA), juvenile rheumatoid arthritis (JRA), gout, and nonidammatory arthropathies. The broken line indicates 766 units,
which is 2 standard deviations above the mean for noninflammatory
fluids.
SHAPLEIGH ET AL
Table 2. Clinical characteristics and synovial fluid parameters for 13 adult rheumatoid patients
Synovial fluid parameters
Diagnosis
Seropositive
rheumatoid
arthritis
Seronegative
rheumatoid
arthritis
Age
Sex
81
46
45
74
50
F
M
M
F
31
62
26
60
75
65
F
M
59
61
Plateletactivating
activity
(units of
PAA)
c3 %/gm
synovial
fluid
protein
IgM RF
(titer)
< 50
Leukocyte
count*
43
5
74
8
7
3
11,439
85
2,630
3,495
1:5,120
1:640
1 : 640
Negative
Negative
16,615
12,470
9,600(65)
F
M
F
M
3,845
5,000
5,495
6,175
7,350
11,880
Negative
Negative
Negative
1: 160
1 : 2,560
Negative
13,680
8,840
9,770
5,000
10,233
8,614
SSO(35)
200(25)
4,000 (25)
40
40
18
4
16
3
18
26,200 (80)
16,600(97)
42
90
11
F
< 50
M
4,760
Negative
Negative
11,980
10,260
17,800(57)
1,250(48)
54
39
< 50
F
9,900(75)
Disease
duration
ESRt (years)
13,150 (46)
10,800
5
1
1
6
Extraarticular
manifestations
None
Nodules
None
Keratoconjunctivitis
sicca
None
Nodules
None
None
Nodule on chest x-ray
None
None
None
* Parentheses indicate percent polymorphonuclear leukocytes.
t Erythrocyte sedimentation rate determined by the Westergren method.
Synovial fluid samples were assayed for immune
complexes by Clq binding and inhibition of ADCC
(Table 3). Rheumatoid synovial fluids contained a significantly higher frequency of immune complexes than
noninflammatory fluids (P < 0.03) as measured by Clq
binding. For all synovial fluids studied, there was a correlation between levels of immune complexes as measured by the Clq binding qssay and units of PAA (rs =
0.40, sample size = 26, P < 0.05) (Figure 4).The level of
PAA was elevated in each of 4 JRA synovial fluids,
whereas Clq binding was increased in only one; conversely, units of PAA were increased in only one gouty
fluid, whereas C l q binding activity was elevated in
three of four. The ADCC inhibition assay was positive
in all patient groups and did not correlate in frequency
or titer with the C l q binding or platelet release assays.
The possible modulation by IgM rheumatoid
factor of platelet activation by IgG complexes in crude
rheumatoid synovial fluid was investigated with purified SF-PAA and purified monoclonal IgM rheumatoid
factor (mRF). SF-PAA was preincubated with buffer
Table 3. Frequency of elevated levels of platelet-activating activity
and immune complexes in synovial fluids
Number of patients with elevated synovial
fluid levels/total number studied
Assay
PAA units*
C Iq binding assayt
Inhibition of ADCC$
Adult
rheumatoid
arthritis
Juvenile
rheumatoid
arthritis
Gout
Noninflammatory arthropathies
9/13
9/12
4/4
1/4
2/4
1/4
3/4
3/4
0/7
1/6
4/6
5/12
* Platelet-activating activity, elevated
*
t Elevated = > 23 pg/ml
0
~
=
> 766 units.
aggregated IgG equivalent.
Inhibition of antibody-dependent cellular cytotoxicity, elevated
> 1.5 pg/ml aggregated IgG equivalent.
=
2000
4000
.**
I
6000
8000
UNKS OF PAA
I
-
10000
12000
Figure 4. Correlation between immune complex levels by C l q binding assay and units of platelet-activating activity (PAA) for synovial
fluids from all patient groups (P < 0.05, Spearman’s rank correlation
coefficient, r, = 0.40, sample size = 26).
PLATELET-ACTIVATING ACTIVITY
alone or with increasing concentrations of mRF at 37°C
for 1 hour and then at 4°C for 1 hour. SF-PAA, in a final concentration of 0.021 mg/ml, stimulated a net I4C
serotonin release of 37%. The percent inhibition of serotonin release with SF-PAA:mRF molar ratios of 128:1,
64:1, 12.8:1, 6.4:1, and 2:l were 0%, lo%, 45%, loo%,
and loo%, respectively. In 3 consecutive experiments,
100% inhibition of serotonin release occurred at molar
ratios ranging from 6.4:1 to 2: 1. Of interest was the finding that 3 of the 11 seropositive rheumatoid synovial
fluids had low levels of PAA, and all three contained
IgM rheumatoid factor, the titers ranging from 1:640 to
15,120. The remaining fluids all contained more than
766 units of PAA, and 6 of 8 had no IgM rheumatoid
factor detectable in the synovial fluid (Table 2).
DISCUSSION
Previous studies have shown that the predominant platelet-activating activity in rheumatoid synovial
fluid (SF-PAA) was an intermediate molecular weight
complex of IgG. This purified material stimulated release of platelet I4C serotonin in a dose-response fashion, whereas crude synovial fluid did not (9). Thus a 5
ml concentrate of the 450,000 molecular weight material
obtained by Sepharose 6B gel filtration of 1 ml of synovial fluid was used to assess PAA in synovial fluids of
patients with inflammatory and noninflammatory arthropathies (Table 1). Additional minor peaks of PAA
were recognized and found to contain less than 10% of
the total PAA (Figure 1). The net percent release equal
to that obtained with 0.08 units/ml thrombin was chosen to define one unit of PAA in order to standardize
the assay from day to day and because I4C serotonin release with this dose was 44 & 11% (mean & SD) and fell
on the dose-response curve for the synovial fluid PAA
(Figure 2). Platelet-activating activity of greater than
766 units of PAA per ml was defined as elevated because this level was 2 standard deviations from the
mean for the noninflammatory synovial fluids (Figure
3).
Reproducibility of the assays was confirmed by
serial assay of the 5 ml concentrate stored at 4°C which
revealed that fluids with or without elevated levels of
PAA retained their initial classification (Figure 2). Stability of PAA after freezing synovial fluid and storing it
for up to 6 months at -70°C was demonstrated for one
rheumatoid fluid which contained 11,880 units of PAA
before and 10,000 units after freezing and for one gouty
and one noninflammatory fluid which each remained
negative.
805
Each of 7 noninflammatory and 3 of 4 gouty
fluids contained <766 units of PAA, whereas 9 of 13
adult rheumatoid fluids contained elevated levels of
PAA (Figure 3). This incidence is consistent with the
Clq binding positivity of this group (Table 3 and Figure 4), and with reports of immune complexes in 7 1% to
95% of rheumatoid synovial fluids in which the Clq
binding assay was used (22-24). Of the 4 rheumatoid
patients with low levels of PAA, one was seronegative
with recent onset of RA, and 3 were seropositive and
had high titers of synovial fluid rheumatoid factor
(Table 2), which would both inhibit SF-PAA and
change its filtration characteristics. Salicylate therapy
would not influence PAA because free or bound salicylate in synovial fluid would be excluded from the 5 ml
concentrate by gel filtration. Synovial fluid PAA does
not correlate with sedimentation rate, hematocrit, synovial fluid leukocyte count, synovial fluid protein, disease
duration, or extraarticular manifestations in patients
with adult rheumatoid arthritis. Although levels of immune complexes in serum, as measured by the Clq
binding assay, correlate with the presence of extraarticular manifestations, synovial fluid levels do not
(20).
Each of the 4 JRA patients with seronegative oligoarticular disease had elevated levels of synovial fluid
PAA, whereas C l q binding was negative in 3 of 4. Although l l of 5 l JRA patients had circulating immune
complexes as measured by the C l q binding assay (25),
immune complexes have not been reported in synovial
fluid of patients with JRA. The presence of complexes
in synovial fluid is suggested by the intraarticular acquired abnormalities of the classic complement pathway in approximately one-half of JRA patients, regardless of subtype or seropositivity (26).
The levels of synovial fluid immune complexes
assessed by Clq binding and units of PAA correlated
(r, = 0.40, n = 26, P < 0.05) (Figure 4) for the whole
group, possibly because the exceptions, namely JRA
and gout, represented only 8 of the 26 patient fluids
studied with both assays. Three of 4 gouty synovial
fluids contained elevated levels of aggregated IgG
equivalents by C l q binding, perhaps because urate
crystals bind IgG (27) and activate complement (28);
only 1 of 4 gouty synovial fluids contained elevated
quantities of PAA. Activation of human platelets occurs
via an Fc receptor as demonstrated by the stimulation
of platelet aggregation and release of serotonin by aggregated IgG, IgG complexes, and aggregated Fc fragments but not aggregated F a b , fragments (6,7). Furthermore, platelet serotonin release by purified SF-PAA
SHAPLEIGH ET AL
806
was inhibitable by monomeric IgG (9). The capacity of
mRF to completely inhibit 14Cserotonin release by SFPAA in molar ratios of SF-PAA:mRF of 2:l to 6:l is
compatible with the finding that IgG complex-induced
platelet aggregation is inhibitable by cryoglobulin IgM
components with rheumatoid factor activity (29).
The findings of local production of IgM rheumatoid factor by rheumatoid synovium (30), the absence of
free rheumatoid factor in rheumatoid synovial eluates
with high levels of IgG complexes, even when these
antibodies were present in serum (3 l), and the inhibition of SF-PAA-induced platelet serotonin release by
purified mRF suggest that one role of IgM rheumatoid
factor in the joint space is the blocking of IgG complexmediated inflammation.
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Rheumatology and Metabolic Bone Disease
The Harvard Medical School Department of Continuing Education will present a course entitled “Rheumatology and Metabolic Bone Disease” October 20-24, 1980 at the Parker House
Hotel, Boston. Sponsored by The Massachusetts General Hospital, the course is designed for
rheumatologists and internists with special interests in rheurnatology and metabolic bone diseases, with emphasis on recent advances in pathogenesis and treatment. Directors for the program are Drs. Dwight R. Robinson, Kurt J. Bloch, Stephen M. Krane, and John A. Mills.
Tuition is $360 (U.S.), and attendance is limited. Harvard Medical School certifies that this
continuing education program meets the criteria for approximately 3 1 credit hours in Category I
of the Physician’s Recognition Award of the AMA.
Application forms and additional information may be obtained from Harvard Medical
School, Department of Continuing Education, 25 Shattuck Street, Boston, MA 02115.
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platelet, patients, gout, activation, arthritis, activity, juvenile, synovial, arthropathies, noninflammatory, rheumatoid, fluid
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