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The occurrence of antibodies to collagen in synovial fluids
CLINICAL
IMMUNOLOGY
AND
3, 567-574 (1975)
IMMUNOPATHOLOGY
The Occurrence of Antibodies to Collagen in Synovial Fluids’ ANDREA CRACCHIOLO, III, Dov MICHAELI,’ LEONARD S. GOLDBERG, AND H. HUGH FUDENBERG Department
of Surgery/Orthopedics and MedicinelRheumatology, UCLA Center for the Health Sciences, and Depurtments of Biochemistry, Surgery, and Medicine. University of Cnliforniu, Sun Francisco, Cu/iforniu
Received May 8, 1974 Synovial fluids and sera from 18 patients with rheumatoid arthritis and 10 subjects with traumatic synovitis were studied for the presence of antibodies to native and denatured collagen. Antibodies to native and denatured collagen were detected in the vast majority of rheumatoid and traumatic synovial fluids. By contrast, these antibodies were present in the sera of less than half of the rheumatoid patients and were rarely found in sera from individuals with traumatic synovitis. In selected instances, antibodies to collagen were eluted from rheumatoid synovial membranes. These data suggest that immune complexes, consisting of collagen and antibody to collagen, may exist in joints affected by chronic synovitis.
Factors which initiate and perpetuate chronic inflammation within joints continue to be investigated intensively. Although multiple mechanisms may be involved in chronic synovitis, considerable importance has been ascribed to immune mechanisms present within joints. The synovial fluids in rheumatoid arthritis have been shown to contain antiglobulins, immunoglobulins, immune complexes, and decreased levels of complement (1). Similar immune factors are also present within rheumatoid synovial membranes (2). These findings have led to the concept that deposition of immune complexes within the synovial membranes may be important in the induction of synovitis. One of the major components of all joint tissue is collagen. Although previously considered as a weak, indifferent antigen, collagen is one of the best characterized of all mammalian protein antigens. Several antigenic determinants of collagen capable of inducing humoral immune responses are well characterized (3-9, and cell-mediated immune reactions to collagen have also been extensively studied (6,7). Many factors important in the antigenicity of collagen have recently been uncovered. Most investigators agree that the antigenic determinants of collagen thus far delineated may not depend on conformation of the intact molecule (5). Antibodies exist to both the native and denatured forms of collagen (8). In a recent study, Michaeli and Fudenberg detected antibodies to denatured collagen in 60% of sera tested from patients with rheumatoid arthritis; ’ Research supported by Grant HL 14759 (D.M.). a USPHS grant HD 05894 and Grant GM 15759 (A.C., L.S.G.). Grant AM 16213, and Research Funds from the Kroc Foundation and the Wilbur D. May Foundation (L.S.G., A.C.). L Recipient of PHS Research Career Development Award K04 AM 50205-04. 567 Copyright PrInted
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1975 United
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Academic States.
Press.
Inc.
All
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in any
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568
CRACCHIOLO
El‘ Al,
20% of the sera contained antibody activity to both native and denatured collagen. Only 8% of normal blood donors showed detectable antibodies to denatured collagen. In the rheumatoid patients, antibody activity was independent of antiglobulin titers and fractionation of two sera showed essentially all anti-collagen activity to be restricted to the IgG fraction. In the present study we determined the presence and titer of these antibodies in synovial fluids; significant titers of antibodies to native and denatured collagen were detected in the synovial fluids and sera of patients with rheumatoid arthritis, and in synovial effusions resulting from chronic traumatic conditions. MATERIALS
AND
METHODS
Matched serum and cell-free synovial fluid specimens were collected from 18 patients with classical rheumatoid arthritis, 10 patients with chronic effusions secondary to a traumatic internal derangement of the knee, and in three other patients (systemic lupus erythematosus, psoriatic arthritis, and osteoarthritis). All specimens were stored at 4°C. IgM was partially isolated by passing sera through Sephadex G-200 columns equilibrated with phosphate-buffered saline, pH 7.35, and IgG fractions were prepared by diethylaminoethyl (DEAE) column chromatography using 0.01 M phosphate buffer, pH 7.5, as the eluant (IO). Synovial membranes from patients with rheumatoid arthritis were prepared and subjected to elution with 2.0 M NaCl and with citrate buffer, pH 3.0 (2). Cesium chloride (CsCl,) density gradient ultracentrifugation was performed as follows (I I): Three synovial fluid specimens containing antibody to native collagen were centrifuged at 10,OOOg for 10 min at 5°C to remove cellular debris. Samples were extensively dialyzed three times against 1 liter of 0.05 M potassium acetate, pH 7.1, at 5°C. All specimens were diluted 1: 2 in 0.05 M potassium acetate buffer, and 1.13 g of CsCl,/ml was added. Cellulose nitrate tubes were sealed with mineral oil and spun in a Beckman L2-65B ultracentrifuge at 100,OOOg for 44 hr at 5°C in a titanium 50 fixed-angle rotor. One-milliliter aliquots were collected by puncturing the bottom of each tube with a 20 gauge needle. Each aliquot was dialyzed and protein concentrations were determined. Aliquots from the top, middle, and lower third of each tube were then separately analyzed for the presence of antibodies to native collagen. Antiglobulin activity was measured simultaneously with latex particles coated with aggregated human IgG, and sheep red blood cells sensitized with rabbit gamma globulin. Heterophile antibody titers were also determined in microtiter plates using a 2% solution of unsensitized sheep erythrocytes. Latex agglutination titers of greater than 1: 80 and differential agglutination titers (sensitized sheep cell agglutination titer minus the heterophile antibody titer) of greater than 4 were considered abnormal. Incubations with 2-mercaptoethanol were performed as previously described ( 12). Total protein concentrations were measured by the method of Lowry (I 3). lmmunoglobulin concentrations were determined by radial immunodiffusion in agar-antibody plates.
ANTI-COLLAGEN
ANTIBODIES
IN SYNOVIAL
FLUID
569
Acid-soluble human skin collagen was prepared by the method of Piez er al. ( 14), as modified by Michaeli et al. (4). Collagen was denatured by heating at 45°C for 10 min. Antibody titers were performed by a microhemagglutination assay utilizing a 2.5% suspension of tanned formalin-treated sheep red blood cells sensitized with native or denatured collagen (4). Rabbit antisera to human collagen (native and denatured) were used as positive control. Normal human serum absorbed with sheep red blood cells was utilized as negative control. All specimens were absorbed with sheep erythrocytes prior to testing for antibodies to collagen. RESULTS Antibodies to native collagen were detected in 15 of 18 rheumatoid synovial fluids, but were present in the serum of only eight patients (Table 1). Six of the seven rheumatoid synovial fluids tested contained antibodies to denatured collagen, while only three of the sera contained this antibody activity. Almost all of the synovial fluids from patients in the trauma group contained antibody to native and denatured collagen, while only one patient, No. 9, showed significant antibody activity in serum (Table 2). Levels of total protein and titers of rheumatoid factor in synovial fluids did not correlate well with the presence of antibodies to either native or denatured collagen in either the rheumatoid or trauma group. For example, the synovial fluid from patient 7 (Table 1) had a low protein concentration, yet it contained one of the highest antibody titers. Although 13 of the rheumatoid synovial fluids demonstrated significant antiglobulin titers, antibodies to collagen were present in the synovial fluid or serum of patients (Nos. 3, 4, 6, 7, 14, 16) who did nor demonstrate significant antiglobulin activity. Only one (No. 4) of the synovial fluids in the trauma group demonstrated significant antiglobulin activity, although in three of the sera low titers were present. None of these three specimens showed antibody activity to either native or denatured collagen. Synovial fluids and matched serum specimens from three other patients (systemic lupus erythematosus, psoriatic arthritis, and osteoarthritis) showed antibody activity against native collagen with the highest titers present in the osteoarthritic fluid; this fluid also contained antibody activity to denatured collagen. Serum from the SLE and the psoriatic patient had low titers of antibody activity, and none was detectable in the osteoarthritic serum. Only the SLE patient had significant antiglobulin activity in both the synovial fluid and serum. In an attempt to localize antibody activity to a specific immunoglobulin class, two sera with high antibody activity were fractionated chromotographically (Table 3). In the first serum (AS), antibody activity to both native and denatured collagen was present in the IgM fraction, but low titer antibody activity to native collagen was also detected in the IgG fraction. IgG and IgM fractions of the second sera (SR) had low titers of antibody activity to both native and denatured collagen. These titers represented a three- and fourfold decrease from the titer seen in the patients’ whole serum. Synovial fluids with high antibody titers were incubated with Z-mercap-
570
CRACCHIOLO TABLE
ET AL. I
ANT~BODIESTOCOLLAGENINRHEUMATOIDSYNOVIALFLUIDSANDSERA
Antinative collagen” I ? 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
SFd S’ SF S SF s SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF s
8 8 0 2 0 I6 0 16 0 8 0 16 4 8 0 2 0 4 0 16 2 4 8 4 8 UD’ 2 2 4 0 4 8 4 0 0
Antidenatured collagen” R 0 4 0 8 0 16 2 8 0 8 2 16
Total protein (g/100 ml) 3.76 6.34 4.14 8.27 2.53 6.71 5.67 6.44 2.43 6.47 3.59 7.246 1.78 5.41 3.70 5.76 3.12 6.85 2.78 6.2 I 2.68 5.77 4.80 7.62 3.13 7.7s 1.67 5.62 4.42 5.98 3.X8 7.25 3.XI 6.M -.
Antiglobulins
..-.
LFT”,” 1,280 640 40,960 80 20 0 I60 0 20 40 0 20 80 40 5,120 320 2.560 160 40 80 I.280 640 1.560 5.120 640 I.280 0 0 40.960 160 ‘I) 70 5.120 640 10.240 5.120
a Expressed as reciprocals of titer. b LFT = latex fixation titer. c DAT = differential agglutination titer. d SF = synovial fluid. c S = serum. ’ UD = undiluted.
toethanol (2-ME) and retested for antibody activity against native and denatured collagen as well as for antiglobulin activity (Table 4). This treatment eliminated all antiglobulin activity and antibody activity to native collagen in four of five rheumatoid synovial fluids. Antibody activity to denatured collagen was not detectable in two patients following 2-ME incubation, but remained in three others.
ANTI-COLLAGEN
ANTIBODIES TABLE
ANTIBODIES
TO COLLAGEN
1 2 3 4 5 6 7 8 Y 10
SF” S” SF S SF S SF S SF S SF S SF S SF S SF S SF S
FLUIDS
TRAUMATIC
Antinative collagen” 16 0 8 0 8 0 16 0 4 0 4 0 4 0 16 0 2 8 2 0
571
FLUID
2
IN SYNOVIAL
WITH
IN SYNOVIAL
AND
SERA
OF PATIENTS
SYNOVITIS
Antidenatured collagen”
Total protein (g/100 ml)
8 0 8 0 8 0 16 0 8 0 4 0 8 2 8 0 0 0 -
3.21 6.28 2.77 6.82 3.62 6.05 3.08 6.69 3.83 7.36 2.44 6.25 3.90 9.20 3.59 6.94 4.21 7.14 5.36 6.92
-
Antiglobulins LFT”,”
-
DAT”.’
20 0 40 40 20 160 20 160 20 160 0 20 20 0 20 80 0 0 0 0
4 0 2 4 4 4 8 8 4 0 0 0 2 0 4 0 0 0 0 0
’ Expressed as reciprocals of titer. ’ LFT = latex fixation titer. c DAT = differential agglutination titer. rl SF = synovial fluid. p S = serum.
TABLE ANTIGLOBULINS
AND
ANTIBODIES
3
TO COLLAGEN
IN
IgM
AND
IgG
FRACTIONS
OF
SERUM
LFTaJ
DAT”)’
Antinative collagen”
Patient A.S. Whole serum kM W
40,960 20,480 <20
64 64 0
16 8 2
16 4 0
Patient S.R. Whole serum W I&
20 <20 <20
4 0 0
32 2 4
32 2 2
R Expressed as reciprocals of titer. * LFT = latex fixation titer. ’ DAT = differential agglutination titer.
Antidenatured collagen”
-
572
CRACCHIOLO
TABLE EFFECTOFZ-MERcAPToETHANOL
ON ANTIBODY
El-
AL..
4 Ac-IIVITIES
flu SYNOV~AL
FLLlM -_
Treated with 2-mercaptoethanul
LFT”,* Rheumatoid synovial fluids 4 5 6 7 11
Antinative collagen”
Antidenatured collagen”
LFT”,”
.hntlnative collagen”
AmIdenaturrd collagen”
160 20 0 80 1280
16 I6 8 16 16
Ih 8 8 ---
20 0 0 0 0
0 0 0 1 II
0 0 16 ih 0
Traumatic synovial fluids 1 3 4 9
20 20 20 0
16 8 16 2
x 8 16 -
0 0 0 0
0 0 I6 0
(! 0 16 il
Osteoarthritis synoviai fluid
20
I6
8
0
0
cl All titer3 expressed as reciprocal of titer. b LFT = latex fixation titer.
When synovial fluids from the trauma patients were incubated with 2-ME, all but one of the specimens showed absence of antibody activity. The single osteoarthritic fluid similarly treated showed a significant reduction of antibody titer. Eluates obtained from four of 19 rheumatoid synovial membranes demonstrated antibody activity to native collagen in low titer. Antibody activity was also present in both the serum and synovial fluid specimens in two patients with one synovial fluid titer being 16. In the third patient the synovial fluid had no activity and no serum was available for analysis: specimens of serum or synovial fluids of the fourth patient were not available for study. In an attempt to determine the influence of hyaluronate on the hemagglutination assay, three synovial fluid specimens with high antibody titers were selected for CsCI, density gradient ultracentrifugation. The highest total protein values were present in the fractions from the upper third of the tubes. The first milliliter aliquot uniformly contained the most protein (44-68.5 mg/ml). The second and third milliliters collected contained much lower amounts of protein (2.9-7.2 mg/ml for the second milliliter and 0.3-3.0 mg/ml for the third). This method has been used effectively to separate hyaluronate from other proteins in synovial fluid (11). Significant quantities of IgM were also detected in the upper third fraction (50- 16 mg/ 100 ml), but were not determined in the other aliquots due to the low levels of total protein present. The highest antibody titers to native collagen (32 and 8) were present in the upper third fractions and could not be detected by our methods in the remaining fractions.
ANTI-COLLAGEN
ANTIBODIES
IN
SYNOVIAL
FLUID
573
DISCUSSION
The results of this study indicate that the synovial fluids obtained from rheumatoid and traumatic joints frequently contain antibodies to collagen. Collagen antibodies are also present in the sera of these patients, but the frequency of these serum antibodies is far less as compared to synovial fluids. Moreover, these antibodies may be sequestered within the synovial tissue as evidenced by our ability to elute this antibody activity from selected synovial tissues. The findings of antibody to collagen in synovial fluid, but not in serum, could be explained by (a) production of these antibodies by immunocytes in the synovial tissue, (b) sequestration of these antibodies within the joint space where abundant collagen antigen is present, or (c) the presence of a serum factor which interferes with the detection of the antibody activity. The finding that synovial fluid contained higher anticollagen titers than sera of the same patients might simply represent a higher concentration of antibody present within a volume of synovial fluid which is many times smaller than the patients’ total serum volume. However, several mechanisms exist which may explain how collagen can be made available as an abundant source of antigen within joints. Chondrocytes can synthesize collagen even in mature tissues such as adult articular cartilage. In addition, collagen turnover is accelerated during periods of reorganization and healing ( 15,16). Mechanical factors may also be involved in the mobilization of collagen components. Collagen fibrils and high levels of hydroxyproline have been detected in synovial fluids of patients with degenerative and traumatic arthritis, possibly reflecting an increased rate of collagen synthesis within these joints (17,18). In addition to these mechanisms, the presence of collagenase in diseased joints may be an important factor in cartilage breakdown, especially in rheumatoid joints. Although the role of collagenase in collagen breakdown has not been clearly established, it may act in combination with other lysosomal enzymes, either extracellularly or intracellularly, and could theoretically contribute to the amount of collagen available as antigen within the joints (19). Thus, the frequent occurrence and high titer of antibodies in the synovial fluid of patients with chronic synovitis may be explained by the local production and/or sequestration of collagen antibodies in response to chronic antigenic stimulation. The antibody activity against both native and denatured collagen appeared to be unrelated to the protein concentration or the presence of other serologic factors such as antiglobulins. This lack of relationship to these factors is probably best illustrated in the trauma group of specimens where detectable antibody activity was confined to the synovial fluid. Since the hemagglutination assay employed could have been affected by the viscosity of the synovial fluid, which appeared to be grossly normal in the trauma group, we considered it necessary to determine if the presence of hyaluronate could be responsible for the elevated antibody titers. It has been shown (11) that cesium chloride density gradient ultracentrifugation localizes the hyaluronate protein to the middle of the gradient at a density of about 1.64 g/ml. Serum proteins sediment at the top of such a gradient, and various unidentified components are present at the bottom after a single ultracentrifugation. The high values of total protein and immunoglobulins
574
CRACCHIOLO
ET AL..
found in the top fractions of our synovial IIuid CsCI, gradients as compared to trace amounts in the middle and lower fractions indicate that the majority of serum proteins were effectively separated from the hyaluronate protein component of synovial fluid. It was also within these top fractions that virtually all antibody activity was detectable. Therefore, it appears that the antibody titers were most likely not affected by the presence of hyaluronate. A preliminary investigation of the antibody to native and denatured collagen in synovial fluid and sera suggests that a heterogenous population of antibodies most likely exists. When synovial fluids with the highest antibody titers were incubated with 2-mercaptoethanol, an agent known to dissociate the disulfide bonds of IgM molecules, antibody activity was reduced or eliminated in most specimens. However, in three specimens (two rheumatoid and one trauma), high antibody titers persisted following 2-ME incubation, although antiglobulin activity was eliminated. Analysis of the two fractionated sera showed antibody activity in both IgM and IgG fractions. Therefore, it appears that antibodies to collagen belong to the IgM and IgG classes. The possibility also exists that a certain fraction of the antibodies to collagen may be firmly bound within the synovial membrane, since four of 19 synovial eluants contained low titer antibody activity to native collagen. In one of these patients antibody activity could not be detected in the synovial fluid. These findings suggest that immune complexes consisting of collagen:antibody to collagen may exist within joints affected by chronic synovitis. The role of such a complex in the inflammatory sequence and its effect, if any, on progressive cartilage destruction are yet to be defined. REFERENCES 1. 2. 3. 4.
Winchester, R. J., Agnello, V., and Kunkel, H. G.. C/i/r. E.x[j. Immrtnol. 6. 145, 1970. Cracchiolo, A., and Goldberg. L. S., A~I. Rherrm. Di.5. 31, 3. 1973. Lindsley, H., Mannik, M., and Barnstein. P., J. E.u/J.bfrd. 133, 1309, 197 I, Michaeli, D., Martin. G.. Kettman, J., Benjamini, E.. Leung, D.. and Bhrtt, B.. S~I’~NC.~166. 1522,
1969.
5. Timpl, B., Wolff, I., Meigel, W.. Pontz, B.. Steffen. C.. and Kuhn, K., ./. Imnmnol. 10.5, I 13 I, 1970. 6.
7. 8. 9. IO. 1 I. 12. 13. 14. 15. 16. 17.
Adelmann, B. C., Kirrane. J. A., and Glynn, L. E., Immrrnolo~~ 23, 723. 1972. Senyk, G., and Michaeli, D., J. Immunol. 111, 1381. 1973. Michaeli, D., Benjamini, E.. and Leung, D. Y. K.. Immun~~c~h~~i.st~~ 8, I, 197 I. Michaeli, D., and Fudenberg, H. H., C/in. Immunol. Immr~noputhol. 2, 153, 1974. Cracchiolo, A., Bluestone, R., and Goldberg, L. S.. C/in. Exll. Immunol. 7, 65. 1970. Scher, I., and Hamerman, D., B&hem. ./. 126, 1075, 1972. Cracchiolo, A., Goldberg, L. S., Barnett, E. V., and Bluestone, R.. Immunology 20, 1067, 1971. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J.. J. Biol. Chem. 193, 265. I95 I. Piez, K. A., Eigner, E. A., and Lewis, M. A., Biochrmi~rr~ 2, 58. 1963. Cooper. D. R., and Russell, A. E., C/in. Orfhop. 67, 188, 1969. Repo, R., and Mitchell, N., J. Bone Joint S~frg. 53B, S41, 1971. Kitridou, R., McCarty, D. J.. Prockop, D. J., and Hummeler, K., Arthritis Rheum. 12, 580. 1969.
18. Bartos. F.. Fiala, O., and Kanth, J., Actrr Orrhop. 19. Woessner. J. F., C/in. Orthop. %, 3 10, 1973.
Sand.
44, 208.
1973.
IMMUNOLOGY
AND
3, 567-574 (1975)
IMMUNOPATHOLOGY
The Occurrence of Antibodies to Collagen in Synovial Fluids’ ANDREA CRACCHIOLO, III, Dov MICHAELI,’ LEONARD S. GOLDBERG, AND H. HUGH FUDENBERG Department
of Surgery/Orthopedics and MedicinelRheumatology, UCLA Center for the Health Sciences, and Depurtments of Biochemistry, Surgery, and Medicine. University of Cnliforniu, Sun Francisco, Cu/iforniu
Received May 8, 1974 Synovial fluids and sera from 18 patients with rheumatoid arthritis and 10 subjects with traumatic synovitis were studied for the presence of antibodies to native and denatured collagen. Antibodies to native and denatured collagen were detected in the vast majority of rheumatoid and traumatic synovial fluids. By contrast, these antibodies were present in the sera of less than half of the rheumatoid patients and were rarely found in sera from individuals with traumatic synovitis. In selected instances, antibodies to collagen were eluted from rheumatoid synovial membranes. These data suggest that immune complexes, consisting of collagen and antibody to collagen, may exist in joints affected by chronic synovitis.
Factors which initiate and perpetuate chronic inflammation within joints continue to be investigated intensively. Although multiple mechanisms may be involved in chronic synovitis, considerable importance has been ascribed to immune mechanisms present within joints. The synovial fluids in rheumatoid arthritis have been shown to contain antiglobulins, immunoglobulins, immune complexes, and decreased levels of complement (1). Similar immune factors are also present within rheumatoid synovial membranes (2). These findings have led to the concept that deposition of immune complexes within the synovial membranes may be important in the induction of synovitis. One of the major components of all joint tissue is collagen. Although previously considered as a weak, indifferent antigen, collagen is one of the best characterized of all mammalian protein antigens. Several antigenic determinants of collagen capable of inducing humoral immune responses are well characterized (3-9, and cell-mediated immune reactions to collagen have also been extensively studied (6,7). Many factors important in the antigenicity of collagen have recently been uncovered. Most investigators agree that the antigenic determinants of collagen thus far delineated may not depend on conformation of the intact molecule (5). Antibodies exist to both the native and denatured forms of collagen (8). In a recent study, Michaeli and Fudenberg detected antibodies to denatured collagen in 60% of sera tested from patients with rheumatoid arthritis; ’ Research supported by Grant HL 14759 (D.M.). a USPHS grant HD 05894 and Grant GM 15759 (A.C., L.S.G.). Grant AM 16213, and Research Funds from the Kroc Foundation and the Wilbur D. May Foundation (L.S.G., A.C.). L Recipient of PHS Research Career Development Award K04 AM 50205-04. 567 Copyright PrInted
0 in the
1975 United
by
Academic States.
Press.
Inc.
All
rights
of
reproduction
in any
form
reserved.
568
CRACCHIOLO
El‘ Al,
20% of the sera contained antibody activity to both native and denatured collagen. Only 8% of normal blood donors showed detectable antibodies to denatured collagen. In the rheumatoid patients, antibody activity was independent of antiglobulin titers and fractionation of two sera showed essentially all anti-collagen activity to be restricted to the IgG fraction. In the present study we determined the presence and titer of these antibodies in synovial fluids; significant titers of antibodies to native and denatured collagen were detected in the synovial fluids and sera of patients with rheumatoid arthritis, and in synovial effusions resulting from chronic traumatic conditions. MATERIALS
AND
METHODS
Matched serum and cell-free synovial fluid specimens were collected from 18 patients with classical rheumatoid arthritis, 10 patients with chronic effusions secondary to a traumatic internal derangement of the knee, and in three other patients (systemic lupus erythematosus, psoriatic arthritis, and osteoarthritis). All specimens were stored at 4°C. IgM was partially isolated by passing sera through Sephadex G-200 columns equilibrated with phosphate-buffered saline, pH 7.35, and IgG fractions were prepared by diethylaminoethyl (DEAE) column chromatography using 0.01 M phosphate buffer, pH 7.5, as the eluant (IO). Synovial membranes from patients with rheumatoid arthritis were prepared and subjected to elution with 2.0 M NaCl and with citrate buffer, pH 3.0 (2). Cesium chloride (CsCl,) density gradient ultracentrifugation was performed as follows (I I): Three synovial fluid specimens containing antibody to native collagen were centrifuged at 10,OOOg for 10 min at 5°C to remove cellular debris. Samples were extensively dialyzed three times against 1 liter of 0.05 M potassium acetate, pH 7.1, at 5°C. All specimens were diluted 1: 2 in 0.05 M potassium acetate buffer, and 1.13 g of CsCl,/ml was added. Cellulose nitrate tubes were sealed with mineral oil and spun in a Beckman L2-65B ultracentrifuge at 100,OOOg for 44 hr at 5°C in a titanium 50 fixed-angle rotor. One-milliliter aliquots were collected by puncturing the bottom of each tube with a 20 gauge needle. Each aliquot was dialyzed and protein concentrations were determined. Aliquots from the top, middle, and lower third of each tube were then separately analyzed for the presence of antibodies to native collagen. Antiglobulin activity was measured simultaneously with latex particles coated with aggregated human IgG, and sheep red blood cells sensitized with rabbit gamma globulin. Heterophile antibody titers were also determined in microtiter plates using a 2% solution of unsensitized sheep erythrocytes. Latex agglutination titers of greater than 1: 80 and differential agglutination titers (sensitized sheep cell agglutination titer minus the heterophile antibody titer) of greater than 4 were considered abnormal. Incubations with 2-mercaptoethanol were performed as previously described ( 12). Total protein concentrations were measured by the method of Lowry (I 3). lmmunoglobulin concentrations were determined by radial immunodiffusion in agar-antibody plates.
ANTI-COLLAGEN
ANTIBODIES
IN SYNOVIAL
FLUID
569
Acid-soluble human skin collagen was prepared by the method of Piez er al. ( 14), as modified by Michaeli et al. (4). Collagen was denatured by heating at 45°C for 10 min. Antibody titers were performed by a microhemagglutination assay utilizing a 2.5% suspension of tanned formalin-treated sheep red blood cells sensitized with native or denatured collagen (4). Rabbit antisera to human collagen (native and denatured) were used as positive control. Normal human serum absorbed with sheep red blood cells was utilized as negative control. All specimens were absorbed with sheep erythrocytes prior to testing for antibodies to collagen. RESULTS Antibodies to native collagen were detected in 15 of 18 rheumatoid synovial fluids, but were present in the serum of only eight patients (Table 1). Six of the seven rheumatoid synovial fluids tested contained antibodies to denatured collagen, while only three of the sera contained this antibody activity. Almost all of the synovial fluids from patients in the trauma group contained antibody to native and denatured collagen, while only one patient, No. 9, showed significant antibody activity in serum (Table 2). Levels of total protein and titers of rheumatoid factor in synovial fluids did not correlate well with the presence of antibodies to either native or denatured collagen in either the rheumatoid or trauma group. For example, the synovial fluid from patient 7 (Table 1) had a low protein concentration, yet it contained one of the highest antibody titers. Although 13 of the rheumatoid synovial fluids demonstrated significant antiglobulin titers, antibodies to collagen were present in the synovial fluid or serum of patients (Nos. 3, 4, 6, 7, 14, 16) who did nor demonstrate significant antiglobulin activity. Only one (No. 4) of the synovial fluids in the trauma group demonstrated significant antiglobulin activity, although in three of the sera low titers were present. None of these three specimens showed antibody activity to either native or denatured collagen. Synovial fluids and matched serum specimens from three other patients (systemic lupus erythematosus, psoriatic arthritis, and osteoarthritis) showed antibody activity against native collagen with the highest titers present in the osteoarthritic fluid; this fluid also contained antibody activity to denatured collagen. Serum from the SLE and the psoriatic patient had low titers of antibody activity, and none was detectable in the osteoarthritic serum. Only the SLE patient had significant antiglobulin activity in both the synovial fluid and serum. In an attempt to localize antibody activity to a specific immunoglobulin class, two sera with high antibody activity were fractionated chromotographically (Table 3). In the first serum (AS), antibody activity to both native and denatured collagen was present in the IgM fraction, but low titer antibody activity to native collagen was also detected in the IgG fraction. IgG and IgM fractions of the second sera (SR) had low titers of antibody activity to both native and denatured collagen. These titers represented a three- and fourfold decrease from the titer seen in the patients’ whole serum. Synovial fluids with high antibody titers were incubated with Z-mercap-
570
CRACCHIOLO TABLE
ET AL. I
ANT~BODIESTOCOLLAGENINRHEUMATOIDSYNOVIALFLUIDSANDSERA
Antinative collagen” I ? 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
SFd S’ SF S SF s SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF S SF s
8 8 0 2 0 I6 0 16 0 8 0 16 4 8 0 2 0 4 0 16 2 4 8 4 8 UD’ 2 2 4 0 4 8 4 0 0
Antidenatured collagen” R 0 4 0 8 0 16 2 8 0 8 2 16
Total protein (g/100 ml) 3.76 6.34 4.14 8.27 2.53 6.71 5.67 6.44 2.43 6.47 3.59 7.246 1.78 5.41 3.70 5.76 3.12 6.85 2.78 6.2 I 2.68 5.77 4.80 7.62 3.13 7.7s 1.67 5.62 4.42 5.98 3.X8 7.25 3.XI 6.M -.
Antiglobulins
..-.
LFT”,” 1,280 640 40,960 80 20 0 I60 0 20 40 0 20 80 40 5,120 320 2.560 160 40 80 I.280 640 1.560 5.120 640 I.280 0 0 40.960 160 ‘I) 70 5.120 640 10.240 5.120
a Expressed as reciprocals of titer. b LFT = latex fixation titer. c DAT = differential agglutination titer. d SF = synovial fluid. c S = serum. ’ UD = undiluted.
toethanol (2-ME) and retested for antibody activity against native and denatured collagen as well as for antiglobulin activity (Table 4). This treatment eliminated all antiglobulin activity and antibody activity to native collagen in four of five rheumatoid synovial fluids. Antibody activity to denatured collagen was not detectable in two patients following 2-ME incubation, but remained in three others.
ANTI-COLLAGEN
ANTIBODIES TABLE
ANTIBODIES
TO COLLAGEN
1 2 3 4 5 6 7 8 Y 10
SF” S” SF S SF S SF S SF S SF S SF S SF S SF S SF S
FLUIDS
TRAUMATIC
Antinative collagen” 16 0 8 0 8 0 16 0 4 0 4 0 4 0 16 0 2 8 2 0
571
FLUID
2
IN SYNOVIAL
WITH
IN SYNOVIAL
AND
SERA
OF PATIENTS
SYNOVITIS
Antidenatured collagen”
Total protein (g/100 ml)
8 0 8 0 8 0 16 0 8 0 4 0 8 2 8 0 0 0 -
3.21 6.28 2.77 6.82 3.62 6.05 3.08 6.69 3.83 7.36 2.44 6.25 3.90 9.20 3.59 6.94 4.21 7.14 5.36 6.92
-
Antiglobulins LFT”,”
-
DAT”.’
20 0 40 40 20 160 20 160 20 160 0 20 20 0 20 80 0 0 0 0
4 0 2 4 4 4 8 8 4 0 0 0 2 0 4 0 0 0 0 0
’ Expressed as reciprocals of titer. ’ LFT = latex fixation titer. c DAT = differential agglutination titer. rl SF = synovial fluid. p S = serum.
TABLE ANTIGLOBULINS
AND
ANTIBODIES
3
TO COLLAGEN
IN
IgM
AND
IgG
FRACTIONS
OF
SERUM
LFTaJ
DAT”)’
Antinative collagen”
Patient A.S. Whole serum kM W
40,960 20,480 <20
64 64 0
16 8 2
16 4 0
Patient S.R. Whole serum W I&
20 <20 <20
4 0 0
32 2 4
32 2 2
R Expressed as reciprocals of titer. * LFT = latex fixation titer. ’ DAT = differential agglutination titer.
Antidenatured collagen”
-
572
CRACCHIOLO
TABLE EFFECTOFZ-MERcAPToETHANOL
ON ANTIBODY
El-
AL..
4 Ac-IIVITIES
flu SYNOV~AL
FLLlM -_
Treated with 2-mercaptoethanul
LFT”,* Rheumatoid synovial fluids 4 5 6 7 11
Antinative collagen”
Antidenatured collagen”
LFT”,”
.hntlnative collagen”
AmIdenaturrd collagen”
160 20 0 80 1280
16 I6 8 16 16
Ih 8 8 ---
20 0 0 0 0
0 0 0 1 II
0 0 16 ih 0
Traumatic synovial fluids 1 3 4 9
20 20 20 0
16 8 16 2
x 8 16 -
0 0 0 0
0 0 I6 0
(! 0 16 il
Osteoarthritis synoviai fluid
20
I6
8
0
0
cl All titer3 expressed as reciprocal of titer. b LFT = latex fixation titer.
When synovial fluids from the trauma patients were incubated with 2-ME, all but one of the specimens showed absence of antibody activity. The single osteoarthritic fluid similarly treated showed a significant reduction of antibody titer. Eluates obtained from four of 19 rheumatoid synovial membranes demonstrated antibody activity to native collagen in low titer. Antibody activity was also present in both the serum and synovial fluid specimens in two patients with one synovial fluid titer being 16. In the third patient the synovial fluid had no activity and no serum was available for analysis: specimens of serum or synovial fluids of the fourth patient were not available for study. In an attempt to determine the influence of hyaluronate on the hemagglutination assay, three synovial fluid specimens with high antibody titers were selected for CsCI, density gradient ultracentrifugation. The highest total protein values were present in the fractions from the upper third of the tubes. The first milliliter aliquot uniformly contained the most protein (44-68.5 mg/ml). The second and third milliliters collected contained much lower amounts of protein (2.9-7.2 mg/ml for the second milliliter and 0.3-3.0 mg/ml for the third). This method has been used effectively to separate hyaluronate from other proteins in synovial fluid (11). Significant quantities of IgM were also detected in the upper third fraction (50- 16 mg/ 100 ml), but were not determined in the other aliquots due to the low levels of total protein present. The highest antibody titers to native collagen (32 and 8) were present in the upper third fractions and could not be detected by our methods in the remaining fractions.
ANTI-COLLAGEN
ANTIBODIES
IN
SYNOVIAL
FLUID
573
DISCUSSION
The results of this study indicate that the synovial fluids obtained from rheumatoid and traumatic joints frequently contain antibodies to collagen. Collagen antibodies are also present in the sera of these patients, but the frequency of these serum antibodies is far less as compared to synovial fluids. Moreover, these antibodies may be sequestered within the synovial tissue as evidenced by our ability to elute this antibody activity from selected synovial tissues. The findings of antibody to collagen in synovial fluid, but not in serum, could be explained by (a) production of these antibodies by immunocytes in the synovial tissue, (b) sequestration of these antibodies within the joint space where abundant collagen antigen is present, or (c) the presence of a serum factor which interferes with the detection of the antibody activity. The finding that synovial fluid contained higher anticollagen titers than sera of the same patients might simply represent a higher concentration of antibody present within a volume of synovial fluid which is many times smaller than the patients’ total serum volume. However, several mechanisms exist which may explain how collagen can be made available as an abundant source of antigen within joints. Chondrocytes can synthesize collagen even in mature tissues such as adult articular cartilage. In addition, collagen turnover is accelerated during periods of reorganization and healing ( 15,16). Mechanical factors may also be involved in the mobilization of collagen components. Collagen fibrils and high levels of hydroxyproline have been detected in synovial fluids of patients with degenerative and traumatic arthritis, possibly reflecting an increased rate of collagen synthesis within these joints (17,18). In addition to these mechanisms, the presence of collagenase in diseased joints may be an important factor in cartilage breakdown, especially in rheumatoid joints. Although the role of collagenase in collagen breakdown has not been clearly established, it may act in combination with other lysosomal enzymes, either extracellularly or intracellularly, and could theoretically contribute to the amount of collagen available as antigen within the joints (19). Thus, the frequent occurrence and high titer of antibodies in the synovial fluid of patients with chronic synovitis may be explained by the local production and/or sequestration of collagen antibodies in response to chronic antigenic stimulation. The antibody activity against both native and denatured collagen appeared to be unrelated to the protein concentration or the presence of other serologic factors such as antiglobulins. This lack of relationship to these factors is probably best illustrated in the trauma group of specimens where detectable antibody activity was confined to the synovial fluid. Since the hemagglutination assay employed could have been affected by the viscosity of the synovial fluid, which appeared to be grossly normal in the trauma group, we considered it necessary to determine if the presence of hyaluronate could be responsible for the elevated antibody titers. It has been shown (11) that cesium chloride density gradient ultracentrifugation localizes the hyaluronate protein to the middle of the gradient at a density of about 1.64 g/ml. Serum proteins sediment at the top of such a gradient, and various unidentified components are present at the bottom after a single ultracentrifugation. The high values of total protein and immunoglobulins
574
CRACCHIOLO
ET AL..
found in the top fractions of our synovial IIuid CsCI, gradients as compared to trace amounts in the middle and lower fractions indicate that the majority of serum proteins were effectively separated from the hyaluronate protein component of synovial fluid. It was also within these top fractions that virtually all antibody activity was detectable. Therefore, it appears that the antibody titers were most likely not affected by the presence of hyaluronate. A preliminary investigation of the antibody to native and denatured collagen in synovial fluid and sera suggests that a heterogenous population of antibodies most likely exists. When synovial fluids with the highest antibody titers were incubated with 2-mercaptoethanol, an agent known to dissociate the disulfide bonds of IgM molecules, antibody activity was reduced or eliminated in most specimens. However, in three specimens (two rheumatoid and one trauma), high antibody titers persisted following 2-ME incubation, although antiglobulin activity was eliminated. Analysis of the two fractionated sera showed antibody activity in both IgM and IgG fractions. Therefore, it appears that antibodies to collagen belong to the IgM and IgG classes. The possibility also exists that a certain fraction of the antibodies to collagen may be firmly bound within the synovial membrane, since four of 19 synovial eluants contained low titer antibody activity to native collagen. In one of these patients antibody activity could not be detected in the synovial fluid. These findings suggest that immune complexes consisting of collagen:antibody to collagen may exist within joints affected by chronic synovitis. The role of such a complex in the inflammatory sequence and its effect, if any, on progressive cartilage destruction are yet to be defined. REFERENCES 1. 2. 3. 4.
Winchester, R. J., Agnello, V., and Kunkel, H. G.. C/i/r. E.x[j. Immrtnol. 6. 145, 1970. Cracchiolo, A., and Goldberg. L. S., A~I. Rherrm. Di.5. 31, 3. 1973. Lindsley, H., Mannik, M., and Barnstein. P., J. E.u/J.bfrd. 133, 1309, 197 I, Michaeli, D., Martin. G.. Kettman, J., Benjamini, E.. Leung, D.. and Bhrtt, B.. S~I’~NC.~166. 1522,
1969.
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Adelmann, B. C., Kirrane. J. A., and Glynn, L. E., Immrrnolo~~ 23, 723. 1972. Senyk, G., and Michaeli, D., J. Immunol. 111, 1381. 1973. Michaeli, D., Benjamini, E.. and Leung, D. Y. K.. Immun~~c~h~~i.st~~ 8, I, 197 I. Michaeli, D., and Fudenberg, H. H., C/in. Immunol. Immr~noputhol. 2, 153, 1974. Cracchiolo, A., Bluestone, R., and Goldberg, L. S.. C/in. Exll. Immunol. 7, 65. 1970. Scher, I., and Hamerman, D., B&hem. ./. 126, 1075, 1972. Cracchiolo, A., Goldberg, L. S., Barnett, E. V., and Bluestone, R.. Immunology 20, 1067, 1971. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J.. J. Biol. Chem. 193, 265. I95 I. Piez, K. A., Eigner, E. A., and Lewis, M. A., Biochrmi~rr~ 2, 58. 1963. Cooper. D. R., and Russell, A. E., C/in. Orfhop. 67, 188, 1969. Repo, R., and Mitchell, N., J. Bone Joint S~frg. 53B, S41, 1971. Kitridou, R., McCarty, D. J.. Prockop, D. J., and Hummeler, K., Arthritis Rheum. 12, 580. 1969.
18. Bartos. F.. Fiala, O., and Kanth, J., Actrr Orrhop. 19. Woessner. J. F., C/in. Orthop. %, 3 10, 1973.
Sand.
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