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Gamma globulin complexes in rheumatoid pericardial fluid
CASE REPORTS
Gamma Globulin Complexes in Rheumatoid Pericardial Fluid
GENE V. BALL, M.D. RALPH SCHROHENLOHER. RAYMOND
Ph.D.
HESTER, Ph.D.’
Birmingham, Alabama
From the Division of Clinical Immunology and Rheumatology, School of Medicine, University of Alabama in Birmingham; and the Veterans Administration Hospital, Birmingham, Alabama. This paper was presented in part at a meeting of the American Rheumatism Association on June 8, 1972. Requests for reprints should be addressed to Dr. Gene V. Ball, Division of Clinical Immunology and Rheumatology, University of Alabama in Birmingham, University Station, Birmingham, Alabama 35294. Manuscript accepted March 1, 1974. Present address: Department of Allergy and Clinical Immunology, National Jewish Hospital and Research Center, Denver, Colorado 80206. l
Cardiac tamponade due to pericarditis occurred in a patient with rheumatoid arthritis. Aspiration afforded us an opportunity to expand the characterization of pericardial fluid. Elevated acid phosphatase levels, decreased whole hemolytic complement and gamma globulin complexes similar to those found in rheumatoid synovial fluid were noted, supporting the concept of a unitary nature of inflammation in rheumatoid disease. As many as 50 per cent of the patients with definite or classic rheumatoid arthritis may have pericarditis at some time. This prevalence was first “confirmed” as an antemortem finding by Bacon and Gibson [ 11, who detected pericardial effusions by echocardiography in 11 of 22 patients with chronic nodular rheumatoid arthritis. A 50 per cent prevalence notwithstanding, Franc0 et al. uncovered in the literature only 58 cases of pericarditis [2] in patients fulfilling the American Rheumatism Association criteria for probable, definite or classic rheumatoid arthritis [3]. Pericardial pain in patients with rheumatoid arthritis may easily be dismissed as “musculoskeletal pain.” The potential danger of pericarditis is well documented and bears emphasis, 34 per cent of such patients having undergone pericardiectomy or died [ 21. In others cardiac tamponade has developed, requiring needle aspiration of fluid [2,4,5]. Drug treatment has controlled the pericarditis in some. Seven of France’s 17 patients were given prednisone in doses varying from 20 mg every other day to 40 mg daily. Signs and symptoms of pericarditis subsided in six of these seven. Two patients showed improvement without receiving corticosteroids, and three underwent pericardiectomy for relief of constriction [2]. Even though corticosteroids may be of value in treating rheumatoid pericarditis, the de novo occurrence of pericarditis in patients taking these drugs has been described [ 21. This is noteworthy in differentiating rheumatoid pericarditis from infectious pericarditis, since patients with rheumatoid arthritis are probably more susceptible to infections than are age-matched controls [S]. The purulent or hemorrhagic appearance of pericardial fluid in patients with rheumatoid arthritis suggests infection [7]. Failure to consider rheumatoid lead
pericarditis
to erroneous
infection
or tuberculosis;
symptomatic doses
and
diagnosis
rheumatoid
uneasiness and
concerning
inappropriate
acceptable pericarditis
immediate would
infection
treatment be
drug
may
for acute
treatment
moderately
of large
of corticosteroids.
January
1975
The American
Journal
of Medicine
Volume
58
123
GAMMA GLOBULIN COMPLEXES IN RHEUMATOID SYNOVIAL FLUID-BALL
TABLE I
Properties of Serum and Pericardial from Patient B.S. Determination
Serum
Sugar level (mg) White blood cells (no./cc whole blood) CHso (units) (normal 30-45 units) Acid phosphatase (units) Electrophoresis (g/100 ml) Total protein Albumin Alpha1 Alpha2 Beta Gamma Fr II latex agglutination Untreated Reduced Precipitation with monoclonal rheumatoid factor
93 10,000 33 0.1 6.5 2.46 0.73 1.20 0.68 1.42 1: 40,960
Fluid
CASE REPORT
Pericardial Fluid 14 88,100
Available descriptions of pericardial effusions in patients with rheumatoid arthritis are incomplete [2,4,8,9]. In contrast, synovial fluid from patients with rheumatoid arthritis has been well characterized,
permitting certain inferences concerning the pathogenesis of synovial inflammation. Our purpose here is to extend the available description of rheumatoid pericardial fluid, obtained here from a patient experiencing cardiac tamponade, and to indicate the unitary nature of inflammation in seropositive rheumatoid arthritis.
Double agar gel diffusion demonstrating preFigure 1. cipitin formation between a monoclonal rheumatoid factor and pericardial fluid B.S. Well 1, serum B.S.; well 2, pericardial fluid B.S.; well 3, aggregated human IgG (5 mg/ ml); and well 4, monoclonal rheumatoid factor (2 mg/ml). The experiment was performed at room temperature using 0.6 per cent agar in pH 7.2 sodium phosphate buffered saline solution [ 121.
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ET AL.
A 52 year old man (B.S.) with classic rheumatoid arthritis of 10 years’ duration was treated in the remote past with corticosteroids and gold, and more recently with aspirin. One week before he was hospitalized, he noticed dull pain in the left anterior portion of his chest. The pain was intensified by coughing or deep breathing; it was slightly worse when he was recumbent and was relieved when he sat up. It became continuous, persisted for a few days and then disappeared. It recurred 1 day later as a sensation of deep, uncomfortable pressure, accompanied by minimal dyspnea. The patient was afebrile on admission and throughout his hospitalization. Numerous subcutaneous nodules and mildly active deforming arthritis were noted. There was no scleritis, and no signs of vasculitis. Unequivocal signs of cardiac tamponade, including neck vein engorgement, a marked fall in blood pressure and decreased urinary output, developed during the first 2 days. An echocardiogram was thought to be diagnostic of pericardial effusion, and pericardiocentesis produced 250 ml of yellowish, turbid fluid, after which signs of tamponade disappeared. The patient was given 30 mg of prednisone daily, and signs or symptoms of pericarditis did not recur over a 4 month follow-up. Fluid was sterile on culture for bacteria, fungi and mycobacteria. Normal cytology preparations were examined carefully because of a lung lesion which, on thoracotomy 1 month later, was found to be an adenocarcinoma. Examination of the pericardial sac at operation revealed no effusion and no evidence of tumor, and a biopsy was performed. The pericardium was fibrotic, slightly thickened and infiltrated with moderate numbers of plasma cells, lymphocytes and a few polymorphonuclear cells. SPECIAL
STUDIES
Studies shown in Table I were begun within a few minutes of pericardiocentesis. The fluid was yellow with many small clumps of material containing white blood cells, decreasing the number available for counting. The white blood cells were almost all polymorphonuclear; many contained intracytoplasmic inclusion bodies, and many were smudged. The sugar level in pericardial fluid was 14 mg/lOO ml. The activity of acid phosphatase, a lysosomal enzyme, was 100 times greater in the pericardial fluid than in the paired serum obtained at the same time. Total hemolytic complement activity was too low to read in the pericardial fluid and within normal limits in serum. Neither fluid nor serum contained antinuclear antibodies. Rheumatoid factor by latex fixation [lo] was present in a titier of 1:40,960 in both serum and pericardial fluid (Table I). Reduction and alkylation [ 1 l] of the serum and fluid by mercaptoethanol and iodoacetamide decreased the rheumatoid factor activity to less than 1:20 titer, locating rheumatoid factor activity in the immunoglobulin M (IgM) fraction. Rheumatoid pericardial fluid, therefore, resembled rheumatoid synovial fluid in its classic elements. Ad-
Volume 58
GAMMA GLOBULIN COMPLEXES IN RHEUMATOID SYNOVIAL FLUID-BALL
0.20
RED. a
I-
R.F.
PERICARDIAL FLUID 150
+
0.15
BLK.
-
ppt.
t
+
+
t
---_-
-
-
-
-
ANTI-IgG
-
+
-
-
ANTI-IgM
PPf I00
ET AL.
MONO.R.F.
0.10
(r9)
0.05 50 : z
lo 4e
‘; 0.2 VOLUME
0.4 OF
NORMAL SERUM _ - -_ __ _
0.6 FLUID
0.0 OR
SERUM
8
_
0.20
1.0
RED. B ALK.
F
(ml.)
F@ure 2. Curve showing precipitation of pericamial f/&Y B.S. with monoctonal rheumatoid factor. Each tube contained 0.1 mg of the monocfonal rheumatoid factor, pericardial f/M and sufficient pH 7.5 sodium phosphate buffer to make 1 ml.
8 0.15 -
0.10
0.05
R. F. CONTROL
-
-
I
2
3
GRADIENT
4
VOLUME
5 (ml)
Figure 3. Density gradient ultracentrifugation of the precipitate formed by mono&M rheumatoti factor and pericardial fluid B.S., solubilized by reduction and alkylation (upper section): and reduced and alkylated monocional rheumatoti factor control (lower section). Centrifugation was performed at 35,000 rpm for 18 hours at 2’ C in linear 12 to 30 per cent sucrose gradients prepared in pH 7.5 sodium phosphate buffer. Double agar gel diffusion reactions between gradient fractions and monoclonal rheumatokf factor and antiserum specific for IgG and IgM are indicated. The posltions of 7s and 19s proteins were determined by human IgG and IgM controls centrifuged under identical conditions.
Flgure 4. Reaction of partially purified gamma globulin complexes from pericardial fluid B.S. and aggregated human IgG with monocfonal rheumatoid factor. Well 1, 0.62 M sodium sulfate fractk?n of pericardial fluti B.S. (2 mg/ml); weft 2, aggregated human IgG (2 mg/ml); well 4, 0.62 M sodium sulfate fraction of pericardtil fluid B.S. (5 mg/ml); and well 5, aggregated human IgG (5 mg/ml). Wells 3 and 6 were empty. The center well contained monoclonal rheumatoki factor (2 mg/m). The experiment was performed as described in Figure 1.
TABLE II
Fractionation
Sodium Sulfate
January 1975
0.36 M 0.62 M 0.81M 0.96 M 1.08 M 1.18 M
of Pericardial “Complexes" Negative 2+ 3+ f Negative Negative
The American Journal 01 Medicine
Fluid RheumatoidFactor 1:80 <1:20 1:640 1:1280 1:640 1:640
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GAMMA GLOBULIN COMPLEXES IN RHEUMATOID SYNOVIAL FLUID-BALL
ditional studies were concerned with defining immunoglobulin complexes and comparing them to those found in rheumatoid synovial fluid. Studies Related to Gamma Globulin Complexes. Figure 1 shows an Ouchterlony agar plate with a precipitin line between pericardial fluid and a monoclonal IgM rheumatoid factor [ 121. Similar precipitation occurred with heat-aggregated immunoglobulin G (IgG) but not with patient’s serum or normal human serum. Reduction and alkylation did not alter the precipitability of the fluid by monoclonal rheumatoid factor, but it did remove the precipitate between the patient’s fluid and aggregated globulin. The precipitation of complexes in the fluid by monoclonal rheumatoid factor was also apparent by quantitative precipitin curve analysis (Figure 2). After incubation for 2 hours at 37OC in pH 7.5 sodium phosphate buffer, the precipitates were washed three times at room temperature with the same buffer, dissolved in dilute sodium hydroxide and analyzed for protein [ 131. Each point on the curve was corrected for the precipitate formed in the absence of the rheumatoid factor. Approximately 300 pg of precipitate/ml fluid was obtained at equivalence in contrast to the low quantity formed using normal human serum as a control. The reaction with the rheumatoid factor was not altered by heating the fluid at 56OC for 30 minutes. Reduction and alkylation solubilized the rheumatoid factor-fluid precipitate: density gradient ultracentrifugation [ 141 of this solubilized precipitate, and reduced and alkylated rheumatoid factor control are shown in Figure 3. Also shown are the positions of purified 7s and 19s immunoglobulins as sedimentation rate markers. The dissociated rheumatoid factor in the control serum and precipitate was in the 7S area. The reactants were heavier components identified antigenically as IgG only. The rheumatoid factor-fluid precipitate was also dissolved in pH 3 buffer and subjected to density gradient centrifugation. IgG was present in the 7s peak only, indicating dissociation of the aggregated materials. Christian’s method of sequential sodium sulfate fractionation was used to isolate and concentrate the gamma globulin complexes [ 151. Shown in Table II is the recovery of complexes in the 0.62 M and 0.60 M fractions with maximum rheumatoid factor activity in fractions of greater molarity. Reaction of identity was obtained with pericardial fluid reactants (fraction 0.62 M) and aggregated IgG (Figure 4). Note that the sodium sulfate complex fraction does not react with the aggregated IgG. COMMENTS
The clinical characteristics of rheumatoid pericarditis have been reviewed by Franc0 et al. [2]. They will
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Volume
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not be discussed beyond the notation that the signs and symptoms displayed by our patient are not uncommon in patients with pericardial effusion and tamponade. Uncommon, though, was the opportunity to study rheumatoid pericardial fluid in detail. In earlier studies of pericardial fluid, wide variability in the numbers of white cells, increased protein content and low sugar levels and increased activity of lactic dehydrogenase have been reported. Franc0 reported the only protein electrophoretic analyses: in three of four fluids tested gamma globulin levels were elevated [2]. CH50 values have been published for three specimens of rheumatoid pericardial fluids [2,4]. As reported, they were below normal, but paired protein values were not furnished, rendering interpretation difficult. None of these components is likely to be of diagnostic specificity; for example, rheumatoid factors have been found in the pleural fluid of patients with bacterial pneumonia, carcinoma and tuberculosis [ 161. The most specific component of rheumatoid fluid reported to date is the gamma globulin complex, presumably an immune complex [ 12,17-211. These complexes can be demonstrated in various ways. Some complexes are detectable by one technic only, whereas other complexes are detectable by more than one technic. Complexes sedimenting between 9s and 14s are found in certain serums and fluids by ultracentrifugal analysis [ 12,17,18]. Some complexes that sediment in the ultracentrifuge at rates greater than 19s will precipitate with polyclonal rheumatoid factors obtained from patients with rheumatoid arthritis [ 19,201. This group of complexes can also be precipitated by the complement component Clq [ 2 11. Gamma globulin complexes have also been detected in the serum and joint fluids of patients with rheumatoid arthritis by interaction with monoclonal rheumatoid factors obtained from patients with lymphoproliferative diseases [ 121. These reagents appear more sensitive than the polyclonal rheumatoid factors in that they react with a wider range of complexes. The gamma globulin complexes present in this pericardial fluid were similar to those isolated by Winchester et al. [ 121 in serum and joint fluids of a patient with rheumatoid arthritis by precipitation with a monoclonal anti-IgG factor. These complexes sedimented over the range of 7s to 20s in the ultracentrifuge and consisted principally of IgG. Those from the pericardial fluid sedimented at approximately 19s. The serum, joint and pericardial fluid complexes dissociated into 7s IgG molecules in acidic buffers and subsequently reassociated on adjustment of the pH to neutrality, but they did not dissociate after reduction and alkylation. These complexes differed
56
GAMMA GLOBULIN COMPLEXES IN RHEUMATOfOSYNOVIAL FLUID-BALL ET AL.
from those detected in joint fluids by polyclonal rheumatoid factors and Clq in that they required higher concentrations of sodium sulfate for precipitation by salt fractionation [ 221. Winchester et al. [ 121 furthermore found these gamma globulin complexes in 70 per cent of the joint fluids from patients with definite or classic rheumatoid arthritis when monoclonal rheumatoid factors were used for their detection. These investigators examined fluids from 36 patients with various types of inflammatory and “traumatic and degenerative” arthritis and found no complexes. In this laboratory gamma globulin complexes reacting with monoclonal rheumatoid factors have been found in fluids (joint, pericardial or pleural) obtained from patients with definite or classic rheumatoid arthritis. Complexes were also found in synovial fluid obtained from a young girl with monarticular arthritis of 1 year’s duration. Synovectomy was performed on the involved knee; the synovial histopathology was “compatible with rheumatoid arthritis,” and she now has polyarthritis. We found no complexes in the pericardial fluids of a patient with “mixed connective tissue disease” and of one with systemic lupus erythematosus. Nor did we find complexes in the pleural fluids of the same two patients and of one other with systemic lupus erythematosus. The role of immune complexes in mediating tissue injury is probably an important one, and the mechanisms whereby immune complexes lead to such injury have been investigated intensively. Phagocytosis of complexes by polymprphonuclear cells results in release of lysosomal enzymes which are injurious to tissue and which can probably cleave C3 and C5, thereby enhancing inflammation [23]. Enzymic activity, represented here by acid phosphatase, appears to be increased in rheumatoid joint fluid in comparison with osteoarthritis joint fluid [24]. Measurements
of enzymic activity in rheumatoid pericardial fluid had not been reported previously. Activation of complement also occurs as a result of its binding with immune complexes. The absence of CH5s activity in rheumatoid pericardial fluid is not unexpected, low levels of CHs0 having been noted frequently in rheumatoid joint and pleural fluid [ 20,251. The evidence linking pericarditis and rheumatoid arthritis has been obtained largely from autopsy studies. Analysis of pericardial fluid reported here provides more direct proof that pericarditis can be a feature of rheumatoid disease. The presence of gamma globulin complexes in pericardlal fluid and their apparent absence in the patient’s serum strongly implies the local production of these complexes. It is tempting to speculate that an antigen circulating in this patient’s blood, even when arthritis was inactive, was “trapped” in the pericardium, invoking a plasma cell and lymphocyte response and local production of antibody. The same process may occur in synovium, pleura and pericardium, prime targets for rheumatoid inflammation. ADDENDUM Since submitting this manuscript, Hunder, Mullen and McDuffie [26] reported evidence that immune mechanisms may also contribute to the development of pericarditis in systemic lupus erythematosus. Total hemolytic complement and the levels of Clq, C3 and C4 were lower in the pericardial fluids of two patients with systemic lupus erythematosus than in pericardial fluids of two control patients with valvular heart disease. Complement-fixing material was present in the fluids from the patients with systemic lupus erythematosus but not in the fluids from the controls. In addition, one of the former fluids formed precipitin lines by agar gel diffusion against a monoclonal rheumatoid factor and Clq.
REFERENCES 1.
2.
3.
4.
5. 6.
Bacon PA, Gibson DG: Cardiac involvement in rheumatoki arthritis-an echocardiograph study (abstract). Arthritis Rheum 15: 49, 1972. Franc0 AE, Levine HD, Hall AP: Rheumatoid pericarditis. Report of 17 cases diagnosed clinically. Ann Intern Med 77: 837, 1972. (a) Ropes MW, Bennett GA, Cobb S, Jacox RF, Jessar RA: Proposed diagnostic criteria for rheumatoid arthritis. Bull Rheum Dis 7: 121, 1956. (b) Ibid. 9: 175, 1958. (a) Metzger AL: Discussion. (Romanoff H. Rozin R, Zlotnick A: Cardiac tamponade in rheumatoid arthritis. A case report and review of the literature). Arthritis Rheumll3:426, 1970. (b) Ibid, p 430. Latham BA: Pericarditis associated with rheumatoid arthritis. Ann Rheum Dis 25; 235, 1966. Uddin J, Kraus AS, Kelly HG: Survivorship and death in
7.
8.
9.
10.
11.
12.
January 1975
rheumatoid arthritis. Arthritis Rheum 13: 125, 1970. Kennedy WPU, Partridge REH, Mathews MB: Rheumatoid pericarditis with cardiac failure treated by pericardiectomy. Br Heart J 28: 692, 1966. Romanoff H, Rozln R, Zlotnick A: Cardiac tamponade in rheumatoid arthritis. A case report and review of the literature. Arthritis Rheum 13: 426, 1970. Rodnan GP: Discussion. (Liss JP, Bachmann WT: Rheumatoid constrictive pericardffis, treated by pericardiectomy. Arthritis Rheum 13: 869, 1970). Arthritis Rheum 13: 674, 1970. Singer JM, Pfotz CM: The latex fixation test. I. Application to the serologic diagnosis or rheumatoid arthritis. Am J Med 21: 888. 1956. Schrohenloher RE, Kunkel HG, Tomasi TB: Activity of dissociated and reassociated 19s anti-y-globulins. J Exp Med 120: 215, 1964. Winchester RJ. Kunkel HG, Agnello V: Occurrence of y-
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13.
14.
15.
16.
17.
18.
19. 20.
128
globulin complexes in serum and joint fluid of rheumatoid arthritis: use of monoclonal rheumatoid factors as reagents for their demonstration. J Exp Med 134: 286% 1971. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265, 1951. Kunkel HG: Macroglobulins and high molecular weight antibodies. The Plasma Proteins (Putnam FW, ed), New York, Academic Press, 1960, p 279. Christian CL: Characterization of the “reactant” (gamma globulin factor) in the FII precipitin reaction and the FII tanned sheep cell agglutination test. J Exp Med 108: 139, 1958. Levine H, Szanto M, Grieble HG, Bach GL, Anderson TO: Rheumatoid factor in non-rheumatoid pleural effusions. Ann Intern Med 69: 487, 1968. Kunkel HG, Muller-Eberhard HJ, Fudenberg HH, Tomasi TB: Gamma globulin complexes in rheumatoid arthritis and certain other conditions. J Clin Invest 40: 117, 1961. Schrohenloher RE: Characterization of the y-globulin complexes present in certain sera having high titers of antiy-globulin activity. J Clin Invest 45: 501, 1966. Hannestad K: Presence of aggregated y-globulin in certain rheumatoid effusions. Clin Exp lmmunol 2: 511, 1967. Winchester RJ, Agnello V, Kunkel HG: Gamma globulin
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1975
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Journal of Medicine
21.
22.
23.
24.
25.
26.
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complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels. Clin Exp lmmunol 6: 689, 1970. Agnello V, Winchester RJ. Kunkel HG: Precipitation reactions of the Clq component of complement with aggregated y-globulin and immune complexes in gel diffusion. Immunology 19: 909, 1970. Winchester RJ, Agnello V, Kunkel HG: The use of sodium sulfate fractionation in the investigation of the y-globulin complexes in the serum and joint fluid of rheumatoid arthritis patients (abstract). Arthritis Rheum 15: 132, 1972. Lepow IH: Biologically active fragments of complement. Progress in Immunology, First International Congress of Immunology (Amos B, ed). New York, Academic Press, 1971, p 579. Muller W, Harwerth HG, Fehr K: Studies in four lysosomal enzymes in rheumatoid arthritis and osteoarthritis, chap 18. Rheumatoid Arthritis, Pathogenetic Mechanisms and Consequences in Therapeutics, New York, Academic Press, 1971. Hunder GG, McDuffie FC, Hepper NGG: Pleural fluid complement in systemic lupus erythematosus and rheumatoid arthritis. Ann Intern Med 76: 357, 1972. Hunder GG. Mullen BJ, McDuffie FC: Complement in pericardial fluid of lupus erythematosus: studies in two patients. Ann Intern Med 80: 453, 1974.
Gamma Globulin Complexes in Rheumatoid Pericardial Fluid
GENE V. BALL, M.D. RALPH SCHROHENLOHER. RAYMOND
Ph.D.
HESTER, Ph.D.’
Birmingham, Alabama
From the Division of Clinical Immunology and Rheumatology, School of Medicine, University of Alabama in Birmingham; and the Veterans Administration Hospital, Birmingham, Alabama. This paper was presented in part at a meeting of the American Rheumatism Association on June 8, 1972. Requests for reprints should be addressed to Dr. Gene V. Ball, Division of Clinical Immunology and Rheumatology, University of Alabama in Birmingham, University Station, Birmingham, Alabama 35294. Manuscript accepted March 1, 1974. Present address: Department of Allergy and Clinical Immunology, National Jewish Hospital and Research Center, Denver, Colorado 80206. l
Cardiac tamponade due to pericarditis occurred in a patient with rheumatoid arthritis. Aspiration afforded us an opportunity to expand the characterization of pericardial fluid. Elevated acid phosphatase levels, decreased whole hemolytic complement and gamma globulin complexes similar to those found in rheumatoid synovial fluid were noted, supporting the concept of a unitary nature of inflammation in rheumatoid disease. As many as 50 per cent of the patients with definite or classic rheumatoid arthritis may have pericarditis at some time. This prevalence was first “confirmed” as an antemortem finding by Bacon and Gibson [ 11, who detected pericardial effusions by echocardiography in 11 of 22 patients with chronic nodular rheumatoid arthritis. A 50 per cent prevalence notwithstanding, Franc0 et al. uncovered in the literature only 58 cases of pericarditis [2] in patients fulfilling the American Rheumatism Association criteria for probable, definite or classic rheumatoid arthritis [3]. Pericardial pain in patients with rheumatoid arthritis may easily be dismissed as “musculoskeletal pain.” The potential danger of pericarditis is well documented and bears emphasis, 34 per cent of such patients having undergone pericardiectomy or died [ 21. In others cardiac tamponade has developed, requiring needle aspiration of fluid [2,4,5]. Drug treatment has controlled the pericarditis in some. Seven of France’s 17 patients were given prednisone in doses varying from 20 mg every other day to 40 mg daily. Signs and symptoms of pericarditis subsided in six of these seven. Two patients showed improvement without receiving corticosteroids, and three underwent pericardiectomy for relief of constriction [2]. Even though corticosteroids may be of value in treating rheumatoid pericarditis, the de novo occurrence of pericarditis in patients taking these drugs has been described [ 21. This is noteworthy in differentiating rheumatoid pericarditis from infectious pericarditis, since patients with rheumatoid arthritis are probably more susceptible to infections than are age-matched controls [S]. The purulent or hemorrhagic appearance of pericardial fluid in patients with rheumatoid arthritis suggests infection [7]. Failure to consider rheumatoid lead
pericarditis
to erroneous
infection
or tuberculosis;
symptomatic doses
and
diagnosis
rheumatoid
uneasiness and
concerning
inappropriate
acceptable pericarditis
immediate would
infection
treatment be
drug
may
for acute
treatment
moderately
of large
of corticosteroids.
January
1975
The American
Journal
of Medicine
Volume
58
123
GAMMA GLOBULIN COMPLEXES IN RHEUMATOID SYNOVIAL FLUID-BALL
TABLE I
Properties of Serum and Pericardial from Patient B.S. Determination
Serum
Sugar level (mg) White blood cells (no./cc whole blood) CHso (units) (normal 30-45 units) Acid phosphatase (units) Electrophoresis (g/100 ml) Total protein Albumin Alpha1 Alpha2 Beta Gamma Fr II latex agglutination Untreated Reduced Precipitation with monoclonal rheumatoid factor
93 10,000 33 0.1 6.5 2.46 0.73 1.20 0.68 1.42 1: 40,960
Fluid
CASE REPORT
Pericardial Fluid 14 88,100
Available descriptions of pericardial effusions in patients with rheumatoid arthritis are incomplete [2,4,8,9]. In contrast, synovial fluid from patients with rheumatoid arthritis has been well characterized,
permitting certain inferences concerning the pathogenesis of synovial inflammation. Our purpose here is to extend the available description of rheumatoid pericardial fluid, obtained here from a patient experiencing cardiac tamponade, and to indicate the unitary nature of inflammation in seropositive rheumatoid arthritis.
Double agar gel diffusion demonstrating preFigure 1. cipitin formation between a monoclonal rheumatoid factor and pericardial fluid B.S. Well 1, serum B.S.; well 2, pericardial fluid B.S.; well 3, aggregated human IgG (5 mg/ ml); and well 4, monoclonal rheumatoid factor (2 mg/ml). The experiment was performed at room temperature using 0.6 per cent agar in pH 7.2 sodium phosphate buffered saline solution [ 121.
124
January 1975
The American Journal of Medicine
ET AL.
A 52 year old man (B.S.) with classic rheumatoid arthritis of 10 years’ duration was treated in the remote past with corticosteroids and gold, and more recently with aspirin. One week before he was hospitalized, he noticed dull pain in the left anterior portion of his chest. The pain was intensified by coughing or deep breathing; it was slightly worse when he was recumbent and was relieved when he sat up. It became continuous, persisted for a few days and then disappeared. It recurred 1 day later as a sensation of deep, uncomfortable pressure, accompanied by minimal dyspnea. The patient was afebrile on admission and throughout his hospitalization. Numerous subcutaneous nodules and mildly active deforming arthritis were noted. There was no scleritis, and no signs of vasculitis. Unequivocal signs of cardiac tamponade, including neck vein engorgement, a marked fall in blood pressure and decreased urinary output, developed during the first 2 days. An echocardiogram was thought to be diagnostic of pericardial effusion, and pericardiocentesis produced 250 ml of yellowish, turbid fluid, after which signs of tamponade disappeared. The patient was given 30 mg of prednisone daily, and signs or symptoms of pericarditis did not recur over a 4 month follow-up. Fluid was sterile on culture for bacteria, fungi and mycobacteria. Normal cytology preparations were examined carefully because of a lung lesion which, on thoracotomy 1 month later, was found to be an adenocarcinoma. Examination of the pericardial sac at operation revealed no effusion and no evidence of tumor, and a biopsy was performed. The pericardium was fibrotic, slightly thickened and infiltrated with moderate numbers of plasma cells, lymphocytes and a few polymorphonuclear cells. SPECIAL
STUDIES
Studies shown in Table I were begun within a few minutes of pericardiocentesis. The fluid was yellow with many small clumps of material containing white blood cells, decreasing the number available for counting. The white blood cells were almost all polymorphonuclear; many contained intracytoplasmic inclusion bodies, and many were smudged. The sugar level in pericardial fluid was 14 mg/lOO ml. The activity of acid phosphatase, a lysosomal enzyme, was 100 times greater in the pericardial fluid than in the paired serum obtained at the same time. Total hemolytic complement activity was too low to read in the pericardial fluid and within normal limits in serum. Neither fluid nor serum contained antinuclear antibodies. Rheumatoid factor by latex fixation [lo] was present in a titier of 1:40,960 in both serum and pericardial fluid (Table I). Reduction and alkylation [ 1 l] of the serum and fluid by mercaptoethanol and iodoacetamide decreased the rheumatoid factor activity to less than 1:20 titer, locating rheumatoid factor activity in the immunoglobulin M (IgM) fraction. Rheumatoid pericardial fluid, therefore, resembled rheumatoid synovial fluid in its classic elements. Ad-
Volume 58
GAMMA GLOBULIN COMPLEXES IN RHEUMATOID SYNOVIAL FLUID-BALL
0.20
RED. a
I-
R.F.
PERICARDIAL FLUID 150
+
0.15
BLK.
-
ppt.
t
+
+
t
---_-
-
-
-
-
ANTI-IgG
-
+
-
-
ANTI-IgM
PPf I00
ET AL.
MONO.R.F.
0.10
(r9)
0.05 50 : z
lo 4e
‘; 0.2 VOLUME
0.4 OF
NORMAL SERUM _ - -_ __ _
0.6 FLUID
0.0 OR
SERUM
8
_
0.20
1.0
RED. B ALK.
F
(ml.)
F@ure 2. Curve showing precipitation of pericamial f/&Y B.S. with monoctonal rheumatoid factor. Each tube contained 0.1 mg of the monocfonal rheumatoid factor, pericardial f/M and sufficient pH 7.5 sodium phosphate buffer to make 1 ml.
8 0.15 -
0.10
0.05
R. F. CONTROL
-
-
I
2
3
GRADIENT
4
VOLUME
5 (ml)
Figure 3. Density gradient ultracentrifugation of the precipitate formed by mono&M rheumatoti factor and pericardial fluid B.S., solubilized by reduction and alkylation (upper section): and reduced and alkylated monocional rheumatoti factor control (lower section). Centrifugation was performed at 35,000 rpm for 18 hours at 2’ C in linear 12 to 30 per cent sucrose gradients prepared in pH 7.5 sodium phosphate buffer. Double agar gel diffusion reactions between gradient fractions and monoclonal rheumatokf factor and antiserum specific for IgG and IgM are indicated. The posltions of 7s and 19s proteins were determined by human IgG and IgM controls centrifuged under identical conditions.
Flgure 4. Reaction of partially purified gamma globulin complexes from pericardial fluid B.S. and aggregated human IgG with monocfonal rheumatoid factor. Well 1, 0.62 M sodium sulfate fractk?n of pericardial fluti B.S. (2 mg/ml); weft 2, aggregated human IgG (2 mg/ml); well 4, 0.62 M sodium sulfate fraction of pericardtil fluid B.S. (5 mg/ml); and well 5, aggregated human IgG (5 mg/ml). Wells 3 and 6 were empty. The center well contained monoclonal rheumatoki factor (2 mg/m). The experiment was performed as described in Figure 1.
TABLE II
Fractionation
Sodium Sulfate
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0.36 M 0.62 M 0.81M 0.96 M 1.08 M 1.18 M
of Pericardial “Complexes" Negative 2+ 3+ f Negative Negative
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Fluid RheumatoidFactor 1:80 <1:20 1:640 1:1280 1:640 1:640
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ditional studies were concerned with defining immunoglobulin complexes and comparing them to those found in rheumatoid synovial fluid. Studies Related to Gamma Globulin Complexes. Figure 1 shows an Ouchterlony agar plate with a precipitin line between pericardial fluid and a monoclonal IgM rheumatoid factor [ 121. Similar precipitation occurred with heat-aggregated immunoglobulin G (IgG) but not with patient’s serum or normal human serum. Reduction and alkylation did not alter the precipitability of the fluid by monoclonal rheumatoid factor, but it did remove the precipitate between the patient’s fluid and aggregated globulin. The precipitation of complexes in the fluid by monoclonal rheumatoid factor was also apparent by quantitative precipitin curve analysis (Figure 2). After incubation for 2 hours at 37OC in pH 7.5 sodium phosphate buffer, the precipitates were washed three times at room temperature with the same buffer, dissolved in dilute sodium hydroxide and analyzed for protein [ 131. Each point on the curve was corrected for the precipitate formed in the absence of the rheumatoid factor. Approximately 300 pg of precipitate/ml fluid was obtained at equivalence in contrast to the low quantity formed using normal human serum as a control. The reaction with the rheumatoid factor was not altered by heating the fluid at 56OC for 30 minutes. Reduction and alkylation solubilized the rheumatoid factor-fluid precipitate: density gradient ultracentrifugation [ 141 of this solubilized precipitate, and reduced and alkylated rheumatoid factor control are shown in Figure 3. Also shown are the positions of purified 7s and 19s immunoglobulins as sedimentation rate markers. The dissociated rheumatoid factor in the control serum and precipitate was in the 7S area. The reactants were heavier components identified antigenically as IgG only. The rheumatoid factor-fluid precipitate was also dissolved in pH 3 buffer and subjected to density gradient centrifugation. IgG was present in the 7s peak only, indicating dissociation of the aggregated materials. Christian’s method of sequential sodium sulfate fractionation was used to isolate and concentrate the gamma globulin complexes [ 151. Shown in Table II is the recovery of complexes in the 0.62 M and 0.60 M fractions with maximum rheumatoid factor activity in fractions of greater molarity. Reaction of identity was obtained with pericardial fluid reactants (fraction 0.62 M) and aggregated IgG (Figure 4). Note that the sodium sulfate complex fraction does not react with the aggregated IgG. COMMENTS
The clinical characteristics of rheumatoid pericarditis have been reviewed by Franc0 et al. [2]. They will
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not be discussed beyond the notation that the signs and symptoms displayed by our patient are not uncommon in patients with pericardial effusion and tamponade. Uncommon, though, was the opportunity to study rheumatoid pericardial fluid in detail. In earlier studies of pericardial fluid, wide variability in the numbers of white cells, increased protein content and low sugar levels and increased activity of lactic dehydrogenase have been reported. Franc0 reported the only protein electrophoretic analyses: in three of four fluids tested gamma globulin levels were elevated [2]. CH50 values have been published for three specimens of rheumatoid pericardial fluids [2,4]. As reported, they were below normal, but paired protein values were not furnished, rendering interpretation difficult. None of these components is likely to be of diagnostic specificity; for example, rheumatoid factors have been found in the pleural fluid of patients with bacterial pneumonia, carcinoma and tuberculosis [ 161. The most specific component of rheumatoid fluid reported to date is the gamma globulin complex, presumably an immune complex [ 12,17-211. These complexes can be demonstrated in various ways. Some complexes are detectable by one technic only, whereas other complexes are detectable by more than one technic. Complexes sedimenting between 9s and 14s are found in certain serums and fluids by ultracentrifugal analysis [ 12,17,18]. Some complexes that sediment in the ultracentrifuge at rates greater than 19s will precipitate with polyclonal rheumatoid factors obtained from patients with rheumatoid arthritis [ 19,201. This group of complexes can also be precipitated by the complement component Clq [ 2 11. Gamma globulin complexes have also been detected in the serum and joint fluids of patients with rheumatoid arthritis by interaction with monoclonal rheumatoid factors obtained from patients with lymphoproliferative diseases [ 121. These reagents appear more sensitive than the polyclonal rheumatoid factors in that they react with a wider range of complexes. The gamma globulin complexes present in this pericardial fluid were similar to those isolated by Winchester et al. [ 121 in serum and joint fluids of a patient with rheumatoid arthritis by precipitation with a monoclonal anti-IgG factor. These complexes sedimented over the range of 7s to 20s in the ultracentrifuge and consisted principally of IgG. Those from the pericardial fluid sedimented at approximately 19s. The serum, joint and pericardial fluid complexes dissociated into 7s IgG molecules in acidic buffers and subsequently reassociated on adjustment of the pH to neutrality, but they did not dissociate after reduction and alkylation. These complexes differed
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from those detected in joint fluids by polyclonal rheumatoid factors and Clq in that they required higher concentrations of sodium sulfate for precipitation by salt fractionation [ 221. Winchester et al. [ 121 furthermore found these gamma globulin complexes in 70 per cent of the joint fluids from patients with definite or classic rheumatoid arthritis when monoclonal rheumatoid factors were used for their detection. These investigators examined fluids from 36 patients with various types of inflammatory and “traumatic and degenerative” arthritis and found no complexes. In this laboratory gamma globulin complexes reacting with monoclonal rheumatoid factors have been found in fluids (joint, pericardial or pleural) obtained from patients with definite or classic rheumatoid arthritis. Complexes were also found in synovial fluid obtained from a young girl with monarticular arthritis of 1 year’s duration. Synovectomy was performed on the involved knee; the synovial histopathology was “compatible with rheumatoid arthritis,” and she now has polyarthritis. We found no complexes in the pericardial fluids of a patient with “mixed connective tissue disease” and of one with systemic lupus erythematosus. Nor did we find complexes in the pleural fluids of the same two patients and of one other with systemic lupus erythematosus. The role of immune complexes in mediating tissue injury is probably an important one, and the mechanisms whereby immune complexes lead to such injury have been investigated intensively. Phagocytosis of complexes by polymprphonuclear cells results in release of lysosomal enzymes which are injurious to tissue and which can probably cleave C3 and C5, thereby enhancing inflammation [23]. Enzymic activity, represented here by acid phosphatase, appears to be increased in rheumatoid joint fluid in comparison with osteoarthritis joint fluid [24]. Measurements
of enzymic activity in rheumatoid pericardial fluid had not been reported previously. Activation of complement also occurs as a result of its binding with immune complexes. The absence of CH5s activity in rheumatoid pericardial fluid is not unexpected, low levels of CHs0 having been noted frequently in rheumatoid joint and pleural fluid [ 20,251. The evidence linking pericarditis and rheumatoid arthritis has been obtained largely from autopsy studies. Analysis of pericardial fluid reported here provides more direct proof that pericarditis can be a feature of rheumatoid disease. The presence of gamma globulin complexes in pericardlal fluid and their apparent absence in the patient’s serum strongly implies the local production of these complexes. It is tempting to speculate that an antigen circulating in this patient’s blood, even when arthritis was inactive, was “trapped” in the pericardium, invoking a plasma cell and lymphocyte response and local production of antibody. The same process may occur in synovium, pleura and pericardium, prime targets for rheumatoid inflammation. ADDENDUM Since submitting this manuscript, Hunder, Mullen and McDuffie [26] reported evidence that immune mechanisms may also contribute to the development of pericarditis in systemic lupus erythematosus. Total hemolytic complement and the levels of Clq, C3 and C4 were lower in the pericardial fluids of two patients with systemic lupus erythematosus than in pericardial fluids of two control patients with valvular heart disease. Complement-fixing material was present in the fluids from the patients with systemic lupus erythematosus but not in the fluids from the controls. In addition, one of the former fluids formed precipitin lines by agar gel diffusion against a monoclonal rheumatoid factor and Clq.
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complexes in synovial fluids of patients with rheumatoid arthritis. Partial characterization and relationship to lowered complement levels. Clin Exp lmmunol 6: 689, 1970. Agnello V, Winchester RJ. Kunkel HG: Precipitation reactions of the Clq component of complement with aggregated y-globulin and immune complexes in gel diffusion. Immunology 19: 909, 1970. Winchester RJ, Agnello V, Kunkel HG: The use of sodium sulfate fractionation in the investigation of the y-globulin complexes in the serum and joint fluid of rheumatoid arthritis patients (abstract). Arthritis Rheum 15: 132, 1972. Lepow IH: Biologically active fragments of complement. Progress in Immunology, First International Congress of Immunology (Amos B, ed). New York, Academic Press, 1971, p 579. Muller W, Harwerth HG, Fehr K: Studies in four lysosomal enzymes in rheumatoid arthritis and osteoarthritis, chap 18. Rheumatoid Arthritis, Pathogenetic Mechanisms and Consequences in Therapeutics, New York, Academic Press, 1971. Hunder GG, McDuffie FC, Hepper NGG: Pleural fluid complement in systemic lupus erythematosus and rheumatoid arthritis. Ann Intern Med 76: 357, 1972. Hunder GG. Mullen BJ, McDuffie FC: Complement in pericardial fluid of lupus erythematosus: studies in two patients. Ann Intern Med 80: 453, 1974.