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Total hemolytic complement CH50 and second component of complement C В╨Ж2hu activity in serum and synovial fluid.

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Total Hemolytic Complement (CH,,) and Second
Component of Complement (C’2hu)Activity in
Serum and Synovial Fluid
By GEORGE
FOSTIROPOULOS,
K. FRANK
AUSTENAND KURTJ. BLOCH
The presence of the second component
of human complement in synovial fluid
was demonstrated by a stoichiometric
titration using EAC’lagp4ap cells; fourth
component and a moiety of third component were identified by immunoelectrophoresis using specific antiserum. The
concentration of total hemolytic complement, C’2, and C‘4 was strikingly diminished in synovial fluid from patients
with rheumatoid arthritis compared to
their concentration in synovial fluid from
patients with traumatic, degenerative or
gouty effusions. These findings suggest
that reduction in total complement activity was due to activation of C’1 with
subsequent utilization of its substrates,
C’2 and C’4.
Le presentia del secunde componente de
complemento human in liquido synovial
esseva demonstrate per un titration
stoichiometric con le us0 de cellulas
EAC’lagp,4gp. Le quarte componente e
un parte del tertie componente esseva
identificate per immunoelectrophorese
con le us0 de antisero specific. Le concentration de complemento hemolytic
total, de C’2, e de C’4 esseva reducite
frappantemente in le liquido synovial de
patientes con arthritis rheumatoide in
comparation con lor concentration in
liquido synovial ab pitientes con effusiones traumatic, degeneratori, o guttose.
Iste constatationes suggere que reductiones in le activitate de complemento
total esseva le effect0 de un activation de
C’l con le utilisation subsequente de su
substratos C’2 e C’4.
c
OMPLEMENT ( C ’ } is required for immune cytoto,xic injury such as immune hemolysis,lg2 the serum bactericidal reaction,.? or ‘the lysis of certain tumor celk4s5 The specificity of the cytotoxic reaction is determined by
the antibody involved. Complement refers to a group of nonspecific serum factors (complement components) which interact in a certain sequence so as to
destroy cells previously sensitized by antibody. Electron microscopic data
indicate that the final step in the cytotoxic reaction is the production of holes
in the cell membrane.6 Complement may also participate in other immunologic phenomena such as phagocytosis,7,8 the Arthus reaction,9J0 and experimental nephritis.ll Available data suggest that phagacytosis and the Arthus
reaction may not require the entire complement sequence involved in cell
lysis.’3-’4
Evidence for the participation of complement in pathogenesis of human disease has usually been obtained by studying changes in serum complement
From the Department of Medicine, Harzjard Madical School and the Medical Services,
Massachusatts General Hospital, Boston, Massachusetts.
This is publication No. 386 of the Robert W. Looett Memorial Group for the Study
of Diseases Causing Deformities, Harvard Medical School, at the Massachusetts General
Hospital.
Supported b y Grants A-3564 and AM-04502 from the National Institute of Arthritis
and Metabolic Diseases, National lnstitutes of Health and by Grant Al-04536 from the
National Institutes of Allergy and Infectious Diseases.
219
ARTHRITIS
AND RHEUMATISM,
VOL. 8, No. 2 (APRIL),1965
220
FOSTIROPOULOS, AUSTEN, BLOCH
activity. The cytotoxic activity of whole serum may be conveniently assayed
using sensitized sheep erythrocytes. The sequence in which the components
of guinea pig complement act in immune hemolysis has been r e v i e ~ e d , ~ J ~ , ~ ~
and will be briefly considered only to introdrrce the necessary terminology and
to stress the theoretical shortcoming of the whole complement titer. Following the union of antibody ( A ) with certain antigenic sites ( S ) on the red
cell ( E ) , these sensitized receptor sites react with the complement system in
a definite sequence. The interaction of the first complement component with
the sensitized red cell (EA) converts the first component from a precursor
(C’lp) to an active form (C’la).Ii The interaction with the fourth complement component (C’4) follows after the firstls forming the EAC’la,4 cell.
The EAC’la,4 cell next reacts with the second complement component ( C 2 ) .
As a result of the enzymatic action of cell bound C‘la on C’21e320an inactive fragment (C’Zi) appears in the fluid phase and an active fragment
(C’Za) is bound to the cell (EAC‘la,4,2a). Once the cell achieves the
EAC’la,4,2a state it can either interact with C’3 factors to yield an irreversibly damaged cell or it can decay back to the EAC’la,4 state.21J2 The C’2a
lost during the decay reaction cannot be recovered as a hemolytically active
component, however the EAC’la,4,2a state can be regenerated by utilizing
additional C’2. In this manner an antigen-antibody complex can deplete the
serum of C’2 without the participation of C‘3 factors. Unless the C’2 titer becomes limiting the reduction in concentration of this component will not be
adequately reflected in the whole complement titer ( C‘H50).23,24
The level of whole complement activity has been shown to be reduced in
systemic lupus erythematosus2b and one of the C’3 factors, PlC globulin has
been identified at the site of the renal lesion in that
In rheumatoid
arthritis the level of whole serum complement is normal or slightly
whereas the titer in rheumatoid joint effusions is strikingly diminished compared to the concentration in synovial fluid of gouty or osteoarthritic joint^.^^,^^
In order to further explore the mechanism of this apparent intraarticular
complement depletion in rheumatoid arthritis, measurement of whole hemolytic complement in blood and synovial fluid was coupled with assays for certain components, C’2, C’4 (PlE globulin) and C’3a ( P l C globulin). The
demonstration of intra-articular depletion of C’2 and C’4 without a reduction
in the concentration of these components in serum, supports the contention
that in certain articular disease there is local activation of the complement
system.
MATERIAL
AND METHODS
Serum and synovial fluid was obtained from 70 patients. Fifty-six patients were included in this study after careful examination and follow-up permitted a definite diagnosis
of their disease. Clinical details of each patient group are presented in the appropriate
.section under Results.
Synovial fluid was obtained by needle aspiration of joint effusions; in most instances
fluid was obtained from the knee joint. Nearly all traumatic and osteoarthritic fluids were
obtained by needle puncture of the joint capsule exposed during surgery. Hemorrhagic
fluids were not tested. Synovial fluids were kept at room temperature for 30-60 minutes
followed by centrifugation at 3000 r.p.m. for 20 minutes, at 0”. Aliquots of the super-
221
CH50 AND C‘2”‘’ ACTIVITY IN SERUM
natant fluid were either tested immediately or stored at -70”. Viscous fluids were centrifuged at 12,000 to 20,000g in order to sediment most of the mucin. In general, venous
blood was obtained from the patients coincident with the joint aspiration and processed
in the same manner as the synovial fluid. Blood for control values was obtained from 20
healthy volunteers.
M e w r e m e n t of the tolaole complement titer (C’H5&. Erythrocytes in Alsever’s buffer
were supplied either by Probio Inc., Nyack, N. Y. or Baltimore Biological Laboratoria
(BBL), and commercial hemolysin was obtained from BBL. Complement titration was performed according to the macro method described by Mayer,ao results are expressed in
terms of 50 per cent hemolytic units (C’H,,). Calcium was omitted from the NaC1-Veronal-gelatin (0.1 per cent) buffer used in this procedure.25 Titrations of hemolytic complement performed with serum initially diluted 1:12(E or 1:160 yielded at least four tubes
in which 20 per cent to 80 per cent of sensitized cells were lysed. In order to obtain
similar results with synovial fluid, it was often necessary to test several dihitions. If a
synovial fluid initially diluted 1 : l O failed to yield significant lysis, then the sample is reported as containing fewer than 2 C’H,, units per ml. The serum C’H,, titer of 20 healthy
volunteers ranged from 32 to 44 units per ml.
iMemrement of the Second Component of Human Complement (C’2kll). The second
component of human complement in serum or synovial fluid was assayed by the two step
procedure of Austen and Beer.23 Human serum or synovial fluid and sensitized sheep
erythrocytes in the EAC’lagp,4gp state werp incubated at 30” for tmax time. The term tmax
refers to the time at which the greatest number of sensitized receptor sites are in the
SAC‘lag~’,4gp,2hl1 state. Lysis of these cells was accomplished by adding guinea pig serum
treated with ethylenediaininetetraacetate as a source of C‘3 factors. The conversion of
SAC’lagg,4-~sites to SAC’lagD,4 gp,2’ll1 sites is stoichometrically related to the relative conZentration of C’2hll in the reaction mixture. The reciprocal of the sernm or synovial fluid
dilution which produces one SAC’lagP.4 gP,211L1 site per cells is arbitrarily defined as the
number of C ‘ 2 h ~units per ml. of serum or synovial fluid. The titration is linear even with
C‘2’1u titcrs as low as 1 per cent of normal or as high as 200 per cent of normal.23
Identical results were obtained in this assay with duplicate serum samples and the
C‘2hu titers of serum aliquots assayed on five consecutive days v‘aried by less than 10
per cent. On the other hand, there was appreciable variation in the absolute C’211u titer of a
standard reference serum meawied with different lots of EAC’lagp,4gp cells.23 For this
reason a standard reference serum was included with each C’211“ assay. The absolute value
for each unknown serum or synovial fluid was corrected for any deviation of the reference
Serum from its assigned value of 650 C‘PUunits per ml. The coirected C’2”u titer of 20
healthy volunteers ranged from 350 to 670 (mean = 510) units per ml. Serum samples
used in the c‘2111‘ assay were generally diluted in two-fold steps from 1:200 to 1:3200;
synovial fluid was diluted 1:50 through 1:1600. Because of the limited quantity available,
lesser dilutions of synovial fluid were not used. Samples failing to give significant lysis at
1:50 were reported as containing fewer than 5 C’2h~unit3 per ml. synovial fluid.
Detection of p l E (C‘4) and p l C Globulin (C’3a) by Immunaelectrophoresis. Specific
rabbit antiserum to B1E and p l C globulin was kindly supplied by Dr. Hans Muller-Eberhard. Both the agar gel and the Verona1 electrophoresis buffer contained 0.01M disodium
ethylenediaminetetraacetate to limit activation of the first component of complement during iminunoelectrophoretic analysis. The latter was performed as described by Scheidegger.31
Additional Procedures. The latex fixation test for rhenmatoid factor was carried out on
serum and synovial fluid by the method of Singer and Pl0t2.32 Estimation of the leukocyte
concentration in synovial fluid and the mucin clot test were performed as described by
Ropes and Bauer.3s Total protein in synovial fluid was determined by biuret reaction.
RESULTS
Trneiniatic Arthritis. Paired synovial fluid and serum specimens were obtained from five male patients with traumatic arthritis (table 1).In cases E. G.
222
FOSTIROPOULOS, AUSTEN, BLOCH
Table 1.-Total Hemolytic Complement and Second Component of Complement
Activity in Serum and Synovial Fluid of Patients with Traumatic
Arthritis and Degenerative Joint Disease
Synovial Fluid
Serum
~
No.
Pt.
1
2
3
4
5
M. L.*
J. W.*
E.G.
J. C.*
S.K.
Duration
of
Effusion
5 wks.
8 wku.
6 days
4 wk s .
1 Vk.
Protein Mnein
9%
Type
5.8
3.8
4.75
5.5
4.2
WBC
per
mm.?
L.F.T.
Xecip. C’Ha
titer u/ml.
Traumntic Arthritis
900
0
1
0
1
640
0
I
250
1
340
0
1
1350
0
~
L.F.T.
Recip.
titer
C‘2
u/ml.
C’Hso
u/ml.
C’2
u/ml.
330
244
314
184
540
344
545
640
N.D.
0
0
0
0
0
810
384
980
660
606
574
475
0
0
0
0
0
0
0
27
29
30
33
36
N.D.
41
43
37
40
45
21
22
23
24
36
37
38
358
232
360
386
214
249
300
42
38
40
40
44
3R
53
Dcgeneratiue .Joint Disease
1
2
3
4
5
6
7
A.B.*
C. D.*
P.R.*
A.D.*
O.M.
D.F.
F.G.
lyear
1 year
6 mo.
5mo.
5 mo.
3 mo.
1 mo.
3.8
3.2
3.0
4.5
3.8
3.7
4.6
1
1
1
1
1
1
2
2000
560
580
2100
460
520
350
0
0
0
0
0
0
0
*Synovial fluid obtained during joint surgery; serum obtained one or two days later.
= not done.
N.D.
and S. K. joint fluid was obtained by aspiration within one week of the OCcurrence of trauma to the knee joint. In the other 3 cases, fluid was obtained
during surgical excision of ruptured menisci. There was no evidence of current
systemic illness or of an antecedent joint disease in these patients. Joint fluids
contained relatively few leukocytes, were highly viscous and had a good
mucin content. The latex fixation test was negative on serum and synovial
fluid. Sera of these patients contained total hemolytic C’ activity ranging from
3745 units per ml. and C’2 activity ranging from 344-640 units per ml.
Total hemolytic complement activity of synovial fluid ranged from 27-36 units
per ml. and second component activity ranged from 184 to 330 units per ml.
Degenerative Joint Disease. The six women and one man in this group had
characteristic osteoarthritis involving either hip or knee joints (table 1 ) . These
patients had no evidence of an active systemic disease and their erythrocyte
sedimentation rate was normal. Synovial fluid characteristics were similar to
those of the preceding group. The latex fixation test was negative in serum and
synovial fluid. With one exception, patient F. G., the number of C’H50units
per ml. of serum fell within the normal range. Two patients, A. B. and P. R.,
had an increased number of C’2 units per ml. of serum. In both cases, serum
was obtained within one or two days of joint surgery and the elevated C‘2
levels probably reflect the acute effect of surgery on serum ~ o m p l e m e n t .In
~~
the patients with osteoarthritis, synovial fluid CH50values ranged from 21 to
38 units per ml. and C’2 from 214 to 386 units per ml.
Gouty Arthritis. Serum and synovial fluid was obtained from six male patients within one week of the onset of an acute attack of gout (table 2 ) . These
synovial fluids had a high concentration of leukocytes and a poor mucin content. All patients had an elevated concentration of serum uric acid and
polariscopic examination of synovial fluid revealed the presence of sodium
~ AND
5
~
~0 ’ ACTIVITY
2 ~
IN~ SERUM
~
223
Table 2.-Total Hemolytic Complement and Second Component of Complement
Activity in Serum and Synovial Fluid of Patients with Acute
Gout and Reiter’s Syndrome
Synovial Fluid
Duration
of
No.
Pt.
Effusion
Protein Mucin
g%
Type
WBC
per
mm.3
__
L.F.T.
Recip. C~HW, C’2
titer u/ml. u/ml.
C‘Hs
u/ml.
Serum
0 2
u/ml.
L.F.T.
Recip.
titer
300
720
650
0
0
0
Acute Gout
H.F.
1 wk.
1 wk.
10 days
I
2
3
4
5
6
A.D.
H.P.
F. B.
F. M.
Zdays
6.4
10 days
1 wk.
6.46
5.45
4
4
3
3
3
3
1
2
T.M.
D. C.
15 days
10 days
5.2
6.0
3
2
s. s.
6.75
4.6
3.7
30300
4000
3850
36000
45700
20050
0
0
0
0
0
21
25
26
29
137
277
290
35
45
N.D.
350
440
55
51
48
45
66
90
39
45
349
325
50
52
N.D.
0
928
1300
0
1100
470
0
0
Reiter’s Syndrome
8000
8050
0
0
urate crystals in phagocytes. In almost every patient in this group, the number
of C’H50 and C’2 units per ml. of serum exceeded the normal range. The
synovial fluid CHoo,2 1 4 5 units per ml., and C‘2 activity, 137440 units per
ml., resembled that of the preceding two groups.
Reiter’s Syndrome. In two young adults manifesting the classical triad of
Reiter’s syndrome, accompanied by recent joint effusions, serum total hemolytic
complement activity again exceeded the normal range (table 2). The C’H50
and C’2 activity of synovial fluid in these two patients was less than that of
serum and resembled the values obtained with synovial fluid in the preceding
groups.
Rheumatoid Arthritis. The seventeen women and seven men included in this
group had classical or definite rheumatoid arthritis as defined by A.R.A.
criteria. All had active disease and joint effusions studied had been present
from two weeks to several months. These patients were all receiving moderate
to large doses of salicylates; in addition, cases E. T., I. M., and S. S. were
receiving small doses of corticosteroids. One patient was receiving phenylbutazone (C.A.) and another (A.F.) chrysotherapy in addition to salicylates.
None of the patients had received intra-articular injections of corticosteroids
for several months prior to the joint aspiration. The titer of whole serum
complement (table 3 ) was within the normal range in 20 patients, slightly
diminished in two, and significantly reduced to 16 C’HC0units in one instance,
patient F. T. The C’2 titer of serum was within the normal range in 16
patients, elevated in two, and significantly reduced in four, the titers in
patients F. T., P. W., A. F., and E. C. being 7.2, 156, 226, and 250 units ml.
respectively.
The elevation in serum C’2 titer in patient E. T. is very likely related to
the presence of a urinary tract infection,24 while the increased titer in the
serum of patient J. M. is probably due to recent surgical trauma.34 The striking
reduction in serum whole complement and C’2 in patient F. T. may be attributable to a m y l o i d o ~ i s ,or~ ~to the presence of considerable serum cryoprot e i n ~ The
. ~ ~ other three patients with moderately reduced serum C’2 titers
224
FOSTIROPOULOS, AUSTEN, BLOCH
Table 3.-Total Hemolytic Complement and Second Component of Complement
Activity in Serum and Synovial Fluid of Patients with
Rheumatoid Arthritis
--.____
No.
Pt.
1
P. W.
J. A.
E. C.
2
3
4
G
fi
7
8
!I
10
11
12
13
14
15
16
17
18
19
20
21
22
23
G. v.
E. T.
F. T.
C . G.
G. A.
I. M.
S. D.
M.N.
w. P.
M. S.
M. G .
0 . c.
M.R.
A. F.
P. F.
s. w.
L. A.
J.M.’
s. s.
C. A .
Duration
of
Effusion
Protein
sr/o
Muein
Type
6mo.
3 mo.
2 mo.
5 mo.
5 mo.
2 mo.
2 wks.
3 mo.
3 mo.
3 mo.
2mo.
2 mo.
6 mo.
3 mo.
2 mo.
12mo.
3 mo.
4 mo.
3 mo.
3 mo.
3mo.
4 mo.
4 mo.
5.0
6.3
5.7
3.8
3.8
3.3
2.4
4.4
3.2
2.6
3.4
4.0
4.2
3.2
3.7
4.0
4.0
5.6
3.5
5.0
4.6
4.5
2.6
4
4
4
4
1
2
2
3
3
3
4
4
2
4
3
3
3
3
2
3
3
4
2
Synovial Fluid
Serum
~
WBC
per
111311.3
12,300
10,400
4,350
5,720
17,250
5,100
10,000
400
13,200
6,300
8,350
26,800
33,550
31,250
17,500
12,600
11,400
10,000
42,850
1,230
16,800
8,500
15.350
L.F.T.
reeip.
titer
1280
12x0
N.D.
5120
0
0
C’HX
u/ml.
C’2
u/ml.
C’Hao
C’2
u/ml. u/ml.
<2
<2
<2
<2
<2
<2
<2
<2
<2
<5
<5
<5
<5
<5
<5
<5
<5
N.D.
<5
<5
<5
<5
16
62
77
52
56
82
86
22
56
445
156
385
250
392
975
16
7.2
44
345
455
40
N.D.
34
45
550
42
342
43
496
39
548
31
435
41
52 0
38
410
226
39
392
39
650
37
595
30
795
39
40
409
43.5 575
0
0
5120
0
640
5120
5120
0
5120
0
0
5120
10
N.D.
5120
5120
5120
5120
10
11.5
12
13
25
<z
3.5
4
4
4.5
7.5
8.0
8.5
33
41
36
38
43
L.F:T.
recip.
titer
5120
12x0
5120
5120
0
640
1280
5120
5120
5120
640
5120
5120
12x0
0
0
10,240
5120
N.D.
5120
N.D.
5120
5120
~
Wynovial fluid obtained during joint surgery; serum obtained one or two days later.
had no unusual clinical or laboratory manifestations distinguishing them from
the remainder of the patients with rheumatoid arthritis.
The concentrations of whole complement and C’2 in synovial fluid of patients with rheumatoid arthritis (table 3 ) were markedly reduced compared
to the preceding disease groups (tables 1 and 2 ) . Patients 1-10 showed no
significant total hemolytic activity in synovial fluid diluted 1:10; C‘2 activity
was barely measurable in several of these fluids under the conditions tested.
Patients 11-14 had minimal hemolytic complement activity. Patients 15-22
had 13 or fewer C’HS0units per ml. and fewer than 90 C’2 units per ml. of
synovial fluid. Only one patient, C. A,, had synovial fluid total hemolytic
complement (25 units) and C’2 (445 units) activity comparable to that of
patients with gouty, osteoarthritic, or traumatic effusions; similar results were
obtained with samples obtained from this patient one year later. There were
no clinical features distinguishing this patient from the remaining patients
with rheumatoid arthritis.
Nearly all rheumatoid synovial fluid tested in this study were obtained
from patients with chronic effusions. Since prolonged stasis might lead to
deterioration of complement components in vivo, it was decided to test
rapidly recurring effusions. Synovial fluid was completely removed from the
swollen knee joint of two rheumatoid patients. Thereafter the patients were
allowed to ambulate and recurrent effusions aspirated at frequent intervals
two to seven days apart. In both patients, initial and subsequent joint fluids
225
CHao AND C’2”‘ ACTIVITY I N SERUM
were found to contain equally low total hemolytic complement and C’2
activity.
Other Joint Diseases. Table 4 lists the findings obtained in patients with
other arthritides. Serum whole complement activity and C’2 titer were significantly diminished in one patient with infectious arthritis due to Nocardia
asteroides (G.D.), presumably on the basis of coexistent liver disease. The
serum C’2 titer was also moderately reduced in the patient with psoriatic arthritis. Otherwise the serum C’Hjo and C’2 titers recorded in table 4 are normal
or elevated.
Total hemolytic complement and C‘2 activity were markedly reduced in
the synovial fluid from single cases of scleroderma, psoriatic arthritis, sarcoidosis with arthritis, in two patients with acute polymyositis accompanied by
joint effusions, and in two patients with septic arthritis due to Staphylacacctls
nureus and Nowrdia asteroidm respectively
Paired synovial fluid-serum samples from two patients with chondrocalcinosis
( pseudogout) yielded unexpected results. Although synovial fluid total hemolytic complement activity was approximately 50 per cent of serum activity, the
C’2 activity of synovial fluid was only 20 per cent of the serum value. Individual cases of juvenile rheumatoid arthritis, ankylosing spondylitis and hypogammaglobulinemia with arthritis had synovial fluid complement activity similar
to that found in traumatic and degenerative arthritis. It should, however, be
noted that five of eight patients with juvenile rheumatoid arthritis examined by
€€edbergS7had low synovial fluid hemolytic complement activity.
Assay of PIE and p l C in Synovlal Fluid. P1E ( C4 )3 *was readily demonstrated in synovial fluid from a patient with traumatic arthritis, osteoarthritis,
and Reiterb syndrome, but there was little or no B1E globulin demonstrable
in five of seven patients with rheumatoid arthritis (table 5, fig. 1).
The p1C globulin component of human complement ( C’3a)39 is readily
converted on storage to an inactive form, P1A globulin, and the processing
and storage conditions used in this study could not be expected to prevent
this conversion. The specific antiserum currently available does not distinguish
the conversion product obtained by storage, plA, from that produced by contact with antigen-antibody aggregates, p1G.40The conversion product identified in this study was arbitrarily designated as PlA. Many synovial fluids subjected to immunoelectrophoresis did contain ,81A globulin, but a more
important finding was the invariable presence of considerable unconverted
PIC globulin in virtually all fluids examined (table 5 ) . This finding implies
that it is the low concentrations of C’2 and C’4 (,HE) rather than C‘3a ( p l C )
which account for the depressed total hemolytic complement activity of certain
synovial fluids.
DISCXJSSIQN
The presence of whole hemolytic complement activity in the synovial fluid
of patients with traumatic, degenerative, and gouty arthritis and Reiter’s
syndrome has been previously ~ b ~ e r v e d .It* ~was
, ~ ~therefore to be expected
4
6
8
9
10
11
12
7
C. B.
c. w.
S. D.
A. T.
H. J.
G. D.
V. B.
c. L.
G. B.
1
2
3
5
Pt.
J. A.
L. z.
M. W.
No.
5.8
4.85
5.4
3.8
3.6
5.2
2.75
2.8
6.2
4.3
7.0
4.0
1wk.
6 wks.
2 wks.
5 days
1wk.
4 days
4 days
3 mo.
3 mo.
2
2
4
4
2
2
3
3
4
1
3
3
Protein Mucin
9%
Type
2 wks.
2 mo.
1wk.
Duration of
Effusion
-~
580
130
139800
176000
9850
500
4250
6250
600
3200
43250
7150
W.B.C.
permm.3
<2
<2
<2
C’Hjo
u/ml.
0
7.0
0
9.0
0
6.8
0
8.3
0
21
0 2 4
5120
21
0
30
0
21
0
5120
0
L.F.T.
Recip.
titer
~___
Synovial Fluid
27
46
23
39
124
130
278
320
230
16
13
N.D.
C’2
u/ml.
Hemolytic Complement and C’2 Activity in Other Joint Diseases
Scleroderma
Psoriatic arthritis
Sarcoidosis with
arthritis
Polymyositis
Polymyositis
Infectious arthritis
Infectious arthritis
“Pseudogout”
“Pseudogout”
Juvenile R.A.
Ankylosing spondylitis
Hypogammaglobulinemia
with arthritis
Diagnosis
Table 4.-Total
885
295
N.D.
565
410
870
232
643
650
855
440
528
42
48.5
46
44
35
53
23
46
43
44
49
47
u/ml.
C”2
u/ml.
C’H~O
Serum
-~
0
0
0
0
0
0
0
0
0
640
0
0
L.F.T.
Recip.
titer
m
!
z
M
2
+
$
$
%0
ii
9
r
227
CH50 AND C’2hU ACTIVITY IN SERUM
Table 5.-PlE
and /3lC Globulins in Synovial Fluid: Correlation with Total
Hemolytic Complement and C‘2 Activitq
Synovial Fluid
Pt.
J. W.
P. R.
D. C.
E. C.
E. T.
F. T.
M. S .
M. R.
P. w.
C. A.
Diagnosis
Traumatic arthritis
Osteoarthritis
Reiter’s snydrome
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
Rheumatoid arthritis
C‘Hm
u/ml.
C’2
u/ml.
29
23
45
<2
<2
<2
4
N.D.
8
<2
43.5
360
325
<5
<5
<5
<5
77
<5
575
B1E
B1E
+++
+++
+++
0
0
0
0
+
++
+++
Conversion
Products
0
0
0
0
0
0
+
0
0
+
++ +++
+++ 0
+++ 0
++
0
++
0
++
+
++ ++
+++ +
+ +++
++ ++
N.D. = not done.
that such fluids contain functionally active C’2 and immunoelectrophoretically
detectable PlE ((2’4) and P1C (C’3a) globulin. The concentration of C’H50
units, 21-38, and C’2 units, 184-386, per ml. of synovial fluid in 12 patients
with traumatic or degenerative arthritis (table 1) was roughly one-half the
serum concentration observed in 20 normal subjects, 32-44 C’H50and 350-670
C’2 units per ml. The synovial fluid values in acute gouty arthritis and Reiter’s
syndrome (table 2) were similar to those observed in traumatic and degenerative arthritis. By comparison with these joint diseases, the concentrations of
whole complement, 2-13 C’HEO
units and C’2 5-86 units per ml. was markedly
diminished in joint fluids from 22 of 23 patients with rheumatoid arthritis
(table 3 ) .
As observed by other^,^^,^^ there did not appear to be a direct relationship
between the synovial fluid leukocyte count and hemolytic activity. Neither
was there an apparent relationship between the protein concentration or mucin
clot test and the complement activity of synovial fluid. Indeed, the leukocyte
count, mucin clot test, and protein concentration were quite comparable in
the patients with gouty or rheumatoid arthritis, and yet the hemolytic activity
in gocty effusions was similar to that in traumatic and degenerative effusions,
while that in rheumatoid fluid was markedly depleted. The complement
activity present in rheumatoid fluids did not correlate with the presence or
absence of rheumatoid factor.
The possible explanations for the relative deficiency of whole complement
activity in the synovial fluid of patients with rheumatoid arthritis include: ( a )
anticomplementary factors in rheumatoid fluid; ( b ) interference with the
transport of complement components into the joint space; ( c ) immunologic
activation of the complement system; or ( d ) non-specific activation of the
complement system by proteolytic enzyme in or released from joint tissue.
Incubation of rheumatoid synovial fluid lacking detectable whole complement and C’2 activity with serum or synovial fluid from patients with gout or
Reiter’s syndrome did not decrease the C’Hjo titer of the latter. Similar obser-
228
Ra anti
FOSTIROPOULOS, AUSTEN, B L W H
PIE
Syn. fl. osteo.
C’H50 23u./ml.
C’2 358u./ml.
Ra anti
PIE
Syn. fl. R.A.
C’H50 < 2u./ml.
C’2
< 5u./ml.
Ra anti
PIE
Fig. 1.-Immunoelectrophoresis of osteoarthritis and rheumatoid synovial fluid.
Wells were filled with 0.02 ml. synovial fluid; troughs were filled with 0.1 ml. of
rabbit anti P1E antiserum. Two precipitin arcs adjacent to the upper well indicate
the presence of P1E globulin; no precipitin arcs were formed with the rheumatoid
synovial fluid below.
vations were made by Pekin and Zvaifler.2SFurthermore, the finding that P1E
(C’4) is physically diminished (table 5, fig. 1) in synovial fluids lacking functional C 2 virtually excludes anticomplementary activity as a valid explanation
for the reduced whole complement activity of these synovial fluids.
Fluid from the supra-patellar pouch of healthy volunteers was noted by
Pekin and Z ~ a i f l e r *to~ contain only one-tenth the hemolytic activity of serum.
This finding may reflect the inability of complement components, some of
which have high molecular
to enter the joint space. It is also
possible that the trauma associated with aspiration of small amounts of fluid
from normal tissue spaces activates proteolytic enzymes capable of destroying
complement. In any event, macromolecular proteins are present in joint
effusion^,^^ and it seems unlikely that complement components would be excluded on the basis of size from rheumatoid but not from traumatic, degenerative, or gouty effusions. The possibility of malfunction of a specific transport
system for complement components in rheumatoid arthritis can neither be
supported nor excluded.
A more attractive explanation is the specific, immunologic, intra-articular
activation of the complement system by antigen-antibody interaction in tissue,
in the fluid phase, or in relation to phagocytosis. The depletion of C’2 and P1E
CHgo AND
C’2’1‘1ACTIVITY IN
229
SERUM
(C’4) is entirely compatible with the activation of C’la. Complement might
be fixed by specific antibody bound to antigens present at the site of the
primary synovial lesion, or by antigen-antibody complexes or gamma globulin
aggregates trapped in the synovial membrane. K z ~ p l a nhas
~ ~found considerable
7s gamma globulin in the synovial lining and in the immediate subsynovial
tissue in rheumatoid arthritis. However it is not known whether this gamma
globulin is aggregated and capable of fixing complement. Alternatively complement could be activated in the fluid phase by immune complexes or gamma
globuin aggregates. The third possibility, namely, utilization in relation to
phagocytosis, could explain the reduced C’2 titer in the septic joints, in the
fluids from patients with “pseudogout,” and even in rheumatoid arthritis since
aggregates of rheumatoid factor and gamma globulin have recently been
demonstrated in synovial fluid l e u k o c y t e ~ . ~ ~Against
,*j
this latter interpretation is the failure to note a consistent reduction in synovial fluid C’2 in gouty
effusions.
Rheumatoid synovial fluid contains increased concentrations of several
hydrolytic enzymes presumably released from the lysosomes of polymorphonuclear leukocytes and lining cells of the synovial membrane,42 and it is
possible that some proteolytic enzyme directly or indirectly inactivates the
complement system. For example, it has been demonstrated by Lepow et al.46
that serum plasmin (fibrinolysin), a proteolytic enzyme, converts the first
component of human complement to its active form, C’la; the latter then
destroys any C’2 and C’4 in the reaction mixture. Such a mechanism would
account for the findings in rheumatoid effusion, i.e., striking depletion of C’2
and C’4 without extensive conversion or disappearance of C’3a. Plasmin
activity has not been detected in synovial fluids which were also low in complement activity.37 This daes not exclude the possibility that other proteolytic
enzymes activate C’l. On the other hand, the failure to note a correlation
between the indices of intra-articular inflammation and reduced hemolytic
activity is against this simple explanation, unless it is assumed that the
postulated proteolytic activity appears in rheumatoid or septic tissue injury
but not in gouty inflammation.
SUMMARY
The observations that total hemolytic complement activity is present in
synovial fluid of patients with traumatic, degenerative, and gouty arthritis
and that it is relatively absent from fluid of patients with rheumatoid arthritis
were confirmed and extended. The presence of the second component of human complement( C’Bhn)in synovial fluid was demonstrated by a stoichiometric
titration using EAC’laSp,4g” cells, while the fourth component (BlE) and a
moiety of the third component ( P1C) were identified by immunoelectrophoretic analysis using specific antiserum. The concentration of second and fourth
components of human complement was strikingly diminished in synovial
fluid from patients with rheumatoid arthritis in comparison to their concentration in synovial fluid from patients with traumatic, degenerative, or gouty
230
FOSTIROPOULOS, AUSTEN, BLOCH
effusions. These findings suggest that the reduction in whole complement
activity was due to activation of the first complement component with subsequent utilization of the second and fourth component of complement. HOWever the mechanism of intra-articular complement fixation or activation in
rheumatoid effusions remains to be determined.
ACKNOWLEDGMENT
The authors are indebted to Drs. Otto E. Aufranc, William N. Jones, Robert S. Pinals
and Theodore B. Bayles for making available several synovial fluids used in this study.
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