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Immunoglobulins IgG and IgM in synovial fluids of swine with erysipelothrix polyarthritis
Veterinary Microbiology, 1 (1976) 467--474
467
Elsevier Scientific Publishing Company, A m s t e r d a m - - Printed in The Netherlands
IMMUNOGLOBULINS IgG AND IgM IN SYNOVIAL FLUIDS OF SWINE WITH EBYSIPELOTHRIX POLYARTHRITIS JOHN F. TIMONEY and URIEL YARKONI
Department of Microbiology, New York State College of V~terinary Medicine, Corneli University, Ithaco~ N. Y. 14853 (U.S.A.) (Received 8 July 1976)
ABSTRACT rimoney, J.F. and Yarkoni, U.~ 1976. Immunoglobulins IgG and IgM in synovial fluids of swine with erysipelothrix polyartbritis. Vet. Microbiol., 1: 467--474. Concentrations of IgG and IgM immunoglobulins in synovial fluids and sera from a group of swine with experimentally produced Erysipelothrix rhusiopathiae polyarthritis were measured to determine if there was local synthesis of the~e immunoglobulins by plasma cells in arthritic synovial tissue. IgG and, to a lesser extent, IgM were significantly higher in arthritic than in normal synovial fluids from the same group of swine and this increase could only pa, tly be explained by the increased permeability of the arthritic synovial membrane to p|asms proteins. When synovial fluid values of IgG and IgM were calculated on the basis of companion serum concentration it was found that 82% of IgG, and 25% of IgM estimations were significantly elevated above levels in normal joints indicatirg that IgG was the dominant immunoglobulin synthesized.
INTRODUCTION
An interesting feature of chronic arthritis caused by Erysipelothrix rhusiopathiae (ER) in swine is the presence of large numbers of plasma cells in synovial tissue (Collins ~ d Goldie, 1940). It is reasonable to assume that at least some of the immunoglobulin synthesize~J by these cells finds its way into the synovial fluid and results in elevated concentrations. In the case of rheumatoid arthritis (RA) in man where there is also a heavy accumulation of plasma cells in synovial tissue, studies on IgG and IgM immunoglobulins in synovial fluids indicate local synthesis in arthritic joints, although some of the increase could be accotmted for by the non-specific effects of arthritis on the permeability of the synovial barrier to plasma proteins (Pruzanski et al., 1973). Furthermore, IgG was found to be increased more than IgM or IgA. In vitro studies have shown that RA synovial tissue was highly productive of IgG and to a much lesser extent of IgM and IgA (Smiley et al., 1968). Immunofluorescence studies on plasma cells in synovial tissue from swine with erysipelothrix arthritis have shown that some of these cells contain
468
antibody to a soluble extract of ER (Timoney and Berman, 1970 a). Schulz et al. (1975) have demonstrated IgG and IgM immunoglobulins in these cells. The extent to which the synthetic products of these plasma cells accumulates in the synovial fluid is, however, unclear. Sikes et al. (1967), Cabral et al. (1968), Ajmal (1969), Timoney and Berman (1970 b) and Timoney (1971) demonstrated significant titers of ER antibody in synovial fluids. However, the titers of many of the fluids examined by these authors were lower than in companion serum samples. Cabral et al. (1968) provided the best evidence of local a c c u m u l a t i o n - using the Wellman growth agglutination test, they were able to detect titers higher than in serum in about a third of synovial fluids examined. The work presented here concerns a quantitative study of IgG and IgM levels in the synovial fluids of arthritic swine in an attempt to resolve the question of local accumulation and to determine which immunoglobulins were being synthesized in greater amount in the arthritic synovial tissue. MATERIALS AND METHODS
Twelve Yorkshire swine aged about 6 months, in which polyarthritis had been produced as described prc,.dously (Timoney, 1976), were used as a source of synovial fluids and tissues. Fluids were collected at necropsy from the hip, stifle, hock, shoulder, elbow, and carpal joints and treated for 15 rain with 75 IU hyaluronidase in 0.1 ml saline per 1.0 ml fluid to reduce viscosity. They were then centrifuged at 2 000 g at 6°C for 30 rain and the supernatants were decanted and stored at--70°C. Cultures of the pellet were made on blood ~U~UI
saline. Joints were classified as normal or m,thntic on the basis of gross and
histopathologic criteria (Collins ~md Goldie, 1940). Because of limitations imposed by small fluid volumes it was not possible to perform all immunoglobulin assays on all samples. Sera were also harvested at necropsy and stored at --70°C. Antisera
Antiserum to porcine IgG was prepared by intravenous hyperimmunization of New Zealand white rabbits with complexes of porcine IgG and heat killed ER. The porcine IgG for this purpose was prepared from the serum of a pig with an agglutinin titer of 1 : 640 to ER. An IgG rich fraction was prepared by gel filtration of the serum on Sephadex 200 and by ion exchange chromatography on QAE Sephadex A 50. A mixture of the IgG preparation (4 rag/ ml), and ER (3 × 109 ceUs/ml) killed by heating to 60°C for I h, was then incubated at 37°C for 2 h to adsorb IgG ,antibodies to the surface of the bacteria. The complexes were then washed and centrifuged three times using phosphate buffered saline (pH 7.2) as washing agent. One tenth of a milliliter of the complexes containing 3 × 109 bacterial cells/ml was then inoculated every 3 days for 4 weeks. Ant!isem were harvested and tested for monospecificity
469
by reaction with whole pig serum after immunoelectrophoresis of the latter. Only antisera with anti-IgG activity alone were saved for further use. Antiserum to porcine IgM was prepared in guinea pigs inoculated in one fore and one hind footpad with 0.1 ml of a homogenate of IgM precipitin arcs mixed with complete Freund's adjuvant. These arcs were harvested by careful dissection following immunoelectrophoresis of an IgM rich fraction and reaction with mouse anti-pig serum. The IgM rich fraction was obtained following gel filtration on Sephadex 200 of the pig serum used as the source of IgG. The guinea pigs were bled 3 weeks after inoculation and their sera tested for monospecificity by reaction with whole pig serum after immunoelectrophoresis of the latter. The "arc harvesting" technique was effective in preventing contamination of the antiserum by antibodies to swine IgG and alpha 2 macroglobulins. Standards
An IgG standard solution was prepared by ion exchange chromatography of normal pig serum on QAE Sephadex A-50. The IgG fraction was concentrated in polyvinylpyrolidone (PVP), run through Sephadex G 200, and concentrated again in PVP to 16.2 mg/ml. The IgM standard solution was obtained by subjecting the IgM preparation used for the production of IgM precipitin arcs to two cycles of gel filtration on Sephadex 200 and concentration in PVP. During each gel filtration only the front shoulder of the first peak to e!ute w ~ h~_rvested. The !gM st~,mdo~d contained 1.25 mg protein/ral. Both standards were ,checked for contamination by immunoelectrophoresis and reaction with mouse antiserum to whole pig serum. The IgG standard had no detectable cont~unination; the IgM standard was contaminated with a barely detectable amount of alpha 2 macroglobulin. Since the only effect of this would be to cause a slight over estimation of the amount of IgM protein, no steps were taken to ~:emove it. Protein estimations
The biuret method was used to assay protein concentration in sera, synovial fluids and in IgG and IgM standard solutions. Radial immuno-diffusion assays
The procedure of Fahey and McKelvey (1965) was followed. Antisera were diluted 1 : 16. Standard curves for each assay were s~t up using IgG and IgM standard solutions appropriately diluted. All samples were assayed in duplicate and repeated when differences in excess of 10% between duplicate sample readings were observed. Percentages of companion serum concentrations of IgG, IgM and protein for each synovial fluid were calcu!ated from the serum
470
values for the pig from which the synovial fluids were collected. A Student's t test was used to determine the significance of the differences between normal and arthritic IgG, IgM, and protein percentages respectively in synovial fluids. RESULTS
There were 59 fluids from arthritic and 26 fluids from histologically normal joints of sufficient volume for examination. ER was isolated from 75% of the arthritic joints. Synovial fluid volume varied from about 0.5 ml in normal joints to as much a~ 30 ml in some arthritic joints. Effusions were more common in the stifle joint than in other joints but some arthritic joints had almost no fluid. Histological examination of arthritic joints revealed infiltration by large numbers of plasma and other mononuclear cells and increased vascularity, fibrosis, hypertrophy and palisading of synoviocytes. In contrast, histologically normal synovial tissue consisted of a loose stroma of areolar tissue with few blood vessels and one or two layers of synoviocytes. The results of protein IgG and IgM estimations are summarized in Table I. Serum protein varied from 59.0 to 96.0 mg/nil, IgG from 11.2 to 63.2 mg/ml and IgM ~'rom 1.7 to 5.0 mg/ml. Serum IgG and IgM varied more than serum protein. Immuno. globulin levels were less variable in normal than in arthritic synovial fluids. TABLE I P r n t e i n T~C'. And ToM lovol~ in ~or~ ~nrl ~ynnvi~! Fhlid~ n f ~win~ w i t h l~.~elnol,~hr;~
rh usiopa thi,~ p o l y a r t h r i t i s J
Protein (mg/ml)
IgG (mg/m|)
IgM (rng/ml)
Serum
77.58 ±1.02 n = 12
24.74 ±9.30 n = 12
2.95 ±0.88 n = 12
Synovi~i f l ~ d s from histologically normal joints
23.40 +-9.50 n = 26
4.95 ±1.68 n ffi 17
0.33 ±0.10 n = 21
Synovial fluids from arthritic joints
31.80 ±8 . 8 0 n = 59
12.11 *-5 . 2 5 n = 44
0.53 ±0 . 2 4 n = 59
V a l u e s are s h o w n ± s t a n d a r d d e v i a t i o n .
IgG and IgM levels in synovial fluids expressed as percentages of companion serum levels are shown in Fig. 1. A summary of statistics for these immunoglobulins and for protein is given in Table II. A highly significant (P < 0.001) difference was detected between IgG percenl~ges in normal and arthritic synovial fluids. There was a smaller, but still significant difference (P ~ 0.05),
471
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120 110
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Fig. 1. Immunoglobulin levels expressed as percentages of companion serum concentration in groups of synovial fluids from histologically normal and arthritic joints. All fluids were collected from 12 swine in which E. rhusiopathiae polyarthritis had been exper;mentally p m d u c o d The vertical bars are standard errors of the mean; e, normal joints; o, arthritic joints.
between IgM percentages in normal and arthritic synovial fluids The difference for the protein percentages was highly significant, (P ~ 0.Co}l). ,~,ig~~ytwo percent of IgG estimations expressed as percentages of compan on ;~rum concentration'~ were significantly increased as compared with 25% c,f IgM estimations. Only 9% of corresponding protein percentages were significantly increased. (A significant increase was taken as one where arthritic values were increased by a figure equal to 2 standard deviations plus the mean of the normal group.) DISCUSSION
The mean serum concentrations of igG and IgM observed in this study were the same order of magnitude as reported by Porter (1.969) and by Curtis and Bourne (1971) for healthy swine. However, there was considerably more variation in both IgG and IgM concentrations than reported by these workers, probably because the group of swine in this work was chronically infected and was manifesting varying degrees of enhanced humoral response. This variation in serum immunoglobulin is an important reason for expressing immunoglobulin
472
TABLE H Protein, IgG and IgM expressed as percentages of companion serum levels in synovial fluids of swine with erysipelothrix polyarthritis
Protein Number of estimations Mean Standard deviation
Normal
Arthritic
21 28.00 8.57
44 40.90 11.34
17 23.10 5.69
44 46.10 16.64
21 10.86 5.95
59 19.59 7.35
Igo Number of estimations Mean Standard deviation
IgM Number of estimations Mean Standard deviation
Significance of differences (a) Normal vs arthritic IgG%, P < 0.001 (b) Normal vs arthritic IgM%, P < 0.05 (c) Normal vs arthritic protein, P < 0 . 0 q l Normal and arthritic synovial fluids were taken from the same group of swine.
levels in synovial fluids as percentages of companion serum concelebrations =ino~, fh,~ t, r m o n n t ~ t l r m
n f a nrnfJain in ~xrnr~vial fltlicl i~ rl~fiarmin~rl nnrt.lv
by its concentration in serum (Decker et al., 1959). Total protein is determined by the degree of synovial permeability present (Kushner and Somerville, 1971), so that one valuable measure of increased synovial permeability is ap increase in total protein concentration assuming there has not been recent release of locally synthesized prot~.~in such as immunog!obulins. The best controls on the non-specific effects of inflammation would be fluids from chronically irritated joints. Unfortunately, such fluids were not available for this study. Still another factor complicating the interpretation of individual protein levels in synovial fluids is that molecules entering the joint fluid can freely escape via lymphatic channels so that products of local synthesis are unlikely to build up bvyond plasma levels (Brown et al., 1969). Total protein expressed as a percentage of the companion serum level increased from 28.0 in normal to 40.9 in arthritic synovial fluids (Table II). This represents an increase of 46% and is doubtless mainly attributable to the greater permeability of the arthritic synovial barrier to plasma proteins. The increases in IgG and IgM over normal values were 100 and 61% r e s p e c t i v e l y more than could be accounted for by increase in total protein. It is unlikely that the greater increase in IgG was because the arthritic synovial barrier was
473 proportionately more permeable to smaller molecules since it is known that changes in permeability affect different molecular weight proteins to the same degree (Kushner and Somerville, 1971). Eighty-two percent of IgG values were increased by an amount equal to 2 standard deviations plus the normal mean as compared with only 25% of IgM and 9% of protein (expressed as percentages of companion serum concentrations). Furthermore, one synovial fluid contained over 120% of the companion serum concentration of IgG -- a level Which is highly suggestive of local synthesis. In Fi~ 1 i t ~ be seen that the number of IgG values which lay within the ~:~,ormal distribution was much less than the number of IgM, another indication that there was more IgG than IgM synthesis in arthritic joints. The occurrence of these "normal" IgG values in some joints is probably explained by the fact that the piece of synovial tissue examined to classify the joint may not have been representative of the general condition of the joint which may, in part, have either been mostly healed or even normal. It is interesting to note that studies in vivo on rheumatoid arthritis in man (Sliwinski and Zwaifler, 1970) on human rheumatoid synovial tissue (Smfley et al., 1968) also indicate that IgG is the dominant immunoglobulin synthesized. An important key to the problem of the pathogenesis of rhe~matoid arthritis in man and of ER arthritis in swine would be knowledge of the specificity of the immunoglobulins being synthesized in arthritic synovial tissue. One specificity of the IgM produced in both diseases is apparently that of rheumatoid factor which has been demonstrated in rheumatoid synovial tissue by Mellors et al. (1959) and in synovial f u i d of arthritic swine by Sikes et al. (1969) and Timoney (1971). In the latter instance, probably only a small part of the IgM has rheumatoid factor activity since titers tended to be low. The specificity of the large amounts of IgG synthesized in rhevmatoid synovium in man is unknown, although a part may have rheumatoid factor activity (Estes et al., 1973). In ER arthritis only a small proportion of plasma cells have been shown to contain antibody to ER (Timoney and Berman, 1970 a). However, Schulz et al. (1976) have found significan ~i~rs of antinuclear antibodies in synovial fluids from arthritic joints of swine, so perhaps some of the plasma cells in chronic synovial tissue are synthesizing antibodies to tissue components. ....
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REFERENCES
Ajmal, M., 1969. Erysipelothrix rhusiopathiae and spontaneous arthritis in pigs. Res. Vet. Sci., 10: 579--582. Browh, D.L., Cooper, A.G. and Bluestone, R., 1969. Exchange of IgM and albumin between pl~ma and synovial fluid in rheumatoid arthritis. Ann. Rheum. Dis., 29: 644--651. Cabral, J.R., Chevalier, H.J., Drommer, W., Mathies, P., Messow, C., Pohlenz, J., Schulz, L.Ci.~ Trautwein, G. and Ueberschar, S., 1968. Zur derseitigen Verlaufsform des ehronischen Rotlaufs bein Schwein. Teil II: Pathologische--anatomische,--histologische, elektronenmikroskopische. Dtsch. Tieriirztl. Wochenschr., 75: 5--23.
474
Collins, D.M. and Goldie, W., 1940. Observations on polyarthritis and on experimental Ery~ipelothrix infection of swine. J. PathoL, 50: 321--353. Curtis, J. and Bourne, F.J., 1971. Immuv+oglobulin quantitation in sow serum, colostrum and milk and the serum of young pigs. Biochim. Biophys. Acta., 236: 319--332. Decker, B., McKensie, B.F., McGucking, W.F. and Slocumb, C.H., 1959. Comparative distribution of proteins and glycoproteins of serum and synovial fluid. Arthritis Rheum., 2: 162--177. Estes, D., Atra, E. and Peltier, A., 1973. An immunofluorescent method for the detection of antigammaglobulin antibodies. Arthritis Rheum., 16: 59--65. Fahey, J.L. and McKelvey, E.M., 1965. Quantitative determination of serum immunoglobulins in antibody agar plates. J. Immunol., 94: 84--90. Kushner, L and Somerville, J.A., 1971. Permeability of human synovial membrane to plasma. Arthritis Rheum., 14: 560--570. Mellors, R.C., Heimer, R., Corcos, J. and Korngold, L., 1959. Cellular origin of rheumatoid factor. J. Exp. Med., 110: 875--886. Porter, P., 1969. Porcine colostral IgA and IgM antibodies of E. coli and their intestinal absorbtion by the neonatal piglet. Immunology, 17: 617--626. Pruzanski, W., Russell, M.L., Gordon, D.A. and Ogryzlo, M.A,, 1973. Serum and synovial fluid proteins in rheumatoid arthritis and degenerative joint diseases. Am. J. Med. Sci., 26 5: 483--490. Schulz, L.-CI., Drommer, W., Seidler, D., Ehard, H., Leimbeck, R. and Weiss, 1~,., 1976. Experimental Erysipelas in different species as a model for systemic connective tissue disease. II. The chronic phase with special reference to polyarthritis. Beitr. Pathol., 154: 27--51. Sikes, D., Fletcher, O.J. and Pan,p, E., 1967. Further studies on the pathologic alterations of tissues of swine given repeated whole blood trans~fusions from swine with rheumatoid like arthritis. Am. J. Vet. Res., 28: 1413--1425. Sikes, D., Crimmins, L.T. and Fletcher, O.J., 1969. Rheumatoid arthritis of swine; A comparative pathologic study of clinical spontaneous remissions and exacerbations. Am. J. Vet. Res., 30: 753--769. Sliwinski, A.J. and Zwaifler, N.J., 1970. In vivo synthesis of IgG by rheumatoid synovium. J. Lab. Clin. Med., 76: 304--310. Smiley, J.D., Sachs, C. and Ziff, M., 1968. In vitro synthesis of immunoglobulins by , .t. . . . . ~. ~i_,,7 rheumatoid synovial m- -e m- ~-r-m'~-. - - - - J , C | , ,"-. ......... ~.. : 62d.-...~.9,9+_ ..... Timoney Jr, J.F., 1971. Antibody and rheumatoid factor in synovia of pigs with Erysipelothrix polyarthritis. J. Comp. Pathol., 81: 243--248. Timoney Jr, J.F., 1976. Erysipelas arthritis in swine: Col~centrations of complement and third component of complement insynovia. Am. J. VeL. Res., 37: 5--8. Timoney Jr, J.F. and Berman, D.T., 1970a. Erysipelothdx arthritis in swine: Bacteriologic and immunopathologic aspects. Am. J. Vet. Res., 31: 1411--1421. Timoney Jr, J.F~ and Berman, D.T., 1970b. Erysipelothz~x arthritis in swine: Serumsynovial fluid gradients for antibody and serum proteins in normal and arthritic joints. Am. J. Vet. Res., 31: 1405--1409.
467
Elsevier Scientific Publishing Company, A m s t e r d a m - - Printed in The Netherlands
IMMUNOGLOBULINS IgG AND IgM IN SYNOVIAL FLUIDS OF SWINE WITH EBYSIPELOTHRIX POLYARTHRITIS JOHN F. TIMONEY and URIEL YARKONI
Department of Microbiology, New York State College of V~terinary Medicine, Corneli University, Ithaco~ N. Y. 14853 (U.S.A.) (Received 8 July 1976)
ABSTRACT rimoney, J.F. and Yarkoni, U.~ 1976. Immunoglobulins IgG and IgM in synovial fluids of swine with erysipelothrix polyartbritis. Vet. Microbiol., 1: 467--474. Concentrations of IgG and IgM immunoglobulins in synovial fluids and sera from a group of swine with experimentally produced Erysipelothrix rhusiopathiae polyarthritis were measured to determine if there was local synthesis of the~e immunoglobulins by plasma cells in arthritic synovial tissue. IgG and, to a lesser extent, IgM were significantly higher in arthritic than in normal synovial fluids from the same group of swine and this increase could only pa, tly be explained by the increased permeability of the arthritic synovial membrane to p|asms proteins. When synovial fluid values of IgG and IgM were calculated on the basis of companion serum concentration it was found that 82% of IgG, and 25% of IgM estimations were significantly elevated above levels in normal joints indicatirg that IgG was the dominant immunoglobulin synthesized.
INTRODUCTION
An interesting feature of chronic arthritis caused by Erysipelothrix rhusiopathiae (ER) in swine is the presence of large numbers of plasma cells in synovial tissue (Collins ~ d Goldie, 1940). It is reasonable to assume that at least some of the immunoglobulin synthesize~J by these cells finds its way into the synovial fluid and results in elevated concentrations. In the case of rheumatoid arthritis (RA) in man where there is also a heavy accumulation of plasma cells in synovial tissue, studies on IgG and IgM immunoglobulins in synovial fluids indicate local synthesis in arthritic joints, although some of the increase could be accotmted for by the non-specific effects of arthritis on the permeability of the synovial barrier to plasma proteins (Pruzanski et al., 1973). Furthermore, IgG was found to be increased more than IgM or IgA. In vitro studies have shown that RA synovial tissue was highly productive of IgG and to a much lesser extent of IgM and IgA (Smiley et al., 1968). Immunofluorescence studies on plasma cells in synovial tissue from swine with erysipelothrix arthritis have shown that some of these cells contain
468
antibody to a soluble extract of ER (Timoney and Berman, 1970 a). Schulz et al. (1975) have demonstrated IgG and IgM immunoglobulins in these cells. The extent to which the synthetic products of these plasma cells accumulates in the synovial fluid is, however, unclear. Sikes et al. (1967), Cabral et al. (1968), Ajmal (1969), Timoney and Berman (1970 b) and Timoney (1971) demonstrated significant titers of ER antibody in synovial fluids. However, the titers of many of the fluids examined by these authors were lower than in companion serum samples. Cabral et al. (1968) provided the best evidence of local a c c u m u l a t i o n - using the Wellman growth agglutination test, they were able to detect titers higher than in serum in about a third of synovial fluids examined. The work presented here concerns a quantitative study of IgG and IgM levels in the synovial fluids of arthritic swine in an attempt to resolve the question of local accumulation and to determine which immunoglobulins were being synthesized in greater amount in the arthritic synovial tissue. MATERIALS AND METHODS
Twelve Yorkshire swine aged about 6 months, in which polyarthritis had been produced as described prc,.dously (Timoney, 1976), were used as a source of synovial fluids and tissues. Fluids were collected at necropsy from the hip, stifle, hock, shoulder, elbow, and carpal joints and treated for 15 rain with 75 IU hyaluronidase in 0.1 ml saline per 1.0 ml fluid to reduce viscosity. They were then centrifuged at 2 000 g at 6°C for 30 rain and the supernatants were decanted and stored at--70°C. Cultures of the pellet were made on blood ~U~UI
saline. Joints were classified as normal or m,thntic on the basis of gross and
histopathologic criteria (Collins ~md Goldie, 1940). Because of limitations imposed by small fluid volumes it was not possible to perform all immunoglobulin assays on all samples. Sera were also harvested at necropsy and stored at --70°C. Antisera
Antiserum to porcine IgG was prepared by intravenous hyperimmunization of New Zealand white rabbits with complexes of porcine IgG and heat killed ER. The porcine IgG for this purpose was prepared from the serum of a pig with an agglutinin titer of 1 : 640 to ER. An IgG rich fraction was prepared by gel filtration of the serum on Sephadex 200 and by ion exchange chromatography on QAE Sephadex A 50. A mixture of the IgG preparation (4 rag/ ml), and ER (3 × 109 ceUs/ml) killed by heating to 60°C for I h, was then incubated at 37°C for 2 h to adsorb IgG ,antibodies to the surface of the bacteria. The complexes were then washed and centrifuged three times using phosphate buffered saline (pH 7.2) as washing agent. One tenth of a milliliter of the complexes containing 3 × 109 bacterial cells/ml was then inoculated every 3 days for 4 weeks. Ant!isem were harvested and tested for monospecificity
469
by reaction with whole pig serum after immunoelectrophoresis of the latter. Only antisera with anti-IgG activity alone were saved for further use. Antiserum to porcine IgM was prepared in guinea pigs inoculated in one fore and one hind footpad with 0.1 ml of a homogenate of IgM precipitin arcs mixed with complete Freund's adjuvant. These arcs were harvested by careful dissection following immunoelectrophoresis of an IgM rich fraction and reaction with mouse anti-pig serum. The IgM rich fraction was obtained following gel filtration on Sephadex 200 of the pig serum used as the source of IgG. The guinea pigs were bled 3 weeks after inoculation and their sera tested for monospecificity by reaction with whole pig serum after immunoelectrophoresis of the latter. The "arc harvesting" technique was effective in preventing contamination of the antiserum by antibodies to swine IgG and alpha 2 macroglobulins. Standards
An IgG standard solution was prepared by ion exchange chromatography of normal pig serum on QAE Sephadex A-50. The IgG fraction was concentrated in polyvinylpyrolidone (PVP), run through Sephadex G 200, and concentrated again in PVP to 16.2 mg/ml. The IgM standard solution was obtained by subjecting the IgM preparation used for the production of IgM precipitin arcs to two cycles of gel filtration on Sephadex 200 and concentration in PVP. During each gel filtration only the front shoulder of the first peak to e!ute w ~ h~_rvested. The !gM st~,mdo~d contained 1.25 mg protein/ral. Both standards were ,checked for contamination by immunoelectrophoresis and reaction with mouse antiserum to whole pig serum. The IgG standard had no detectable cont~unination; the IgM standard was contaminated with a barely detectable amount of alpha 2 macroglobulin. Since the only effect of this would be to cause a slight over estimation of the amount of IgM protein, no steps were taken to ~:emove it. Protein estimations
The biuret method was used to assay protein concentration in sera, synovial fluids and in IgG and IgM standard solutions. Radial immuno-diffusion assays
The procedure of Fahey and McKelvey (1965) was followed. Antisera were diluted 1 : 16. Standard curves for each assay were s~t up using IgG and IgM standard solutions appropriately diluted. All samples were assayed in duplicate and repeated when differences in excess of 10% between duplicate sample readings were observed. Percentages of companion serum concentrations of IgG, IgM and protein for each synovial fluid were calcu!ated from the serum
470
values for the pig from which the synovial fluids were collected. A Student's t test was used to determine the significance of the differences between normal and arthritic IgG, IgM, and protein percentages respectively in synovial fluids. RESULTS
There were 59 fluids from arthritic and 26 fluids from histologically normal joints of sufficient volume for examination. ER was isolated from 75% of the arthritic joints. Synovial fluid volume varied from about 0.5 ml in normal joints to as much a~ 30 ml in some arthritic joints. Effusions were more common in the stifle joint than in other joints but some arthritic joints had almost no fluid. Histological examination of arthritic joints revealed infiltration by large numbers of plasma and other mononuclear cells and increased vascularity, fibrosis, hypertrophy and palisading of synoviocytes. In contrast, histologically normal synovial tissue consisted of a loose stroma of areolar tissue with few blood vessels and one or two layers of synoviocytes. The results of protein IgG and IgM estimations are summarized in Table I. Serum protein varied from 59.0 to 96.0 mg/nil, IgG from 11.2 to 63.2 mg/ml and IgM ~'rom 1.7 to 5.0 mg/ml. Serum IgG and IgM varied more than serum protein. Immuno. globulin levels were less variable in normal than in arthritic synovial fluids. TABLE I P r n t e i n T~C'. And ToM lovol~ in ~or~ ~nrl ~ynnvi~! Fhlid~ n f ~win~ w i t h l~.~elnol,~hr;~
rh usiopa thi,~ p o l y a r t h r i t i s J
Protein (mg/ml)
IgG (mg/m|)
IgM (rng/ml)
Serum
77.58 ±1.02 n = 12
24.74 ±9.30 n = 12
2.95 ±0.88 n = 12
Synovi~i f l ~ d s from histologically normal joints
23.40 +-9.50 n = 26
4.95 ±1.68 n ffi 17
0.33 ±0.10 n = 21
Synovial fluids from arthritic joints
31.80 ±8 . 8 0 n = 59
12.11 *-5 . 2 5 n = 44
0.53 ±0 . 2 4 n = 59
V a l u e s are s h o w n ± s t a n d a r d d e v i a t i o n .
IgG and IgM levels in synovial fluids expressed as percentages of companion serum levels are shown in Fig. 1. A summary of statistics for these immunoglobulins and for protein is given in Table II. A highly significant (P < 0.001) difference was detected between IgG percenl~ges in normal and arthritic synovial fluids. There was a smaller, but still significant difference (P ~ 0.05),
471
,.I ILl b.I
120 110
..J
100
X G: tkl
90
(n Z C)
z
8O
70
o
<[
Q" =[
60
0
(~
50
o
tl.
o Z tM t,J r~,
£
40 30 2O
o
o o o o
• ee
0% °0% I#,,
2~
iX
10
Ig6
Ii IgM
Fig. 1. Immunoglobulin levels expressed as percentages of companion serum concentration in groups of synovial fluids from histologically normal and arthritic joints. All fluids were collected from 12 swine in which E. rhusiopathiae polyarthritis had been exper;mentally p m d u c o d The vertical bars are standard errors of the mean; e, normal joints; o, arthritic joints.
between IgM percentages in normal and arthritic synovial fluids The difference for the protein percentages was highly significant, (P ~ 0.Co}l). ,~,ig~~ytwo percent of IgG estimations expressed as percentages of compan on ;~rum concentration'~ were significantly increased as compared with 25% c,f IgM estimations. Only 9% of corresponding protein percentages were significantly increased. (A significant increase was taken as one where arthritic values were increased by a figure equal to 2 standard deviations plus the mean of the normal group.) DISCUSSION
The mean serum concentrations of igG and IgM observed in this study were the same order of magnitude as reported by Porter (1.969) and by Curtis and Bourne (1971) for healthy swine. However, there was considerably more variation in both IgG and IgM concentrations than reported by these workers, probably because the group of swine in this work was chronically infected and was manifesting varying degrees of enhanced humoral response. This variation in serum immunoglobulin is an important reason for expressing immunoglobulin
472
TABLE H Protein, IgG and IgM expressed as percentages of companion serum levels in synovial fluids of swine with erysipelothrix polyarthritis
Protein Number of estimations Mean Standard deviation
Normal
Arthritic
21 28.00 8.57
44 40.90 11.34
17 23.10 5.69
44 46.10 16.64
21 10.86 5.95
59 19.59 7.35
Igo Number of estimations Mean Standard deviation
IgM Number of estimations Mean Standard deviation
Significance of differences (a) Normal vs arthritic IgG%, P < 0.001 (b) Normal vs arthritic IgM%, P < 0.05 (c) Normal vs arthritic protein, P < 0 . 0 q l Normal and arthritic synovial fluids were taken from the same group of swine.
levels in synovial fluids as percentages of companion serum concelebrations =ino~, fh,~ t, r m o n n t ~ t l r m
n f a nrnfJain in ~xrnr~vial fltlicl i~ rl~fiarmin~rl nnrt.lv
by its concentration in serum (Decker et al., 1959). Total protein is determined by the degree of synovial permeability present (Kushner and Somerville, 1971), so that one valuable measure of increased synovial permeability is ap increase in total protein concentration assuming there has not been recent release of locally synthesized prot~.~in such as immunog!obulins. The best controls on the non-specific effects of inflammation would be fluids from chronically irritated joints. Unfortunately, such fluids were not available for this study. Still another factor complicating the interpretation of individual protein levels in synovial fluids is that molecules entering the joint fluid can freely escape via lymphatic channels so that products of local synthesis are unlikely to build up bvyond plasma levels (Brown et al., 1969). Total protein expressed as a percentage of the companion serum level increased from 28.0 in normal to 40.9 in arthritic synovial fluids (Table II). This represents an increase of 46% and is doubtless mainly attributable to the greater permeability of the arthritic synovial barrier to plasma proteins. The increases in IgG and IgM over normal values were 100 and 61% r e s p e c t i v e l y more than could be accounted for by increase in total protein. It is unlikely that the greater increase in IgG was because the arthritic synovial barrier was
473 proportionately more permeable to smaller molecules since it is known that changes in permeability affect different molecular weight proteins to the same degree (Kushner and Somerville, 1971). Eighty-two percent of IgG values were increased by an amount equal to 2 standard deviations plus the normal mean as compared with only 25% of IgM and 9% of protein (expressed as percentages of companion serum concentrations). Furthermore, one synovial fluid contained over 120% of the companion serum concentration of IgG -- a level Which is highly suggestive of local synthesis. In Fi~ 1 i t ~ be seen that the number of IgG values which lay within the ~:~,ormal distribution was much less than the number of IgM, another indication that there was more IgG than IgM synthesis in arthritic joints. The occurrence of these "normal" IgG values in some joints is probably explained by the fact that the piece of synovial tissue examined to classify the joint may not have been representative of the general condition of the joint which may, in part, have either been mostly healed or even normal. It is interesting to note that studies in vivo on rheumatoid arthritis in man (Sliwinski and Zwaifler, 1970) on human rheumatoid synovial tissue (Smfley et al., 1968) also indicate that IgG is the dominant immunoglobulin synthesized. An important key to the problem of the pathogenesis of rhe~matoid arthritis in man and of ER arthritis in swine would be knowledge of the specificity of the immunoglobulins being synthesized in arthritic synovial tissue. One specificity of the IgM produced in both diseases is apparently that of rheumatoid factor which has been demonstrated in rheumatoid synovial tissue by Mellors et al. (1959) and in synovial f u i d of arthritic swine by Sikes et al. (1969) and Timoney (1971). In the latter instance, probably only a small part of the IgM has rheumatoid factor activity since titers tended to be low. The specificity of the large amounts of IgG synthesized in rhevmatoid synovium in man is unknown, although a part may have rheumatoid factor activity (Estes et al., 1973). In ER arthritis only a small proportion of plasma cells have been shown to contain antibody to ER (Timoney and Berman, 1970 a). However, Schulz et al. (1976) have found significan ~i~rs of antinuclear antibodies in synovial fluids from arthritic joints of swine, so perhaps some of the plasma cells in chronic synovial tissue are synthesizing antibodies to tissue components. ....
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REFERENCES
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