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IMMUNOELECTROPHORETIC ASSAY FOR SYNOVIAL FLUID C3 WITH CORRECTION FOR SYNOVIAL FLUID GLOBULIN
PETER HASSELBACHER
Synovial fluid C3 was measured by electroimmunoassay. When C3 was expressed as mg/ml, the amounts found in Reiter’s disease, psoriatic arthritis, gout, and systemic lupus erythematosus were significantly different from degenerative arthritis. When C3 was corrected for total protein, the levels for rheumatoid arthritis, Reiter’s disease, psoriatic arthritis, and systemic lupus were significantly different from degenerative arthritis. When C3 was corrected for synovial fluid globulin, only rheumatoid arthritis and systemic lupus were significantly different from degenerative arthritis. Correction of C3 for globulin increases the difference between rheumatoid arthritis and degenerative arthritis. A proportion of gouty fluids with a relative decrease in C3 is demonstrated. It is argued that correction of C3 for globulin is more meaningful than correction for total From the University of Pennsylvania School of Medicine, Philadelphia, PA 19 104. Supported in part by Clinical Investigator award No. KO8 AM00385 from the National Institutes of Health, a Postdoctoral Fellowship of the Arthritis Foundation, and grants from the Philadelphia Chapter of the American Rheumatism Association and the Lupus Foundation of Northeast Philadelphia. Peter Hasselbacher, MD: Presently Assistant Professor of Medicine, Dartmouth Medical School, and Associate Director of the Dartmouth-Hitchcock Arthritis Center, Hanover, New Hampshire. Address reprint requests to Peter Hasselbacher, MD, Department of Medicine, Connective Tissue Disease Section, Dartmouth Medical School, Hanover, New Hampshire 03755. Submitted for publication December 30, 1977; accepted in revised form October 23. 1978. Arthritis and Rheumatism, Vol. 22, No. 3 (March 1979)
protein. While many nonrheumatoid inflammatory effusions demonstrate split products of C3, the majority of fluids from patients with systemic lupus have none. Measurements of hemolytic complement activity and of individual complement proteins in synovial fluid have been used as investigational tools in studies of the pathogenesis of joint inflammation and have been advocated as useful in the diagnosis of arthritis (1-14). The first studies of synovial fluid complement activity in disease observed that to make meaningful comparisons, it was necessary to make a correction for total synovial fluid protein (1,2). Thus it was described that corrected complement activity in synovial fluids of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) were lower than predicted by total protein (1,2). These observations have been used as supporting evidence of immune complex-mediated inflammation in the arthritis of RA and SLE. Additional studies of complement activity and individual complement proteins have confirmed these findings (3-1 1) and have shown that the level of individual complement proteins correlates well with hemolytic activity (8,9,12). Although the mean levels of corrected complement proteins may be lower in seropositive RA fluids, the range of values overlaps with that of degenerative joint disease (DJD), and there is no significant difference between seronegative RA and DJD (6-8,lO). Corrected complement levels in fluids from patients
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with gout and Reiter’s disease are said to be the same or greater than those of DJD (1-3,5,9,11,13,14). Any sizable overlap of ranges diminishes the usefulness of complement determinations for diagnosis or for identification of new syndromes in which complement activation plays a pathogenetic role. The appearance of specific serum proteins in synovial fluid is a function of molecular weight. Noninflammatory fluids contain predominantly albumin and little or no high molecular weight proteins such as alpha, macroglobulin or IgM (15-17). As the degree of articular inflammation increases, so does the proportion of the higher molecular weight serum globulins. Because C3 and other complement proteins have high molecular weights compared to albumin (18), it is physiologically more meaningful to correct synovial fluid C3 levels for globulin than for total protein. This report describes an electroimmunoassay for C3 protein and demonstrates that when C3 is corrected for synovial fluid globulin the difference between fluids from RA and other diseases increases, and that differences among nonrheumatoid fluids are altered.
MATERIALS AND METHODS Clinical material. One hundred twenty-seven synovial fluids that were aspirated by the author for routine diagnostic or therapeutic purposes and fluids referred by other members of the rheumatology division were studied. A definite diagnosis was not possible in some and conditions for collection were not met in others. Blood tinged fluids were excluded. For purposes of analysis in this report, fluids studied included 23 DJD, 31 RA, 8 gout, 9 SLE, 5 Reiter’s, and 5 psoriatic arthritis. The diagnosis of all patients studied was agreed upon by 2 or more observers and met usual diagnostic criteria. No questionable cases were included in the analysis. Almost all the rheumatoid patients had established disease, and only 3 seronegative patients were identified (although recent tests for rheumatoid factor were not available in all patients). For the purpose of this study, no attempt was made to quantitate the clinical disease activity of the individual rheumatoid patients. Most had active systemic disease and all had individual joint inflammation sufficient to prompt arthrocentesis. All patients with gout had inflammatory fluids containing urate crystals. Not all patients with Reiter’s disease had the typical triad, but all had sufficient genitourinary, cutaneous, opthalmologic, oral, and skeletal manifestations to establish the diagnosis. The patients with psoriatic arthritis had asymmetric, oligoarticular, inflammatory arthritis with or without axial skeletal disease. All the patients with SLE had 4 or more of the preliminary American Rheumatism Association criteria. One of these had end stage renal disease, but all were edema free. When measured near the time of study, serum C3 levels in
most were normal, although one had undetectable serum levels. Collection of fluids. Immediately following aspiration, portions of the fluids were placed in Vacutainers (Becton, Dickenson and Co.) containing potassium EDTA such that the final concentration of EDTA varied from 0.005 to 0.01M. A few specimens were chelated with sodium EDTA, 0.005M. The tubes were repeatedly inverted, spun in a clinical centrifuge to remove cells and gross debris, and frozen at -20°C for up to 2 weeks before use. Some fluids were studied immediately and again after storage to assure stability over this period. Before use, sera were gradually thawed and allowed to come to room temperature. Tubes were mixed by inversion and not centrifuged. Electroimmunoassay (EIA) of C3. C3 was determined by the quantitative EIA method of Laurell, the so-called rocket assay (19). A rabbit was sensitized with a highly purified preparation of human C3 (a generous gift of Dr. Carlos Arroyave). Unadsorbed antisera was monospecific to native C3 by Ouchterlony analysis and by both simple and crossed immunoelectrophoresis which used varying proportions of human and rabbit sera. Antiserum from a single bleeding was used for all studies described below. One ml of this early antisera precipitated 0.15 mg of C3 at equivalence in a quantitative immunoprecipitation test. Agarose plates containing 7.5% antisera were prepared on glass lantern slides (92 X 101 mm) by using 15 ml of 1% agarose (Agarose ME, Marine Colloids, Inc.) in veronal buffer (0.05M, pH 8.6) containing 0.005M sodium EDTA and 0.5 gm/liter azide. A row of wells 2.5 mm in diameter and 5 mm apart were prepared. Samples were diluted 1:5 before use in phosphate buffered saline (0.14MNaCI, 0.01M phosphate, pH 7.4) (PBS), containing hyaluronidase (Wydase) 3.75 TR units/ml, and 0.005M sodium EDTA. These dilutions were incubated at room temperature for 30 minutes before use. Pretreatment of synovial fluids with hyaluronidase was necessary to prevent the formation of double peaked rockets in the EIA assay. Control dilutions of human sera were incubated at room temperature for 6 hours to test for possible proteolytic degradation of C3 by hyaluronidase. A single reference serum was stored in multiple aliquots at -20°C. This serum was made 0.005Msodium EDTA and was initially calibrated with a commercial radial immunodiffusion assay kit (Hyland). All manipulations of synovial fluid and serum were done using Eppendorf pipettes with the reverse mode method (20) that eliminates the considerable and variable error caused by adherence of viscous fluids to the pipette tip. With this technique, the pipette is allowed to fill beyond its calibrated volume by initially depressing the plunger beyond its first stop before filling, and delivering the measured amount by depressing only to the first stop with the tip immersed in buffer. While a low voltage is applied to the plate, 5 pl of diluted samples were placed in the wells using Lang-Levy double constriction pipettes (Bio-Rad) which yielded less than a 1% variation in delivery. It was noted that a 5 p1 Eppendorf pipette used in the reverse mode yielded a similar reproducibility (less than 0.5% variation) but with greater ease of operation. Electrophoresis was then continued on a water cooled platform for 4 hours at 7.5 V/cm using the veronal-EDTA-
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SYNOVIAL FLUID C3
azide buffer described above. (The direction of rocket formation is toward the positive electrode.) The plate is then pressed, washed, dried, and stained with Coomassie blue (21). The height of the rocket is estimated to the nearest 0.1 mm and a standard curve prepared. Because the standard curve was not linear, at least 5 points were used in its construction over a range of diluted sample values from 0.036 to 0.30 mg/ ml. Rocket heights from replicate samples in adjacent wells had a coefficient of variation of less than 1%, but due to nonhomogeneities in the gel, the electric field, or the rate of cooling, the coefficient of variation of rockets up to 100 mm distant was up to 3.5%. C3 was expressed as mg/ml, as percent of total protein (C3/TP X loo), or as percent globulin (C3/ globulin X 100). Crossed immunoelectrophoresis. To examine fluids and sera for evidence of C3 activation, crossed immunoelectrophoresis was performed by using the reagents defined above as described by Laurel (22) and modified by Clarke and Freeman (23). When activated C3 was studied, two discrete peaks were observed corresponding to what are conventionally termed beta,C (native C3) and beta,A globulins. Beta,A globulin corresponds to C3c and C3d (24). A decrease in area of the beta,C peak is accompanied by a 1.5 fold increase in the area of the beta,A peak under the conditions above. This technique was not available during the entire period of this study and therefore a systematic analysis of all specimens was not possible. Because the results of selected fluids were of interest and pertinent to the discussion, semiquantitative data are presented below. Radial immunodiffusion assay (RID) for C3. To serve as a control for the EIA assay, Research Products immunodiffusion plates (Miles) were prepared with 7.5 ml of 1% agarose in PBS containing 7.5% antisera as well as 0.005M Na EDTA and 0.5 @liter sodium azide. Five or 10 1.11 of the identical dilutions of sample or standard used in the EIA assay were placed in 3 mm wells and incubated at room temperature until maximal diameter of immunodiffusion rings had occurred. When synovial fluid is studied by RID, it is important that it be pretreated with hyaluronidase. The effects of hyalu-
ronic acid (HA) on the rate of immunoprecipitin ring enlargement and ultimate size is complex. By delaying fluid absorption by the gel through its osmotic activity, HA can decrease the rate of ring enlargement. For some proteins the ultimate ring size is increased, perhaps due to enhanced diffusion or altered antibody-antigen equivalence ratio. Unless the sample and standards are similar in HA content or highly diluted, error has been observed (25). The author is not aware that this effect is understood or described. Hyaluronidase exposure of human serum without HA did not affect ring development under the conditions of this assay which suggests that the C3 molecule was not altered. Miscellaneous methods. Total protein was determined by using the biuret reagent (26). Albumin was measured directly with a modification of a dye displacement assay (27). To avoid formation of a much clot in the acid buffer of the assay, the synovial fluid was pretreated with hyaluronidase exactly as described above using the same dilutions for the C3 assay. Globulin was calculated by subtracting albumin from total protein. A serum protein standard containing 50 mg/ml albumin and 30 mg/ml globulin (Sigma) was used. The coefficient of variation of these chemical assays was routinely less than 3%. Leukocyte count and differential were done manually by using standard techniques. Viscosity was measured as previously described (28) with white blood cell diluting pipettes manufactured without a mixing bead, the generous gift of the Clay Adams Co. Statistics. The means of different samples were compared using the two-tailed Student’s t test (29).
RESULTS Under the conditions of collection and storage used, there was no detectable conversion of C3 in serum. Care must be taken because significant conversion may occur if the fluids are allowed to react with glass surfaces at room temperature before EDTA is added. The C3 reference sera used showed a minimal
Table 1. Synovial fluid total protein, globulin, and C3
Synovial fluid* DJD RA Reiter’s Psoriatic Reiter’s + psoriatic Gout SLE
Number of fluids 23 31 5 5 10 8 9
* DJD = degenerative joint disease; RA
Total protein mg/ml 31.90 40.08 43.93 47.06 45.20 47.08 28.86
f 7.35t f 10.36* f 4.86 f 5.74 f 5.528 f 10.658 f 13.00
Globulin mg/ml 4.98 12.44 11.88 11.90 11.18 13.32 9.30
f 2.21 f 6.788 5.33 f 4.84 f 4.498 f 10.82* f 7.0211
*
C3 mg/ml 0.51 0.48 0.98 1.02 1.00 0.79 0.17
0.20 0.27 0.32 0.41 f 0.345 f 0.3311 f 0.148
f f f f
= rheumatoid arthritis; SLE = systemic lupus erythematosus. standard deviation. # Significant at P 5 0.01 compared to DJD. 8 Significant at P c 0.001 compared to DJD. 11 Significant at P I0.05 compared to DJD.
t Mean f
C3 %total
c3 %
protein
globulin
1.56 1.16 2.23 2.13 2.18 1.65 0.61
f 0.36 0.52$ f 0.50 f 0.68 f 0.565 f 0.54 f 0.478
*
11.55 4.37 10.61 9.01 9.84 9.71 2.62
f 6.90 f 2.548 f 4.73 f 3.07 k 3.85 f 6.89 f 2.679
HASSELBACHER
246
purposes of statistical analysis. As expected, total protein levels were significantly higher than DJD in RA, Reiter’s, psoriatic arthritis, and gout; but DJD was not significantly different from SLE. Synovial fluid globulins in DJD were significantly lower than RA, Reiter’s, psoriatic arthritis, gout, and SLE. Figure 1 shows the distribution of uncorrected C3 values. The mean value for DJD was not significantly higher than RA, but it was significantly lower than Reiter’s and psoriasis (P I0.001) and gout (P I 0.05) and was higher than SLE (P I0.001). Although the means of some of the groups were significantly different, there was considerable overlap of individual values in all five groups. When corrected for total protein (Figure 2) the mean value for DJD was significantly higher than RA (P I0.01) and SLE (P I0.001), and significantly lower than Reiter’s and psoriasis (P 5 0.001) but there was no significant difference from gout. Although the mean values of DJD and RA were significantly different, the dis-
DJD
RA
GOUT
B-27
SLE
Figure 1. Synovial fluid C3 in various diseases expressed as mg/ml. DJD = degenerativejoint disease; RA = rheumatoid arthritis; B-27 = Reiter’s disease (closed circles) and psoriatic arthritis (open circles); SLE = systemic lupus erythernatosus. The bars depict the standard deviation of the mean.
0 0 0 0
0
0 0 0
change by crossed immunoelectrophoresis after 8 months of storage. There was no discernable alteration of native serum C3 when exposed to the hyaluronidase preparation in the concentrations and times used in this study. For 33 fluids studied by both the EIA and RID assays, the agreement was good, with the rocket assay measuring an average of 0.033 mg/ml (* 0.075) less than the RID assay. Table 1 summarizes the relevant data. Because the data for patients with Reiter’s disease and psoriasis were identical and because of the similarity of these seronegative syndromes, they were combined for the
DJD
RA
GOUT
6-27
SLE
Figure 2. Synovial fluid C3 in various diseases expressed as percent of total synovial fluid protein.
SYNOVIAL FLUID C3
247
0
8
DJD
RA
GOUT
8-27
effusions where the area under the beta,A curve was often equal to or greater than that under the betalc curve. All the rheumatoid patients studied had more than small amounts of conversion (i.e., beta,A area greater than 10% of the combined area under the beta,A and betalc curves). However, in fluids from patients with gout, Reiter’s disease, and psoriatic arthritis, more than small amounts of conversion were noted. In a few such fluids the beta,A area equaled the betalc area. No C3 activation was noted in the 3 DJD fluids collected directly into tubes containing EDTA and studied the same day without freezing. A few random DJD fluids collected and stored showed small amounts of C3 split products. Of interest, in none of the 7 SLE fluids studied was more than 9% of total area under the beta,A curve. A single exception (50%) had both SLE and gonococcal arthritis. In at least 4 fluids from patients with joint symptoms, active disease, and normal serum C3, there was no evidence of C3 split products. Viscosity. The mean relative synovial fluid viscosity for DJD = 25.9 times water, RA= 25.4, Reiter’s = 8.9, psoriasis = 13.6, gout = 24.1, and SLE = 56.4. Only the viscosity of SLE fluids was significantly different from any other group (P 5 0.05).
SLE
Figure 3. Synovial fluid C3 in various diseases expressed as percent of total synovial fluid globulin.
DISCUSSION
tribution of values was very similar with only 4 RA values lower than the lowest DJD. These relationships between the different diseases were identical with previously reported studies of synovial fluid complement (1-14). When corrected for globulin (Figure 3), the mean value for DJD was significantly higher than RA (P 5 0.001) and SLE (P 5 0.001) but was not different from Reiter’s, psoriasis, or gout. The separation of RA fluids from DJD was marked, with only 4 RA values greater than the lowest DJD measurement. Three patients with gout, one with Reiter’s disease, and all but one with SLE had values less than 6%, the lowest DJD value. C3 split products. Study of several typical fluids from each disease group demonstrated that in only some fluids from patients with DJD and SLE there was absolutely no evidence of C3 conversion noted. The greatest conversion to- beta,A was seen in rheumatoid
It is well recognized that normal synovial fluid has a high a1bumin:globulin ratio compared to serum, and that this ratio falls as the degree of joint inflammation and total protein increases (15-17). The present study confirms that the a1bumin:globulin ratio varies widely, even within a single diagnostic group. Most of the influx of protein in inflammatory fluids is probably due to an increase in capillary permeability. It is likely, however, that in normal and noninflammatory fluids at least some portion of serum protein is excluded from the articular cavity because of inability to penetrate the domain of the high molecular weight hyaluronic acid (30). The molecular weight of C3 is 185,000 daltons (18), making it one of the larger of the serum proteins. Therefore, to facilitate comparison of C3 levels in different synovial fluids, it is reasonable to attempt a correction for globulin. Hedburg has shown that for degenerative fluids, total hemolytic activity correlated as well with globulin as with total protein (6). However, he made no comparison among other pathologic fluids in this regard.
248
The data presented here confirm that for uncorrected C3 levels in synovial fluids there is such an extensive overlap of ranges of values in different diagnostic groups as to preclude diagnostic usefulness or allow meaningful statements about pathogenesis. When C3 was corrected from total protein, as is the standard practice, there was a previously described tendency for rheumatoid arthritis (RA) fluids to have lower C3 levels than degenerative joint disease (DJD);gout and the seronegative spondylarthropathies were the same or higher than DJD.There remained, however, such similarity of individual values that measurement of C3 corrected in this way offered little discriminatory value. Many RA values and most systemic lupus erythematosus (SLE) values were very low. These observations have been used to support the hypothesis of immune complex-mediated inflammation in these disorders. However, by the criteria of C3 levels alone (as % total protein) many RA fluids would be said not to demonstrate immunologic disturbance. The somewhat higher levels seen in Reiter’s and psoriatic arthritis have been considered diagnostically useful and imply some special causative circumstance. Because gouty fluids are the same or higher than DJD,a role for complement-mediated inflammation in this disorder has been discounted. When corrected for globulin, the difference between RA and DJD became greatly magnified, with almost all RA fluids showing a relative depression of C3. This is compatible with the observation of Perrin et a1 (7) who found split products of C3 in virtually all of the RA fluids they studied, independent of the presence of rheumatoid factor in the serum, with no measurable split products in DJD fluids. For all rheumatoid fluids studied here, there was no statistical correlation by linear regression analysis of C3 (as % globulin) with total leukocyte count (r = 0.14). The only significant available correlate for lower C3 levels in rheumatoid fluids was the presence of insoluble aggregates of IgG and IgM seen in extracellular fluid and intracellular inclusions. Twelve of 28 whole rheumatoid fluids studied with immunoperoxidase techniques had such particulate aggregates, always containing both IgG and IgM. None of 50 control fluids had aggregates. Aggregatepositive fluids had a lower C3 as % of globulin of 3.29 f 2.00% (mean f SD) compared to aggregate-negative fluids, 5.29 f 2.77 (P I0.05). The aggregate-positive fluids had significantly higher leukocyte counts, percent neutrophils, and total protein and globulin (3 1).
HASSELBACHER
It was also apparent that the globulin-corrected C3 levels in Reiter’s disease and psoriatic arthritis did not differ significantly from DJD in either direction. Thus the increased globulin levels alone can explain the higher levels of C3 in these disorders. If complement activation is important, it would appear to differ in a qualitative or quantitative way from mechanisms operative in rheumatoid arthritis. Of interest is the presence of C3 levels less than 6% globulin in 3 of 8 cases of gout sera. This is compatible with previously demonstrated activation of the complement cascade and C3 by crystals of monosodium urate (32,33), and with observations of diminished urate-induced inflammation in decomplemented animals (34). The role of complement in gout remains uncertain. Figure 3 demonstrates a tendency to skew the highest values in an upward direction. These were the fluids with the lowest globulin content. Following the completion of the experimental work reported here, Speicher et a1 have reported that the bromcresol green technique tends to overestimate serum albumin (35). This would cause an underestimation of globulin and at very low levels would increasingly exaggerate calculated C3 levels. This possible source of error does not affect the interpretation of results discussed here. In future studies, albumin can be measured by other means, such as electroimmunoassay. Albumin and C3 may be measured simultaneously on the same plate which simplifies the procedure further. It is known that there may be appreciable local synthesis of immunoglobulin by the rheumatoid synovial membrane. This would falsely lower the corrected C3. It has been calculated that a mean of 17.2%of total rheumatoid fluid IgG is synthesized locally (36). Assuming that 20% of active rheumatoid synovial fluid protein is gammaglobulin (1,16) and extrapolating the amount of local IgG to total gammaglobulin, 3.4% of total protein in RA is locally produced. In the present series, the mean total protein in RA was 40.08 mg/ml, 1.4 mg/ml of which might be of local origin. Thus the mean rheumatoid fluid globulin of 12.44 mg/ml - 1.37 mg/ml = 1 1.07 mg/ml of globulin which should be used to correct C3. Using the mean C3 of 0.48 mg/ml, the corrected C3 is 4.34%. This is only 0.48%of globulin higher than would otherwise have been calculated and would not alter the present conclusions. An increased loss of immunoglobulin in the form of immune complexes would tend to reduce the possible error of local
249
SYNOVIAL FLUID C3
production. Formation of cryoglobulins during storage might also tend to lower synovial fluid globulin. However, these were not removed prior to study and at least a portion would have redissolved. It has been shown that an average rheumatoid synovial fluid cyroprecipitate may contain 0.1 mg/ml (37). As demonstrated above, such a small amount would have little effect on the results. It is clear that simple static measurement of C3 in synovial fluid is inadequate to describe dynamic intraarticular events. In serum, both biosynthesis and catabolic rates of C3 must be known to fully appreciate the meaning of serum C3 levels (38). In synovial fluid, local synthesis of C3 has been suggested but its magnitude remains uncertain (39). Activated split products of C3 remain in synovial fluid and can be detected with a variety of techniques (6,7,9,40). Although these split products are nonfunctional in a hemolytic assay (9), they are measured as immunoreactive C3 and thus interfere with estimates of native C3 to the variable degree that a given reagent antisera will cross react. The simple presence of C3 split products in synovial fluid does not necessarily imply the presence of immune complexes. It was noted in this study and in others that split products may be observed in at least some fluids of almost every major type of arthritis (6,40). Proteolytic enzymes will cause formation of C3 split products (24,41) and may in part explain their presence in inflammatory fluids. The effect of collection and storage of native C3 must be carefully controlled if measurement of split products is to be meaningful. In most nonrheumatoid fluids in which C3 split products have been described, the total C3 was not low. This may result from a different distribution of C3 between the soluble and insoluble protein. If C3 is fixed to immune complexes in RA fluids and precipitated from solution, less C3 or its split products will remain in the fluid phase for measurement. The previously discussed finding of insoluble particulate immunoglobulin aggregates supports such an hypothesis. The observation of a low C3 does not necessarily imply that intraarticular complement consumption has occurred. If the serum C3 is low, as demonstrated by one of the SLE patients above, the synovial fluid C3 will necessarily be low unless local synthesis is appreciable. However, in at least 4 additional SLE patients with active disease and normal serum C3, there was no evidence of C3 split products in synovial fluid despite symptoms of arthritis. It therefore is premature to pos-
tulate that most of the effusions of SLE are due to immune complex disease unless some explanation for the complement findings is found. A word of additional caution is in order here. Although corrected SLE C3 levels were lower than DJD, degenerative fluids can not be considered completely normal. With regard to relative viscosity, the SLE effusions were more “normal” than the DJD fluids. Until more is known of truly normal C3 levels and of the factors which influence the appearance of C3 in synovial fluid, the meaning of a low C3 in SLE fluids is yet unsettled. Although it remains to be proved, the use of corrected C3 levels in synovial fluid in this laboratory appears to be useful in identifying additional forms of arthritis in which immune mechanisms may be present.
ACKNOWLEDGMENTS The author wishes to thank the generous help of Dr. Joseph L. Hollander and other members of the Arthritis Section of the University of Pennsylvania for referring synovial fluids for study. The assistance of Ms Esther Lobb and Ms Chris Funk in the preparation of this manuscript is also gratefully acknowledged.
REFERENCES 1. Hedberg H: Studies on the depressed hemolytic complement activity of synovial fluid in adult rheumatoid arthritis. Acta Rheum Scand 9:165-193, 1963 2. Pekin TJ, Zvaifler NJ: Hemolytic complement in synovial fluid. J Clin Invest 43:1372-1382, 1964 3. Bunch TW, Hunder GG, McDuffie FC, O’Brien PC, Markowitz H: Synovial fluid complement determination as a diagnostic aid in inflammatory joint disease. Mayo Clin Proc 49:715-720, 1974 4. Bunch TW, Hunder GG, Offord K, McDuffie FC: Synovial fluid complement: usefulness in diagnosis and classification of rheumatoid arthritis. Ann Intern Med 81:3235, 1974 5. Fostiropoulos G, Austen KF, Block, KJ: Total hemolytic complement (CH50) and second component of complement activity in serum and synovial fluid. Arthritis Rheum 8:219-232, 1965 6. Hunder GG, McDuffie FC, Mullen BJ: Activation of complement components C3 and factor B in synovial fluids. J Lab Clin Med 89:160-171, 1977 7. Perrin LH, Nydegger UE, Zubler RH, Lambert PH, Meischer PA: Correlation between levels of breakdown products of C3, C4 and properdin factor B in synovial fluids from patients with rheumatoid arthritis. Arthritis Rheum 20:647-652, 1977
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8. Ruddy S, Austen KF: The complement system in rheumatoid arthritis. I. An analysis of complement component activities in rheumatoid synovial fluids. Arthritis Rheum 13:713-723, 1970 9. Rynes RI, Ruddy S, Schur PH, Spragg J, Austen KF: Levels of complement components, properdin factors, and kininogen in patients with inflammatory arthritis. J Rheumatol 1:413427, 1974 10. Ruddy S, Fearson DT, Austen KF: Depressed synovial fluid levels of properdin and properdin factor B in patients with rheumatoid arthritis. Arthritis Rheum 18:289295, 1975 11. Townes AS, Sowa JM: Complement in synovial fluid. Johns Hopkins Med J 127:23-37, 1970 12. Lundh B, Hedberg H, Laurell AB: Studies of the third component of complement in synovial fluid from arthritic patients. I. Immunochemical quantitation and relation to total complement. Clin Exp Immunol6:407411, 1970 13. Hedberg H: Studies on synovial fluid in arthritis. I. The total complement activity. Acta Med Scand Suppl 479:l78, 1967 14. Pekin TJ, Malinin TI, Zvaifler NJ: Unusual synovial fluid findings in Reiter’s syndrome. Ann Intern Med 66:677684, 1967 15. Kushner I, Somerville JA: Permeability of human synovial membrane to plasma proteins. Arthritis Rheum 14560-570, 1971 16. Decker B, McKenzie BF, McGuckin WF, Slocumb CH: Comparative distribution of proteins and glycoproteins of serum and synovial fluid. Arthritis Rheum 2:162-177, 1959 17. Schur PH, Sandson J: Immunologic studies of the proteins of human synovial fluid. Arthritis Rheum 6: 115-129, 1963 18. Ruddy S, Gigli I, Austen KF: Complement system in man. N Engl J Med 287:489495, 1972 19. Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:45-52, 1966 20. BioRad Laboratories Bulletin 1046, December 1976 21. Axelsen NH, Kroll J, Weeke B: A manual of quantitative immunoelectrophoresis: methods and applications. Scand J Immunol 2:l-36, 1973 22. Laurell CB: Antigen-antibody crossed electrophoresis. Anal Biochem 10358-361, 1965 23. Clarke HGM, F~~~~~~ T: ~ ~ immune- ~ electrophoresis of human serum proteins. Clin Sci 35:403413, 1668 24. Arroyave CM, Tan EM: Detection of complement activation by counterimmunoelectrophoresis. J Immunol Meth 13:101-112, 1976
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25. Hasselbacher P Unpublished observation 26. Gornall AG, Bardawill CJ, David MM: Determination of serum proteins by means of biuret reaction. J Biol Chem 177~751-766, 1949 27. Bauer JD, Ackerman PG, Toro G: Clinical Laboratory Methods. Eighth edition. C.V. Mosby, St. Louis, 1974, pp 403-404 28. Hasselbacher P Measuring synovial fluid viscosity with a white blood cell diluting pipette: a simple, rapid, and reproducible method. Arthritis Rheum 19:1358-1362, 1976 29. Colton T: Statistics in Medicine. Little, Brown and Co, Boston, 1974, pp 127-142 30. Ogston AG, Phelps CF: The partition of solutes between buffer solutions containing hyaluronic acid. Biochem J 78:827-833, 1960 3 I . Hasselbacher P Particulate immunoglobulin aggregates in rheumatoid synovial fluids. Proceedings of Southeastern Regional Am Rheum Assoc, Dec 1977 32. Hasselbacher P: Activation of C3 by monosodium urate, potassium urate, and steroid crystals. Arthritis Rheum 21:565, 1978 33. Naff GB, Byers PH: Complement as a mediator of inflammation in acute gouty arthritis. I. Studies on the reaction between human serum complement and sodium urate crystals. J Lab Clin Med 81: 747-760, 1973 34. Kellermeyer RW, Naff GB: Chemical mediators of inflammation in acute gouty arthritis. Arthritis Rheum 18~765-770, 1976 35. Speicher CE, Widish JR, Gaudot FJ, Helper BR: An evaluation of the overestimation of serum albumin by bromcresol green. Am J Clin Pathol69:347--350, 1978 36. Slivinski AJ, Zvaifler NJ: In vivo synthesis of IgG by rheumatoid synovium. J Lab Clin Med 76:304-310, 1970 37. Cracchiolo A, Goldberg LS, Barnett EV, Bluestone R: Studies of cryoprecipitates from synovial fluid of rheumatoid patients. Immunology 20:1067-1077, 197 1 38. Petz LD, Powers R, Fries JR, Cooper HR: The in vivo metabolism of the third component of complement in systemic lupus erythematosus. Arthritis Rheum 2013041313, 1977
39. Ruddy s, Colten HR: Rheumatoid arthritis: biosynthesis of complement proteins by synovial tissues. N Engl J Med 290:12841288, 1974 40. Zwaifler productsi of C3 in human syno- ~ ~ NJ: Breakdown ~ ~ vial fluids. J Clin Invest 48:1532-1542, 1969 41. Spitzer RE, Stitzel AE, Pauling BL, Davis NC, West CD: The antigenic and molecular alterations of C3 in the fluid phase during an immune reaction in normal human serum. J Exp Med 134656-680, 1971
IMMUNOELECTROPHORETIC ASSAY FOR SYNOVIAL FLUID C3 WITH CORRECTION FOR SYNOVIAL FLUID GLOBULIN
PETER HASSELBACHER
Synovial fluid C3 was measured by electroimmunoassay. When C3 was expressed as mg/ml, the amounts found in Reiter’s disease, psoriatic arthritis, gout, and systemic lupus erythematosus were significantly different from degenerative arthritis. When C3 was corrected for total protein, the levels for rheumatoid arthritis, Reiter’s disease, psoriatic arthritis, and systemic lupus were significantly different from degenerative arthritis. When C3 was corrected for synovial fluid globulin, only rheumatoid arthritis and systemic lupus were significantly different from degenerative arthritis. Correction of C3 for globulin increases the difference between rheumatoid arthritis and degenerative arthritis. A proportion of gouty fluids with a relative decrease in C3 is demonstrated. It is argued that correction of C3 for globulin is more meaningful than correction for total From the University of Pennsylvania School of Medicine, Philadelphia, PA 19 104. Supported in part by Clinical Investigator award No. KO8 AM00385 from the National Institutes of Health, a Postdoctoral Fellowship of the Arthritis Foundation, and grants from the Philadelphia Chapter of the American Rheumatism Association and the Lupus Foundation of Northeast Philadelphia. Peter Hasselbacher, MD: Presently Assistant Professor of Medicine, Dartmouth Medical School, and Associate Director of the Dartmouth-Hitchcock Arthritis Center, Hanover, New Hampshire. Address reprint requests to Peter Hasselbacher, MD, Department of Medicine, Connective Tissue Disease Section, Dartmouth Medical School, Hanover, New Hampshire 03755. Submitted for publication December 30, 1977; accepted in revised form October 23. 1978. Arthritis and Rheumatism, Vol. 22, No. 3 (March 1979)
protein. While many nonrheumatoid inflammatory effusions demonstrate split products of C3, the majority of fluids from patients with systemic lupus have none. Measurements of hemolytic complement activity and of individual complement proteins in synovial fluid have been used as investigational tools in studies of the pathogenesis of joint inflammation and have been advocated as useful in the diagnosis of arthritis (1-14). The first studies of synovial fluid complement activity in disease observed that to make meaningful comparisons, it was necessary to make a correction for total synovial fluid protein (1,2). Thus it was described that corrected complement activity in synovial fluids of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) were lower than predicted by total protein (1,2). These observations have been used as supporting evidence of immune complex-mediated inflammation in the arthritis of RA and SLE. Additional studies of complement activity and individual complement proteins have confirmed these findings (3-1 1) and have shown that the level of individual complement proteins correlates well with hemolytic activity (8,9,12). Although the mean levels of corrected complement proteins may be lower in seropositive RA fluids, the range of values overlaps with that of degenerative joint disease (DJD), and there is no significant difference between seronegative RA and DJD (6-8,lO). Corrected complement levels in fluids from patients
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with gout and Reiter’s disease are said to be the same or greater than those of DJD (1-3,5,9,11,13,14). Any sizable overlap of ranges diminishes the usefulness of complement determinations for diagnosis or for identification of new syndromes in which complement activation plays a pathogenetic role. The appearance of specific serum proteins in synovial fluid is a function of molecular weight. Noninflammatory fluids contain predominantly albumin and little or no high molecular weight proteins such as alpha, macroglobulin or IgM (15-17). As the degree of articular inflammation increases, so does the proportion of the higher molecular weight serum globulins. Because C3 and other complement proteins have high molecular weights compared to albumin (18), it is physiologically more meaningful to correct synovial fluid C3 levels for globulin than for total protein. This report describes an electroimmunoassay for C3 protein and demonstrates that when C3 is corrected for synovial fluid globulin the difference between fluids from RA and other diseases increases, and that differences among nonrheumatoid fluids are altered.
MATERIALS AND METHODS Clinical material. One hundred twenty-seven synovial fluids that were aspirated by the author for routine diagnostic or therapeutic purposes and fluids referred by other members of the rheumatology division were studied. A definite diagnosis was not possible in some and conditions for collection were not met in others. Blood tinged fluids were excluded. For purposes of analysis in this report, fluids studied included 23 DJD, 31 RA, 8 gout, 9 SLE, 5 Reiter’s, and 5 psoriatic arthritis. The diagnosis of all patients studied was agreed upon by 2 or more observers and met usual diagnostic criteria. No questionable cases were included in the analysis. Almost all the rheumatoid patients had established disease, and only 3 seronegative patients were identified (although recent tests for rheumatoid factor were not available in all patients). For the purpose of this study, no attempt was made to quantitate the clinical disease activity of the individual rheumatoid patients. Most had active systemic disease and all had individual joint inflammation sufficient to prompt arthrocentesis. All patients with gout had inflammatory fluids containing urate crystals. Not all patients with Reiter’s disease had the typical triad, but all had sufficient genitourinary, cutaneous, opthalmologic, oral, and skeletal manifestations to establish the diagnosis. The patients with psoriatic arthritis had asymmetric, oligoarticular, inflammatory arthritis with or without axial skeletal disease. All the patients with SLE had 4 or more of the preliminary American Rheumatism Association criteria. One of these had end stage renal disease, but all were edema free. When measured near the time of study, serum C3 levels in
most were normal, although one had undetectable serum levels. Collection of fluids. Immediately following aspiration, portions of the fluids were placed in Vacutainers (Becton, Dickenson and Co.) containing potassium EDTA such that the final concentration of EDTA varied from 0.005 to 0.01M. A few specimens were chelated with sodium EDTA, 0.005M. The tubes were repeatedly inverted, spun in a clinical centrifuge to remove cells and gross debris, and frozen at -20°C for up to 2 weeks before use. Some fluids were studied immediately and again after storage to assure stability over this period. Before use, sera were gradually thawed and allowed to come to room temperature. Tubes were mixed by inversion and not centrifuged. Electroimmunoassay (EIA) of C3. C3 was determined by the quantitative EIA method of Laurell, the so-called rocket assay (19). A rabbit was sensitized with a highly purified preparation of human C3 (a generous gift of Dr. Carlos Arroyave). Unadsorbed antisera was monospecific to native C3 by Ouchterlony analysis and by both simple and crossed immunoelectrophoresis which used varying proportions of human and rabbit sera. Antiserum from a single bleeding was used for all studies described below. One ml of this early antisera precipitated 0.15 mg of C3 at equivalence in a quantitative immunoprecipitation test. Agarose plates containing 7.5% antisera were prepared on glass lantern slides (92 X 101 mm) by using 15 ml of 1% agarose (Agarose ME, Marine Colloids, Inc.) in veronal buffer (0.05M, pH 8.6) containing 0.005M sodium EDTA and 0.5 gm/liter azide. A row of wells 2.5 mm in diameter and 5 mm apart were prepared. Samples were diluted 1:5 before use in phosphate buffered saline (0.14MNaCI, 0.01M phosphate, pH 7.4) (PBS), containing hyaluronidase (Wydase) 3.75 TR units/ml, and 0.005M sodium EDTA. These dilutions were incubated at room temperature for 30 minutes before use. Pretreatment of synovial fluids with hyaluronidase was necessary to prevent the formation of double peaked rockets in the EIA assay. Control dilutions of human sera were incubated at room temperature for 6 hours to test for possible proteolytic degradation of C3 by hyaluronidase. A single reference serum was stored in multiple aliquots at -20°C. This serum was made 0.005Msodium EDTA and was initially calibrated with a commercial radial immunodiffusion assay kit (Hyland). All manipulations of synovial fluid and serum were done using Eppendorf pipettes with the reverse mode method (20) that eliminates the considerable and variable error caused by adherence of viscous fluids to the pipette tip. With this technique, the pipette is allowed to fill beyond its calibrated volume by initially depressing the plunger beyond its first stop before filling, and delivering the measured amount by depressing only to the first stop with the tip immersed in buffer. While a low voltage is applied to the plate, 5 pl of diluted samples were placed in the wells using Lang-Levy double constriction pipettes (Bio-Rad) which yielded less than a 1% variation in delivery. It was noted that a 5 p1 Eppendorf pipette used in the reverse mode yielded a similar reproducibility (less than 0.5% variation) but with greater ease of operation. Electrophoresis was then continued on a water cooled platform for 4 hours at 7.5 V/cm using the veronal-EDTA-
245
SYNOVIAL FLUID C3
azide buffer described above. (The direction of rocket formation is toward the positive electrode.) The plate is then pressed, washed, dried, and stained with Coomassie blue (21). The height of the rocket is estimated to the nearest 0.1 mm and a standard curve prepared. Because the standard curve was not linear, at least 5 points were used in its construction over a range of diluted sample values from 0.036 to 0.30 mg/ ml. Rocket heights from replicate samples in adjacent wells had a coefficient of variation of less than 1%, but due to nonhomogeneities in the gel, the electric field, or the rate of cooling, the coefficient of variation of rockets up to 100 mm distant was up to 3.5%. C3 was expressed as mg/ml, as percent of total protein (C3/TP X loo), or as percent globulin (C3/ globulin X 100). Crossed immunoelectrophoresis. To examine fluids and sera for evidence of C3 activation, crossed immunoelectrophoresis was performed by using the reagents defined above as described by Laurel (22) and modified by Clarke and Freeman (23). When activated C3 was studied, two discrete peaks were observed corresponding to what are conventionally termed beta,C (native C3) and beta,A globulins. Beta,A globulin corresponds to C3c and C3d (24). A decrease in area of the beta,C peak is accompanied by a 1.5 fold increase in the area of the beta,A peak under the conditions above. This technique was not available during the entire period of this study and therefore a systematic analysis of all specimens was not possible. Because the results of selected fluids were of interest and pertinent to the discussion, semiquantitative data are presented below. Radial immunodiffusion assay (RID) for C3. To serve as a control for the EIA assay, Research Products immunodiffusion plates (Miles) were prepared with 7.5 ml of 1% agarose in PBS containing 7.5% antisera as well as 0.005M Na EDTA and 0.5 @liter sodium azide. Five or 10 1.11 of the identical dilutions of sample or standard used in the EIA assay were placed in 3 mm wells and incubated at room temperature until maximal diameter of immunodiffusion rings had occurred. When synovial fluid is studied by RID, it is important that it be pretreated with hyaluronidase. The effects of hyalu-
ronic acid (HA) on the rate of immunoprecipitin ring enlargement and ultimate size is complex. By delaying fluid absorption by the gel through its osmotic activity, HA can decrease the rate of ring enlargement. For some proteins the ultimate ring size is increased, perhaps due to enhanced diffusion or altered antibody-antigen equivalence ratio. Unless the sample and standards are similar in HA content or highly diluted, error has been observed (25). The author is not aware that this effect is understood or described. Hyaluronidase exposure of human serum without HA did not affect ring development under the conditions of this assay which suggests that the C3 molecule was not altered. Miscellaneous methods. Total protein was determined by using the biuret reagent (26). Albumin was measured directly with a modification of a dye displacement assay (27). To avoid formation of a much clot in the acid buffer of the assay, the synovial fluid was pretreated with hyaluronidase exactly as described above using the same dilutions for the C3 assay. Globulin was calculated by subtracting albumin from total protein. A serum protein standard containing 50 mg/ml albumin and 30 mg/ml globulin (Sigma) was used. The coefficient of variation of these chemical assays was routinely less than 3%. Leukocyte count and differential were done manually by using standard techniques. Viscosity was measured as previously described (28) with white blood cell diluting pipettes manufactured without a mixing bead, the generous gift of the Clay Adams Co. Statistics. The means of different samples were compared using the two-tailed Student’s t test (29).
RESULTS Under the conditions of collection and storage used, there was no detectable conversion of C3 in serum. Care must be taken because significant conversion may occur if the fluids are allowed to react with glass surfaces at room temperature before EDTA is added. The C3 reference sera used showed a minimal
Table 1. Synovial fluid total protein, globulin, and C3
Synovial fluid* DJD RA Reiter’s Psoriatic Reiter’s + psoriatic Gout SLE
Number of fluids 23 31 5 5 10 8 9
* DJD = degenerative joint disease; RA
Total protein mg/ml 31.90 40.08 43.93 47.06 45.20 47.08 28.86
f 7.35t f 10.36* f 4.86 f 5.74 f 5.528 f 10.658 f 13.00
Globulin mg/ml 4.98 12.44 11.88 11.90 11.18 13.32 9.30
f 2.21 f 6.788 5.33 f 4.84 f 4.498 f 10.82* f 7.0211
*
C3 mg/ml 0.51 0.48 0.98 1.02 1.00 0.79 0.17
0.20 0.27 0.32 0.41 f 0.345 f 0.3311 f 0.148
f f f f
= rheumatoid arthritis; SLE = systemic lupus erythematosus. standard deviation. # Significant at P 5 0.01 compared to DJD. 8 Significant at P c 0.001 compared to DJD. 11 Significant at P I0.05 compared to DJD.
t Mean f
C3 %total
c3 %
protein
globulin
1.56 1.16 2.23 2.13 2.18 1.65 0.61
f 0.36 0.52$ f 0.50 f 0.68 f 0.565 f 0.54 f 0.478
*
11.55 4.37 10.61 9.01 9.84 9.71 2.62
f 6.90 f 2.548 f 4.73 f 3.07 k 3.85 f 6.89 f 2.679
HASSELBACHER
246
purposes of statistical analysis. As expected, total protein levels were significantly higher than DJD in RA, Reiter’s, psoriatic arthritis, and gout; but DJD was not significantly different from SLE. Synovial fluid globulins in DJD were significantly lower than RA, Reiter’s, psoriatic arthritis, gout, and SLE. Figure 1 shows the distribution of uncorrected C3 values. The mean value for DJD was not significantly higher than RA, but it was significantly lower than Reiter’s and psoriasis (P I0.001) and gout (P I 0.05) and was higher than SLE (P I0.001). Although the means of some of the groups were significantly different, there was considerable overlap of individual values in all five groups. When corrected for total protein (Figure 2) the mean value for DJD was significantly higher than RA (P I0.01) and SLE (P I0.001), and significantly lower than Reiter’s and psoriasis (P 5 0.001) but there was no significant difference from gout. Although the mean values of DJD and RA were significantly different, the dis-
DJD
RA
GOUT
B-27
SLE
Figure 1. Synovial fluid C3 in various diseases expressed as mg/ml. DJD = degenerativejoint disease; RA = rheumatoid arthritis; B-27 = Reiter’s disease (closed circles) and psoriatic arthritis (open circles); SLE = systemic lupus erythernatosus. The bars depict the standard deviation of the mean.
0 0 0 0
0
0 0 0
change by crossed immunoelectrophoresis after 8 months of storage. There was no discernable alteration of native serum C3 when exposed to the hyaluronidase preparation in the concentrations and times used in this study. For 33 fluids studied by both the EIA and RID assays, the agreement was good, with the rocket assay measuring an average of 0.033 mg/ml (* 0.075) less than the RID assay. Table 1 summarizes the relevant data. Because the data for patients with Reiter’s disease and psoriasis were identical and because of the similarity of these seronegative syndromes, they were combined for the
DJD
RA
GOUT
6-27
SLE
Figure 2. Synovial fluid C3 in various diseases expressed as percent of total synovial fluid protein.
SYNOVIAL FLUID C3
247
0
8
DJD
RA
GOUT
8-27
effusions where the area under the beta,A curve was often equal to or greater than that under the betalc curve. All the rheumatoid patients studied had more than small amounts of conversion (i.e., beta,A area greater than 10% of the combined area under the beta,A and betalc curves). However, in fluids from patients with gout, Reiter’s disease, and psoriatic arthritis, more than small amounts of conversion were noted. In a few such fluids the beta,A area equaled the betalc area. No C3 activation was noted in the 3 DJD fluids collected directly into tubes containing EDTA and studied the same day without freezing. A few random DJD fluids collected and stored showed small amounts of C3 split products. Of interest, in none of the 7 SLE fluids studied was more than 9% of total area under the beta,A curve. A single exception (50%) had both SLE and gonococcal arthritis. In at least 4 fluids from patients with joint symptoms, active disease, and normal serum C3, there was no evidence of C3 split products. Viscosity. The mean relative synovial fluid viscosity for DJD = 25.9 times water, RA= 25.4, Reiter’s = 8.9, psoriasis = 13.6, gout = 24.1, and SLE = 56.4. Only the viscosity of SLE fluids was significantly different from any other group (P 5 0.05).
SLE
Figure 3. Synovial fluid C3 in various diseases expressed as percent of total synovial fluid globulin.
DISCUSSION
tribution of values was very similar with only 4 RA values lower than the lowest DJD. These relationships between the different diseases were identical with previously reported studies of synovial fluid complement (1-14). When corrected for globulin (Figure 3), the mean value for DJD was significantly higher than RA (P 5 0.001) and SLE (P 5 0.001) but was not different from Reiter’s, psoriasis, or gout. The separation of RA fluids from DJD was marked, with only 4 RA values greater than the lowest DJD measurement. Three patients with gout, one with Reiter’s disease, and all but one with SLE had values less than 6%, the lowest DJD value. C3 split products. Study of several typical fluids from each disease group demonstrated that in only some fluids from patients with DJD and SLE there was absolutely no evidence of C3 conversion noted. The greatest conversion to- beta,A was seen in rheumatoid
It is well recognized that normal synovial fluid has a high a1bumin:globulin ratio compared to serum, and that this ratio falls as the degree of joint inflammation and total protein increases (15-17). The present study confirms that the a1bumin:globulin ratio varies widely, even within a single diagnostic group. Most of the influx of protein in inflammatory fluids is probably due to an increase in capillary permeability. It is likely, however, that in normal and noninflammatory fluids at least some portion of serum protein is excluded from the articular cavity because of inability to penetrate the domain of the high molecular weight hyaluronic acid (30). The molecular weight of C3 is 185,000 daltons (18), making it one of the larger of the serum proteins. Therefore, to facilitate comparison of C3 levels in different synovial fluids, it is reasonable to attempt a correction for globulin. Hedburg has shown that for degenerative fluids, total hemolytic activity correlated as well with globulin as with total protein (6). However, he made no comparison among other pathologic fluids in this regard.
248
The data presented here confirm that for uncorrected C3 levels in synovial fluids there is such an extensive overlap of ranges of values in different diagnostic groups as to preclude diagnostic usefulness or allow meaningful statements about pathogenesis. When C3 was corrected from total protein, as is the standard practice, there was a previously described tendency for rheumatoid arthritis (RA) fluids to have lower C3 levels than degenerative joint disease (DJD);gout and the seronegative spondylarthropathies were the same or higher than DJD.There remained, however, such similarity of individual values that measurement of C3 corrected in this way offered little discriminatory value. Many RA values and most systemic lupus erythematosus (SLE) values were very low. These observations have been used to support the hypothesis of immune complex-mediated inflammation in these disorders. However, by the criteria of C3 levels alone (as % total protein) many RA fluids would be said not to demonstrate immunologic disturbance. The somewhat higher levels seen in Reiter’s and psoriatic arthritis have been considered diagnostically useful and imply some special causative circumstance. Because gouty fluids are the same or higher than DJD,a role for complement-mediated inflammation in this disorder has been discounted. When corrected for globulin, the difference between RA and DJD became greatly magnified, with almost all RA fluids showing a relative depression of C3. This is compatible with the observation of Perrin et a1 (7) who found split products of C3 in virtually all of the RA fluids they studied, independent of the presence of rheumatoid factor in the serum, with no measurable split products in DJD fluids. For all rheumatoid fluids studied here, there was no statistical correlation by linear regression analysis of C3 (as % globulin) with total leukocyte count (r = 0.14). The only significant available correlate for lower C3 levels in rheumatoid fluids was the presence of insoluble aggregates of IgG and IgM seen in extracellular fluid and intracellular inclusions. Twelve of 28 whole rheumatoid fluids studied with immunoperoxidase techniques had such particulate aggregates, always containing both IgG and IgM. None of 50 control fluids had aggregates. Aggregatepositive fluids had a lower C3 as % of globulin of 3.29 f 2.00% (mean f SD) compared to aggregate-negative fluids, 5.29 f 2.77 (P I0.05). The aggregate-positive fluids had significantly higher leukocyte counts, percent neutrophils, and total protein and globulin (3 1).
HASSELBACHER
It was also apparent that the globulin-corrected C3 levels in Reiter’s disease and psoriatic arthritis did not differ significantly from DJD in either direction. Thus the increased globulin levels alone can explain the higher levels of C3 in these disorders. If complement activation is important, it would appear to differ in a qualitative or quantitative way from mechanisms operative in rheumatoid arthritis. Of interest is the presence of C3 levels less than 6% globulin in 3 of 8 cases of gout sera. This is compatible with previously demonstrated activation of the complement cascade and C3 by crystals of monosodium urate (32,33), and with observations of diminished urate-induced inflammation in decomplemented animals (34). The role of complement in gout remains uncertain. Figure 3 demonstrates a tendency to skew the highest values in an upward direction. These were the fluids with the lowest globulin content. Following the completion of the experimental work reported here, Speicher et a1 have reported that the bromcresol green technique tends to overestimate serum albumin (35). This would cause an underestimation of globulin and at very low levels would increasingly exaggerate calculated C3 levels. This possible source of error does not affect the interpretation of results discussed here. In future studies, albumin can be measured by other means, such as electroimmunoassay. Albumin and C3 may be measured simultaneously on the same plate which simplifies the procedure further. It is known that there may be appreciable local synthesis of immunoglobulin by the rheumatoid synovial membrane. This would falsely lower the corrected C3. It has been calculated that a mean of 17.2%of total rheumatoid fluid IgG is synthesized locally (36). Assuming that 20% of active rheumatoid synovial fluid protein is gammaglobulin (1,16) and extrapolating the amount of local IgG to total gammaglobulin, 3.4% of total protein in RA is locally produced. In the present series, the mean total protein in RA was 40.08 mg/ml, 1.4 mg/ml of which might be of local origin. Thus the mean rheumatoid fluid globulin of 12.44 mg/ml - 1.37 mg/ml = 1 1.07 mg/ml of globulin which should be used to correct C3. Using the mean C3 of 0.48 mg/ml, the corrected C3 is 4.34%. This is only 0.48%of globulin higher than would otherwise have been calculated and would not alter the present conclusions. An increased loss of immunoglobulin in the form of immune complexes would tend to reduce the possible error of local
249
SYNOVIAL FLUID C3
production. Formation of cryoglobulins during storage might also tend to lower synovial fluid globulin. However, these were not removed prior to study and at least a portion would have redissolved. It has been shown that an average rheumatoid synovial fluid cyroprecipitate may contain 0.1 mg/ml (37). As demonstrated above, such a small amount would have little effect on the results. It is clear that simple static measurement of C3 in synovial fluid is inadequate to describe dynamic intraarticular events. In serum, both biosynthesis and catabolic rates of C3 must be known to fully appreciate the meaning of serum C3 levels (38). In synovial fluid, local synthesis of C3 has been suggested but its magnitude remains uncertain (39). Activated split products of C3 remain in synovial fluid and can be detected with a variety of techniques (6,7,9,40). Although these split products are nonfunctional in a hemolytic assay (9), they are measured as immunoreactive C3 and thus interfere with estimates of native C3 to the variable degree that a given reagent antisera will cross react. The simple presence of C3 split products in synovial fluid does not necessarily imply the presence of immune complexes. It was noted in this study and in others that split products may be observed in at least some fluids of almost every major type of arthritis (6,40). Proteolytic enzymes will cause formation of C3 split products (24,41) and may in part explain their presence in inflammatory fluids. The effect of collection and storage of native C3 must be carefully controlled if measurement of split products is to be meaningful. In most nonrheumatoid fluids in which C3 split products have been described, the total C3 was not low. This may result from a different distribution of C3 between the soluble and insoluble protein. If C3 is fixed to immune complexes in RA fluids and precipitated from solution, less C3 or its split products will remain in the fluid phase for measurement. The previously discussed finding of insoluble particulate immunoglobulin aggregates supports such an hypothesis. The observation of a low C3 does not necessarily imply that intraarticular complement consumption has occurred. If the serum C3 is low, as demonstrated by one of the SLE patients above, the synovial fluid C3 will necessarily be low unless local synthesis is appreciable. However, in at least 4 additional SLE patients with active disease and normal serum C3, there was no evidence of C3 split products in synovial fluid despite symptoms of arthritis. It therefore is premature to pos-
tulate that most of the effusions of SLE are due to immune complex disease unless some explanation for the complement findings is found. A word of additional caution is in order here. Although corrected SLE C3 levels were lower than DJD, degenerative fluids can not be considered completely normal. With regard to relative viscosity, the SLE effusions were more “normal” than the DJD fluids. Until more is known of truly normal C3 levels and of the factors which influence the appearance of C3 in synovial fluid, the meaning of a low C3 in SLE fluids is yet unsettled. Although it remains to be proved, the use of corrected C3 levels in synovial fluid in this laboratory appears to be useful in identifying additional forms of arthritis in which immune mechanisms may be present.
ACKNOWLEDGMENTS The author wishes to thank the generous help of Dr. Joseph L. Hollander and other members of the Arthritis Section of the University of Pennsylvania for referring synovial fluids for study. The assistance of Ms Esther Lobb and Ms Chris Funk in the preparation of this manuscript is also gratefully acknowledged.
REFERENCES 1. Hedberg H: Studies on the depressed hemolytic complement activity of synovial fluid in adult rheumatoid arthritis. Acta Rheum Scand 9:165-193, 1963 2. Pekin TJ, Zvaifler NJ: Hemolytic complement in synovial fluid. J Clin Invest 43:1372-1382, 1964 3. Bunch TW, Hunder GG, McDuffie FC, O’Brien PC, Markowitz H: Synovial fluid complement determination as a diagnostic aid in inflammatory joint disease. Mayo Clin Proc 49:715-720, 1974 4. Bunch TW, Hunder GG, Offord K, McDuffie FC: Synovial fluid complement: usefulness in diagnosis and classification of rheumatoid arthritis. Ann Intern Med 81:3235, 1974 5. Fostiropoulos G, Austen KF, Block, KJ: Total hemolytic complement (CH50) and second component of complement activity in serum and synovial fluid. Arthritis Rheum 8:219-232, 1965 6. Hunder GG, McDuffie FC, Mullen BJ: Activation of complement components C3 and factor B in synovial fluids. J Lab Clin Med 89:160-171, 1977 7. Perrin LH, Nydegger UE, Zubler RH, Lambert PH, Meischer PA: Correlation between levels of breakdown products of C3, C4 and properdin factor B in synovial fluids from patients with rheumatoid arthritis. Arthritis Rheum 20:647-652, 1977
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8. Ruddy S, Austen KF: The complement system in rheumatoid arthritis. I. An analysis of complement component activities in rheumatoid synovial fluids. Arthritis Rheum 13:713-723, 1970 9. Rynes RI, Ruddy S, Schur PH, Spragg J, Austen KF: Levels of complement components, properdin factors, and kininogen in patients with inflammatory arthritis. J Rheumatol 1:413427, 1974 10. Ruddy S, Fearson DT, Austen KF: Depressed synovial fluid levels of properdin and properdin factor B in patients with rheumatoid arthritis. Arthritis Rheum 18:289295, 1975 11. Townes AS, Sowa JM: Complement in synovial fluid. Johns Hopkins Med J 127:23-37, 1970 12. Lundh B, Hedberg H, Laurell AB: Studies of the third component of complement in synovial fluid from arthritic patients. I. Immunochemical quantitation and relation to total complement. Clin Exp Immunol6:407411, 1970 13. Hedberg H: Studies on synovial fluid in arthritis. I. The total complement activity. Acta Med Scand Suppl 479:l78, 1967 14. Pekin TJ, Malinin TI, Zvaifler NJ: Unusual synovial fluid findings in Reiter’s syndrome. Ann Intern Med 66:677684, 1967 15. Kushner I, Somerville JA: Permeability of human synovial membrane to plasma proteins. Arthritis Rheum 14560-570, 1971 16. Decker B, McKenzie BF, McGuckin WF, Slocumb CH: Comparative distribution of proteins and glycoproteins of serum and synovial fluid. Arthritis Rheum 2:162-177, 1959 17. Schur PH, Sandson J: Immunologic studies of the proteins of human synovial fluid. Arthritis Rheum 6: 115-129, 1963 18. Ruddy S, Gigli I, Austen KF: Complement system in man. N Engl J Med 287:489495, 1972 19. Laurell CB: Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Anal Biochem 15:45-52, 1966 20. BioRad Laboratories Bulletin 1046, December 1976 21. Axelsen NH, Kroll J, Weeke B: A manual of quantitative immunoelectrophoresis: methods and applications. Scand J Immunol 2:l-36, 1973 22. Laurell CB: Antigen-antibody crossed electrophoresis. Anal Biochem 10358-361, 1965 23. Clarke HGM, F~~~~~~ T: ~ ~ immune- ~ electrophoresis of human serum proteins. Clin Sci 35:403413, 1668 24. Arroyave CM, Tan EM: Detection of complement activation by counterimmunoelectrophoresis. J Immunol Meth 13:101-112, 1976
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25. Hasselbacher P Unpublished observation 26. Gornall AG, Bardawill CJ, David MM: Determination of serum proteins by means of biuret reaction. J Biol Chem 177~751-766, 1949 27. Bauer JD, Ackerman PG, Toro G: Clinical Laboratory Methods. Eighth edition. C.V. Mosby, St. Louis, 1974, pp 403-404 28. Hasselbacher P Measuring synovial fluid viscosity with a white blood cell diluting pipette: a simple, rapid, and reproducible method. Arthritis Rheum 19:1358-1362, 1976 29. Colton T: Statistics in Medicine. Little, Brown and Co, Boston, 1974, pp 127-142 30. Ogston AG, Phelps CF: The partition of solutes between buffer solutions containing hyaluronic acid. Biochem J 78:827-833, 1960 3 I . Hasselbacher P Particulate immunoglobulin aggregates in rheumatoid synovial fluids. Proceedings of Southeastern Regional Am Rheum Assoc, Dec 1977 32. Hasselbacher P: Activation of C3 by monosodium urate, potassium urate, and steroid crystals. Arthritis Rheum 21:565, 1978 33. Naff GB, Byers PH: Complement as a mediator of inflammation in acute gouty arthritis. I. Studies on the reaction between human serum complement and sodium urate crystals. J Lab Clin Med 81: 747-760, 1973 34. Kellermeyer RW, Naff GB: Chemical mediators of inflammation in acute gouty arthritis. Arthritis Rheum 18~765-770, 1976 35. Speicher CE, Widish JR, Gaudot FJ, Helper BR: An evaluation of the overestimation of serum albumin by bromcresol green. Am J Clin Pathol69:347--350, 1978 36. Slivinski AJ, Zvaifler NJ: In vivo synthesis of IgG by rheumatoid synovium. J Lab Clin Med 76:304-310, 1970 37. Cracchiolo A, Goldberg LS, Barnett EV, Bluestone R: Studies of cryoprecipitates from synovial fluid of rheumatoid patients. Immunology 20:1067-1077, 197 1 38. Petz LD, Powers R, Fries JR, Cooper HR: The in vivo metabolism of the third component of complement in systemic lupus erythematosus. Arthritis Rheum 2013041313, 1977
39. Ruddy s, Colten HR: Rheumatoid arthritis: biosynthesis of complement proteins by synovial tissues. N Engl J Med 290:12841288, 1974 40. Zwaifler productsi of C3 in human syno- ~ ~ NJ: Breakdown ~ ~ vial fluids. J Clin Invest 48:1532-1542, 1969 41. Spitzer RE, Stitzel AE, Pauling BL, Davis NC, West CD: The antigenic and molecular alterations of C3 in the fluid phase during an immune reaction in normal human serum. J Exp Med 134656-680, 1971
