CORRELATION BETWEEN LEVELS OF BREAKDOWN PRODUCTS OF C3, C4, AND PROPERDIN FACTOR B IN SYNOVIAL FLUIDS FROM PATIENTS WITH RHEUMATOID ARTHRITIS

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1 647 CORRELATION BETWEEN LEVELS OF BREAKDOWN PRODUCTS OF C3, C4, AND PROPERDIN FACTOR B IN SYNOVIAL FLUIDS FROM PATIENTS WITH RHEUMATOID ARTHRITIS L. H. PERRIN, U. E. NYDEGGER, R. H. ZUBLER, P. H. LAMBERT, and P. A. MIESCHER Synovial fluids from 31 patients with seropositive rheumatoid arthritis (RA) and 23 patients with seronegative RA had significantly increased levels of breakdown products of C3, C4, and properdin factor B when compared to patients with osteoarthritis (OA) (P <.1). The same patients exhibited a considerable overlap of native C3, C4, and properdin factor B levels when their results were compared with those of OA patients. The parallel increase of C3d, C4d, and Ba levels in patients with RA suggests an activation of the complement system rather than a nonspecific enzymatic breakdown in synovial fluids. From the World Health Organization immunology Research and 'Training Center. Geneva Blood Center, and the Department of Medicine. University of Geneva. Switzerland. Supported by Swiss National Foundation grant no by the World Health Organization. and by the Dubois-Ferrikre- Dinu Lipatti Foundation. Luc H. Perrin. M.D.: Assistant. WHO Immunology Research and Training Center. HBpital Cantonal; Urs E. Nydegger. M.D.: Assistant. Department of Medicine, University of Geneva. HBpital Cantonal: Rudolf H. Zubler. M.D.: Assistant, WHO Irnmunology Research and Training Center. HBpital Cantonal; Paul H. Lambert. M.D.: Associate Professor of Medicine. University of Geneva, and Head. WHO Immunology Research and Training Center: Peter A. Miescher. M.D.: Professor of Medicine, Department of Medicine. University of Geneva. and Head, Geneva Blood Center, HBpital Cantonal. Address reprint requests to Paul H. Lambert. M.D., WHO Immunology Research and Training Center, HBpital Cantonal. I21 1 Geneva 4, Switzerland. Submitted for publication May : accepted August Whole hemolytic complement activity (CH5) is usually normal or elevated in serum from patients suffering from rheumatoid arthritis (l,2), although a few RA patients have had episodes of hypocomplementemia, usually associated with an exacerbation of the disease (3). However the synovial fluid from patients with seropositive RA exhibits a decreased complement activity when compared to that measured in patients with osteoarthritis (43). Activation of the complement system proceeds through at least two pathways-the classic and the alternative. Both lead to the activation of C3 and the late complement components (6). Activation of native C3 results in the generation of C3b and C3a. C3b is involved in the activation of the late-acting complement components and also triggers an amplification system through activation of factor D and factor B, which leads to a further activation of native C3 (6,7). This process is modulated by C3b inactivator, which cleaves C3b into C3c and C3d (7). In the classic complement pathway, activation of CI by immune complexes promotes the generation of a C3 convertase (CZ), which initiates activation of C3. During this process C4 is first cleaved into C4a and C4b, and further cleavage of C4b leads to the generation of fragments C4c and C4d (8). Activation of the alternative pathway, which can be triggered by nonimmune reactants, also leads to the generation of a C3 convertase. Activation of factor B usually results in its cleavage into a large fragment, Bb, and a small one, Ba (6). The complement profile observed in synovial fluid Arthritis and Rheumatism, Vol. 2, No. 2 (March 1977)

2 648 PERRIN ET AL from patients with seropositive rheumatoid arthritis suggests an activation of the classic complement pathway. However the slight decrease of factor B and properdin levels may indicate an activation of the alternative pathway as well as that of the C3b amplification system (9). The involvement of the complement system in rheumatoid arthritis is usually estimated in synovial fluid by measuring the level of complement components by hemolytic or immunologic methods (4,lO). Unfortunately an increased synthetic rate can mask an increased catabolism of complement components, and such static studies are of limited practical value in this disease. Turnover studies performed in vivo with radiolabeled complement components allow for a better estimation of the catabolism of complement components (1 1). An alternative approach to investigate the involvement of the complement system in various clinical conditions is to detect the presence of breakdown products of complement components in plasma or synovial fluid. Such products have been demonstrated in synovial fluids from patients with RA by means of analytic methods based on the changes in physicochemical properties and on the antigenic constitution of complement components occurring during complement activation (12-14). Recently methods have been developed to directly quantitate C3d, C4d, and Ba fragments resulting from the breakdown of C3, C4, and factor B respectively (15,16). These techniques have been applied to the investigation of patients with systemic lupus erythematosus and various types of glomerulonephritis (15). In the present investigation the hypercatabolism of C3, C4, and factor B was investigated in synovial fluids from patients with RA and other articular diseases through quantitation of the corresponding breakdown products. Furthermore, it is known that C3 exhibits several antigens, among them a native antigen present only on the native molecule of C3, and an antigen (the main antigen of C3c) present on native C3 as well as on C3b and C3c fragment (17,18). The level of C3 has been measured in synovial fluids by means of either a specific anti-native C3 antiserum or an anti-c3c antiserum to estimate the amount of C3 that has undergone cleavage. MATERIALS AND METHODS Patient Population. Patients were selected on the basis of availability of sufficient synovial fluid (SF) for aspiration. The SF from 71 patients was studied, including 31 with seropositive rheumatoid arthritis (RA+), 23 with seronegative rheumatoid arthritis (RA-), and 17 with osteoarthritis (OA). RA patients satisfied the American Rheumatism Association s criteria for classic or definite rheumatoid arthritis. IgM rheumatoid factor was screened by the Waaler-Rose hemagglutination technique, and patients were considered seropositive when a titer of 1/64 or more was observed. OA patients satisfied generally accepted radiologic, clinical, and biologic criteria. RA+ A c3 nat. RA - c3 nat. OA c3 tot. - A Fig 1. Sj~noriul //id c~~itt(~i~titrutiott,~ o/ notice C3, c 3 nirasured bj, anti-c3c antigen (total C31, and the r/(fliwnc~c, hc,titwn ~,iiftc,i,fitratiiiti.s defected with anfi-c3c, anfigen and anri-narirr C3 anti.wra fs 1 in parienrs with.wropi~.sitiiv rhcwnafoicl arthritis (RA + 1,.serrinegati~,e rheumatoid arthritis (RA- 1, and asteoarthriiis f OA 1. Shaded area represrnt.s nieati f I SD.

3 C3d, C4d, AND Ba FRAGMENTS IN SYNOVIAL FLUIDS 649 The synovial fluids, mostly from the knee joint, were drawn on tubes containing EDTA (final concentration: 2 mm). They were centrifuged at 15 X g for 15 minutes at room temperature, and the supernatants were frozen at -7 C in small aliquots. Preparation of Antisera. Antisera were raised in rabbits by injecting native C3, C ~C, C3d, factor B, and Bb (l5,19). The C3 antisera were rendered specific for the main antigens of native C3, C ~C, and C3d respectively. The factor B antiserum reacted with both Bb and Ba fragments; part of this antiserum was made specific for the Ba antigen present on factor B and Ba fragment. A goat antiserum reacting with C4, C4b, and C4d was also used (16). A rabbit anti-human C4 reacting with both native C4 and C4b was purchased from Behringwerke, Marburg, Germany. Quantitation of C3, C4, and Factor B. Synovial fluid C3 levels were measured by radial immunodiffusion with either anti-native C3 or anti-c3c antisera. Factor B was quantitated similarly, with either anti-bb or anti-ba antisera. C4 levels were measured by radial immunodiffusion using the rabbit anti-human C4 serum. Standard reference curves were determined by using various concentrations of purified C3 and factor B or serial dilutions of a calibrated EDTA plasma pool for C4. Therefore the values for the various C3 and factor B concentrations are expressed in mg%, and those for C4 in percentage of a normal plasma pool. Quantitation of C3d, C4, and Ba Fragments. A twostep procedure was used for the dosage of small molecular weight fragments (C3d, C4d, and Ba) as previously described (15.16). In the first step the native proteins (C3, C4, and factor B) as well as the high molecular weight fragments (C3b. C4b, and most of the C3c and Bb fragments) were precipitated by a concentration of polyethylene glycol (DAB7 Siegfried, Zofingen, Switzerland, MW 6). In the second step concentrations of the fragments were determined by radial immunodiffusion using an anti-c3d antigen antiserum for C3d, anti-ba antigen antiserum for Ba, and goat antlc4 for C4d. Precipitation with polyethylene glycol was performed by mixing 2 pl of synovial fluid with 2 MI of normal rabbit serum (inactivated for 6 minutes at 56 C) to adjust the protein concentrations of the samples. Then 4 pl of polyethylene glycol dissolved in.1 M EDTA and.1 M borate buffer, ph 8.3, were added. The samples were mixed, left at 4 C for 2 hours, and centrifuged at 12 X g for 3 minutes. The supernates were then collected for dosage by radial immunodiffusion. The final concentration of polyethylene glycol to be used was determined as previously described (l5,16); it was 1 I % for C3d. 12% for C4d, and 18% for Ba fragments. The standard reference curves were determined by using various concentrations of C3d and Ba or calibrated serum pool previously activated by aggregated human immunoglobulin (5 mg/ml, 1 hour at 37 C). and treated with the same concentrations of polyethylene glycol as those used for patients samples. It has been shown that in vitro generation of breakdown products preceding the analysis hardly ever occurs under the conditions used for storage and handling of test samples (15). All measurements were made in duplicate and were repeated if the variation exceeded 1%. Statistical Evaluation. Statistical evaluation was carried out by the student s I test and by linear regression analysis by the method of the least squares. RESULTS Concentration of C3 in Synovial Fluid Measured with Anti-Native C3 and Anti-C3c Antisera. C3 concentration was measured by directing antisera against either native C3 or C3c to evaluate the proportion of C3 that had undergone structural changes leading to the loss of the native antigen. Levels of C3 were 34 f 21 mg% (mean 1 SD) for the RA+ patients, 26 f 12 mg% for the RA- patients, and 25 f 14 mg% for the OA patients when anti-native C3 antiserum was used (Figure 1). Levels found with the anti-c3c antiserum were 42 f 23 mg% for the RA+ patients, 33 f 13 mg% for the RApatients, and 26 f 13 mg% for the OA patients. Anti- C3c antiserum reacts with native C3, C3b, and C3c. The values found with the anti-native C3 antiserum were subtracted from those found with the anti-c3c antiserum, on the assumption that the difference between these values reflects the fraction of C3 that has lost its native antigen during activation. The mean difference determined by this subtraction was significantly larger in the patients with either seropositive RA (8.7 f 6.6 mg%) (+ 1 SD) or seronegative RA (7.1 f 4.5 mg%), when compared to patients with OA (< 2 mg%) (P <.1 ). C3d Levels in Synovial Fluids. The mean level of C3d was 3.9 f 1.8 mg% (* 1 SD) in RA+ patients, 2.9 f 1.3 mg% in RA- patients, and.2 f.8 mg% in OA patients (Figure 2). The mean ratio of C3d/native C3 was significantly higher (P <.1) in RA+ patients (.143 k.89) and RA- patients (.123 *.66), (mgo ol l;*l I C3d/native C3 I : l I Fig 2. Left. Swouial,fluid C3d 1eoel.v in patienrs nith seropositicc~ rheutitaroirl arrhriri.~ (RA + 1, seronegarioe rheun~aroid arrhriris (RA- 1. and ostroarrhritis (OAI. Right. Ratio o/ C3d/natirv C3 in the sattie groups of parients. Shaded area represents inean f I SD.

4 65 PERRIN ET AL when compared to OA patients (.8 f.4). Generally, patients with low native C3 levels had greatly increased C3d levels. Four RA+ and 2 RA- patients with a native C3 less than 2 mg% had C3d levels corresponding to a breakdown of more than one-fifth of their total C3. Three RA+ and 1 RA- patient exhibited native C3 levels above the normal range associated with C3d levels of more than 4 mg%. C4 and C4d Levels in Synovial Fluids. The mean levels of C4 were lower in patients with RA+ (14 f 1%) (*I SD) and RA- (17 f 15%) compared to patients with OA (2 f 12%). The mean levels of C4d in both RA+ patients (31 f 17%) and RA- patients (18 f 17%) were significantly higher (P <.1) when compared to the mean values in OA patients (< 5%). There was no significant correlation between the C4 and the C4d levels in individual patients with RA. B and Ba Levels in Synovial Fluids. The synovial fluid concentrations of factor B and Ba fragment were measured in 16 RA+, 14 RA-, and 17 OA patients from whom sufficient fluid had been acquired. The values found in individual patients are shown on Figure 3. The mean levels of factor B were 15 & 7 mg% (f I SD) in the RA+ patients, 1 k 6 mg% in the RA- patients, and 9 f 4 mg% in the OA patients. The mean level of the Ba fragments was significantly higher (P <.1) in both RA+ patients (2.7 f 1.O mg%) and RA- patients (2.1 f.7 mg%), when compared to OA patients (.3 f.8 mg%). Generally, RA patients with a normal factor B level had a Ba level similar to or higher than that of RA patients with an increased factor B level. The mean ratio Ba/B was significantly higher (P <,1) in both RA+ patients (.152 f. 68) and RA- patients (.235 f.116) compared to OA patients (.44 k.17). Correlation Between Levels of C3d, C4d, and Ba. By linear regression analysis, there was a significant correlation between the levels of C3d and Ba in RA+ patients (P <.5) and RA- patients ( P <.5) (Figure 4). C3d levels also correlated significantly with C4d levels in patients with RA- (P <.5), but the correlation was not significant in patients with RA+ ( >.5). DISCUSSION The concentration of complement components in synovial fluid depends on the filtration and reabsorption of plasma proteins, but it is also directly influenced by the local synthesis of some components and by the catabolism of these components in the joint spaces. Indeed a biosynthesis of C2, C3, C4, and C5 by synovial tissues has been demonstrated in vitro (2), and there is good evidence for an active catabolism of complement components in some pathologic conditions (13,16,21). Because an increased catabolism of complement components can be balanced by an increased synthesis or an increased filtration rate of complement components, it is not surprising that the measurement of the concentration of complement components in synovial fluids often does not reflect the intensity of the immune reactions within the joint spaces. In synovial fluid from patients with rheumatoid arthritis, the hemolytic complement activity, the level of I Properdin factor B I Ba fragment I L :%!* 1 - Fig 3. Synooial fluid levels of properdin /actor B (left), Ba fragment (middle), and Ba/properdin factor B rutio (right) iri pa/ierrt.v ~Yth seropositicr (RA + I arid.varoriegutite f RA - / rhrurmtoid arthritis and osteourthri/i.v f OA 1. Shaded area reprrsents itic un &I SD.

5 C3d, C4d, AND Ba FRAGMENTS IN SYNOVIAL FLUIDS 65 1 C3d (mg %I 8l 4 I&&?/ - m RA+ RA- A OA I I I I 1 I Ba (mg%i Fig 4. C'ortipari.sorr (I/' C.3d 1e~'rI.c with Bri 1ecel.c iti itidicidual.satiip/e.c /rotti porirtrt s itvt/i,srropositiri~ rhrutiratoid arthritis (.),.seronegatioe rlit~iitttatoid artlrriti.s (), and osrroarrhriri.e (A). C3, and particularly the C4 level, are generally decreased with respect to the total protein concentration (1,21), but individual results are usually difficult to interpret. Moreover the complement levels are frequently depressed to a lesser extent or may even be normal in seronegative patients. Using a quantitative method, we were able to demonstrate more precisely breakdown products in synovial fluid from patients with rheumatoid arthritis. This finding agrees with previous reports that used qualitative or semi-quantitative methods (l3,14). In addition, the direct quantitation of C3d, C4d. and Ba fragments allowed an estimation of the catabolism of the corresponding components through calculation of the ratio of concentration of C fragment/concentration of native C molecule, independent of the protein concentration in synovial fluid. Although this ratio may still be influenced by diffusion in and out of the joint space, the results strongly suggest an increased catabolism of C3, C4, and factor B in the synovial fluid from patients with RA. The significant correlation between the levels of C3d and Ba fragments in individual synovial fluids indicates that a similar mecha- nism is responsible for the increased catabolism of each of these components. Under the conditions of the test used to measure the levels of complement fragments, the in vitro generation of breakdown products is largely inhibited (15). Therefore it can be considered that the increased levels of C3d, C4d, and Ba in synovial fluids actually reflect an in vivo phenomenon. One cannot exclude the possibility that proteolytic enzymes would nonspecifically cleave complement components in the joint spaces. However the parallel occurrence of C3d, C4d, and Ba fragments in synovial fluid suggests that an activation of the complement system is indeed responsible for the increased catabolism of C3, C4, and factor B. The parallel increase of the levels of C3d and C4d in patients with rheumatoid arthritis suggests an activation of the classic complement pathway, and this observation is consistent with the finding of immune complexes in such synovial fluids (22,23). The occurrence of Ba fragments may reflect an activation of the alternative pathway, as well as an involvement of the C3b amplification system. Although the levels of C3d. C4d, and Ba allow a clear distinction between patients with rheumatoid arthritis and patients with osteoarthritis, similar immunologic conditions leading to complement activation may be found in the synovial fluids from some patients with other inflammatory arthritis (22). The value of the quantitation of complement breakdown products in follow-up studies of patients with rheumatoid arthritis should be considered. For such purposes, this method is easier than a measurement of hemolytic activity/protein ratios for individual complement components such as C2 or C4 (1). Furthermore, although activity/protein ratios are usually decreased in seropositive RA, they may often be normal in seronegative RA. ACKNOWLEDGMENTS The authors thank Dr K. Fehr, Universitaets-Rheumaklinik. Kantonsspital. Zurich, Switzerland. Dr R. Gabay. Division de Rheumatologie. Universitk de Genkve. Switzerland, and Dr J. N. McCormick, Rheumatic Disease Unit. Northern General Hospital, Edinburgh, Scotland for providing synovial fluids and clinical data. Ms. Friedrun Machnik is gratefully acknowledged for expert technical assistance. The secretarial assistance of Ms. Jean Ringrose is appreciated. REFERENCES I. Vaughan JH, Bayles TB, Favour CB: Serum complement in rheumatoid arthritis. Am J Med Sci222: , 1951

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