The pathogenesis of chronic inflammation in experimental antigen-induced arthritis. I. The role of antigen on the local immune response
код для вставкиСкачатьarthritis and rheumatism Official Journal of The American Rheumatism Association Section of The Arthritis Foundation The Pathogenesis of Chronic Inflammation in Experimental Antigen- Induced Arthritis. 1. The Role of Antigen on the Local Immune Response T.Derek Cooke and Hugo E. Jasin Using the model described by Dumonde and Glynn in which a chronic synovitis is induced by the intraarticular injection of antigen in previously immunized rabbits, marked chronic local synthesis of immunoglobulin comparable to that of lymph nodes and spleen was found as long as 6 weeks after the arthritis was induced. Between 30 and 40% of this newly synthesized immunoglobulin was accounted for as specific antibody t o . the locally injected antigen. Very low levels of synthesis were found in the spleen, control synovia and regional lymph nodes not draining immunized sites. The circulating antibody levels at the time of induction showed a positive correlation ( I = 0.66) with the severity of the subsequent histologic findings in the synovium. The fate of the intraarticular antigen was studied in arthritic and control animals. There was a selective local retention of antigen in animals previously immunized with the homologous antigen. The retained intraarticular antigen was eliminated very slowly, with a half-life of over 20 days. These data indicate that the chronic synovial inflammatory response is associated with a chronic local immune response in which prolonged active synthesis of immunoglobulin and specific antibody directed against the locally retained inducing antigen takes place. The antigen-induced arthritis described in the rabbit by Dumonde and Glynn ( 1 ) has certain features in common with rheumatoid arthritis which suggest that it may serve as a sat- isfactory model for this disease. The synovitis is associated with a pronounced local response in which lymphocyte and plasma-cell infiltration is prominent (2); i t is chronic, often lasting From the Department of Internal Medicine, Rheumatic Diseases Unit, University of Texas Southwestern Medical School, Dallas, Texas This work was presented at the Interim Session of the American Rheumatism Association, January 8, 197 1 . This work was supported in part by National Institutes of Health Research Grant AM 09989 and Training Grant AM 05154. T. DEREK COOKE, MD: Traveling Fellow, McLaughlin Foundation, Ontario, Canada; HUGO E. JASIN, MD: Postdoctoral Fellow, The Arthritis Foundation; The University of Texas Southwestern Medical School, 5323 Harry Hines Blvd, Dallas, Texas 75235. Reprint requests should be addressed to: Dr. Hugo E. Jasin. Submitted for publication Nov 23, 1971; arrepted Jan 31, 1972. Arthritis and Rheumatism,Vol. 15, No. 4 (July-August 1972) 327 COOKE AND JASlN several months after a single injection of antigen into the joint (1-3). Glynn (2-4) has considered two mechanisms for the chronicity of the arthritis: a) a maintained local response to a persisting antigen and b) the induction of an immune response to an autoantigen (2, 3, 5), possibly a byproduct of the local inflammatory reaction. Active synthesis of immunoglobulin (Ig) has been demonstrated in vitro ( 6 ) and in vivo (7) in rheumatoid synovial membrane. It has been suggested that this represents an immune response to an as yet unidentified local antigen. The synovial response is believed to depend, in addition, on the formation of complexes between IgG and rheumatoid factors of both the IgG and IgM varieties (8-1 1). In view of the similarities noted between the antigen-induced arthritis and rheumatoid arthritis, it was of interest to characterize the antibody response in the inflamed rabbit synovial membrane, and to investigate the fate of the intraarticular inducing antigen as well as its role in the production of the local inflammatory response. The data reported here indicate that a local immune response takes place in the involved synovial membrane in which lymphoid infiltrates maintain intense, prolonged and largely specific antibody synthesis directed at the inducing antigen. They also show that the inducing antigen is selectively retained within the joint and eliminated at a very slow rate, suggesting a causal relationship between the persistence of antigen and the prolonged local synthesis of antibody in the synovial membrane. MATERIALS AND METHODS Animals. New Zealand-White rabbits of either sex weighing between 2.5 and 3 kg were used. They were fed regular Purina chow and water ad libitum. Materials. lC-amino acids (Schwartz-Mann BioRrsearch, Orangeburg, NY) consisted of a mixture of Larginine (125 mCi/mM), Lleucine (103 mCi/mM), L lysine (180 inCi/mM) and L-valine (143 mCi/mM) at a concentration of 250 pCi/ml for each of the four amino acids. Carrier and reductant-free lZsI was obtained from 328 New England Nuclear Corp, Boston, Mass. Egg albumin (EA), five times recrystallized, was obtained from Pentex Corp, Kankakee, Ill, and bovine serum albumin (BSA) from Armour Pharmaceutical Co, Chicago, 111. Dried Mycobacterium tuberculosis, H37 Ra, and incomplete Freund's adjuvant were both obtained from Difco Labs, Detroit, Mich. Rabbit anti-EA and anti-BSA antisera were obtained by methods previously described (12). Goat antiserum to rabbit immunoglobulins were obtained from Hyland Laboratories, Los Angeles, Calif. Induction of the arthritis. Rabbits were immunized with 2 ml of an emulsion of EA or BSA and complete Freund's adjuvant (CFA) in a ratio of 1:1 v/v containing 5 mg of EA or BSA and 2 mg of dried tubercle bacilli. The emulsion was injected into the footpads, glutei and multiple intradermal sites in the neck. Seventeen days later, the animals were challenged with 2.5 mg of the respective antigen in 0.5 ml of pyrogen-free saline solution (Ambot", Cutter Labs, Berkeley, Calif) sterilized by micropore filtration, injected into each knee joint under aseptic conditions through the patellar tendon. Control animals immunized in the same way were challenged 17 days later with 5 mg of soluble antigen intraperitoneally and 0.5 ml of saline in each knee. Preparation of tissues and culture procedure. Animals were sacrificed by exsanguination and air embolism at intervals of 2, 4, 6, or 8 weeks after intraarticular challenge. The areas to be dissected were carefully shaved and cleaned with alcohol. Using an aseptic technic, synovectomy was performed by vertically dividing the skin and soft tissues directly over the knee joint. The synovium was exposed by a transverse incision through the quadriceps retinaculum and patellar tendon. The synovial membrane, including the posterior compartment, was removed completely. The synovium was placed in freshly prepared, sterile modified Eagle's Minimum Essential Medium (MEM) and cut into pieces about 2 x 3 mm. The M E M lacked L-arginine, L-leucine, L-lysine and L-valine, added later as the radioactive amino acid mixture, and contained 5% inactivated normal rabbit serum, vitamins, glutamine and penicillin. Samples of synovium, 300 mg in wet weight, were cultured in 2 ml of M E M containing 10 pCi of IFlabeled amino acids. Cultures were incubated on shaker platforms (Micromix, Cordis Laboratories, Miami, Fla) at 37OC for 21 hours in 5 ml Petri dishes (Falcon Plastics, Oxnard, Calif) in an atmosphere of 95% air and 5%COz. Spleen, popliteal and axillary lymph nodes, obtained by aseptic dissection, were sliced and teased gently in MEM. The debris was allowed to settle and the cell suspensions collected and washed once in M E M by centrifugation at 600 g. Leukocyte counts were taken and the volumes adjusted to provide 5 x 10' nucleated cells. These were spun again, resuspended in 2 ml of M E M containing 10 pCi of lC-labeled amino acids and incubated in identical fashion Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) ANTIGEN-INDUCED ARTHRITIS. I to the synovial cultures. After the incubation period, the supernatants were removed a n d the remaining cells separated by centrifugation at 600 g for 10 minutes. Supernatants were centrifuged again at 77,000 g for 1 hour to remove cell debris, dialyzed against four changes of phosphate-buffered saline (PBS), p H 7.2, over a 36- to 48-hour period and stored at -20" C until assayed. Iodination of EA and BSA. N a Iz51 was used to iodinate EA by a modification of the ICI technic of McFarlane (13). IZ5I-BSA was prepared by the method of A. Taurog (14), using human thyroid peroxidase kindly supplied by Dr. Taurog. After dialysis of the free iodine, the radioactive antigens were at least 87% precipitable by their respective antisera. In vitro incorporation of "C-lobeled omino o c i d s into protein ond immunoglobulin. T h e thawed supernatants of synovial, lymph node and spleen cell cultures were centrifuged at 2000 g for 20 minutes and 50 rliter samples were removed for total radioactivity and trichloracetic acid (TCA) precipitable radioactivity determinations. Incorporation of radioactive amino acids into the tissue culture supernatant immunoglobulin was assayed in 0.1 ml aliquots by immune precipitation using goat antirabbit Ig in excess. T h e 5% rabbit serum in the M E M provided sufficient carrier protein to yield a precipitate of about 0.5 mg of protein. In vitro synthesis of anti-EA was assayed on a 0.1 ml aliquot of the radioactive supernatant by immune precipitation, using carrier EA and rabbit anti-EA at equivalence. T h e amounts of carrier antigen and antibody used were such that immune precipitates of about 0.5 mg were obtained. Nonspecific coprecipitable radioactivity was determined using an unrelated antigen-antibody system. In this procedure, sufficient BSA and anti-BSA to yield 0.5 mg of protein at equivalence were precipitated with a 0.1 ml aliquot of culture supernatant. T h e immune precipitates were incubated at 37" C for 2 hours followed by overnight incubation at 4" C. They were then centrifuged at 2000 g, washed three times with 1 ml of cold PBS and dissolved in 0.5 ml of 0.5 N NaOH. T h e samples taken for measurement of total radioactivity, T C A precipitable radioactivity and the radioactivity of the immune precipitates were transferred to glass vials to which 15 ml of Bray's phosphor containing a thixotropic gel (Cab-0-SiP, Packard Instruments Co, Downers Grove, Ill) were added. Radioactivity was measured with a Beckman scintillation spectrometer. T h e results were expressed as counts per minute (counts/min) after quench correction using the external standard technic. T h e small amounts of radioactivity nonspecifically coprecipitated by the BSA anti-BSA system, amounting from 2 to 5% of the total, were subtracted from the radioactivity measured in the immune precipitates. All the excised synovium, including the sample used for tissue culture, was dessicated to constant weight under vacuum, and the dry weights obtained. Histologic preparation of joint tissues. Specimens of synovium, cartilage and ligament were fixed in 10% buffered formalin solution. They were stained with hematoxylin and eosin (H and E). T h e histologic findings were graded blindly by one of us (HEJ) on a 0 to 4 point basis for the degree of a) lining layer hyperplasia and b) mononuclear cell infiltration. T h e maximum score for the two knees of each animal examined would therefore he 16 points. Induction of arthritis in robbits using '251-labeled ontigen. Groups of rabbits, immunized according to the scheme described above with either EA or BSA in CFA, were challenged 2 to 3 weeks later with bilateral IA injections of the respective antigen. Either i*51-labeled EA or BSA was injected into the right knees at the same time. The detailed schedule is outlined below. Rabbits were sacrificed at 2, 4 and 6 weeks after induction of arthritis. They received Lugol's solution in the drinking water 24 hours before injection and during the period in which IZ'I-antigens were being measured. Measurements of Iz5I-EA or I2jI-BSA tissue radioactivity. T h e animals were exsanguinated under pentobarbital anesthesia and killed by air embolism. Both knee joints were completely removed. Two milliliter volumes of minced tissue were counted in a Gamma-guard 150 Spertromatic well-scintillation counter (Tracerlab, Oakland, Calif). Counts per minute in the right knee tissue were corrected by subtracting the small number of counts per minute in the corresponding tissue from the left knee. External measurements of joint radioactivity and precordium, using a gamma scintillation external probe SPA-3 and Radiation Monitor R M 15 (Eberline Instrument Corp, Santa Fe, NM), were made at 1- to 3-day intervals after the injection of the '*jI-antigens. Measurement of circuloting anti-EA titers. These were measured by a modification of Farr's technic (15). Sera obtained just prior to IA challenge and at sacrifice were stored frozen at - 2 P until tested. Standard solutions of IZ5I-EA containing 6.25 pg EA (1 N/ml) were allowed to react with serial dilutions of the antisera and the gammaglobulin then precipitated by the addition of an equal volume of saturated ammonium sulphate solution. Serum antibody titers were expressed as the antigen binding capacity (ABC-33), the amount of antigen nitrogen bound per milliliter of undiluted serum, using as an end-point 33% precipitation of the antigen. Least square regression analysis was applied to ABC-33 serum titers and histology grades. RESULTS Histologic appearance of the arthritis induced by EA. T h e arthritis induced by intraarticular injection of 2.5 mg EA in saline solution in previously immunized rabbits was Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) 329 C W K E AND JASlN Fig 1. Histologic appearance of synovium obtained from a rabbit immunized with EA and injected IA with 2.5 m g EA in saline solution 4 weeks previously. Note the lining layer hyperplasia and the infiltration with mononuclear cells resembling a lymphoid follicle. Histology grade: 7 ( x 130). Fig2. Synovium of immunized rabbit 8 weeks after challenge with 2.5 m g EA intraarticularly. Note the cell aggregates composed predominantly of lymphocytes and plasma cells. Histology grade: 6 ( x 800). 330 Arthritis and Rheumatism, Vol. 15, No. 4 (July-August1972) ANTIGEN-INDUCED ARTHRITIS. I WEEKS AFTER INTRAARTICULAR CHALLENGE Fig 3. 14C-amino acid incorporated into Ig in 21 hour cultures of synovium. popliteal and axillary lymph nodes and spleen expressed as percent of TCA precipitable counts/min: 2,4 and 6 weeks after IA challenge. 0 = Saline-injected joints; W = Antigen-injected joints; illl = Spleen cell suspen= Lymph node cell suspensions. sions and Arthritis was induced by IA injection of 2.5 m g EA in rabbits previously immunized with EA in the four foot pads, glutei and neck. Control animals, also immune, were challenged with 5 m g EA intraperitoneally and IA saline solution. Figures in parenthesis represent number of animals in each group where all three tissues were cultured simultaneously. radioactivity. It can be seen that Ig synthesis by these inflamed synovial tissues comprised 15 to 30% of the newly synthesized TCA precipitable proteins released into the supernatant. It is also apparent that the synovial Ig synthesis compares well with that of the lymph nodes and spleen at 2, 4 and 6 weeks after challenge. In fact, at 6 weeks the percentages in these three tissues were similar in magnitude. It is of interest that the saline-injected control synovium synthesized some Ig, as shown by the small but reproducible radioactivity in the immunoprecipitates. Synovial cultures from two normal rabbits, not shown in the figure, did not synthesize any detectable Ig. When these data were expressed as counts per minute incorporated into Ig by all the syn- r "t 3.6 2.8 'f 2.4- 2 X ~ present in animals killed up to 8 weeks after the injection (Figures 1 and 2). It was characterized histologically by hyperplasia of the lining layer and infiltration of the deeper layers with lymphocytes and plasma cells. T h e cell infiltration was diffuse, but focal accumulations resembling lymphoid follicles were often seen (Figure 1). The joint fluids contained variable numbers of cells, with polymorphonuclear neutrophiles as the main cell type. T h e synovitis produced by IA injection of BSA in BSA-immunized animals was indistinguishable from the synovitis induced by EA in EA-immunized animals. In vitro Ig synthesis by synovium and lymphoid cells. Figure 3 shows the incorporation of '4C-amino acids into Ig by inflamed and control synovial tissues, popliteal and axillary lymph node and spleen from the same animals, expressed as percent of TCA precipitable 2.0- W .-Ec 0 1.6- \ c c s V ... A 0 1.2- 0.8- . 0 .. . -r tl 0.4 1 WEEKS AFTER INTRAARTICULAR CHALLENGE Fig 4. 14C-amino acid incorporation into Ig i n 21 hour cultures of synovium from immune rabbits obtained 2, 4 and 6 weeks after IA challenge with 2.5 m g EA or 0.5 m l saline solution. = Anttgeninjected and 0 = Saline-injected. The latter animals were challenged with 5 mg EA intraperitoneally. Results are expressed as c o u n t s h i n incorporated into Ig by all the synovium excised from each joint. Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) 331 COOKE AND JASIN pressed as percent of radioactivity incorporated into Ig is shown in Figure 5. At 2 , 4 and 6 weeks, between 30 to 40% of the Ig synthesized in the synovium was anti-Ea. In comparison, the control synovia and the spleen produced less than 5%. T h e popliteal and axillary lymph nodes, all of which were draining immunized paws, synthesized between 15 and 20% of Ig as specific antibody. 50r (71 (6) Influence of site of primary immunizat i o n o n p r o d u c t i o n of specific a n t i body. To examine this question, two rabbits were immunized in the paws, gluteus muscle and intradermally on one side only. T w o weeks later they were challenged in both knees with antigen. Tissues were obtained 4 weeks after inducing the IA injection. A marked difference in the production of specific antibody was found in the lymph node cultures of each side. T h e specific antibody synthesis in these tissues, expressed as percent of newly synthesized Ig, is seen in Figure 6. Both popliteal and the axillary lymph nodes draining the immunized paws produced anti-EA at similar levels. O n the unimmunized side, the lymph nodes were smaller and the cultured popliteal nodes did not synthesize detectable anti-EA. Specific antibody synthesis by spleen was also undetectable. However, the synovium from the knees of both immunized and unimmunized sides demonstrated marked synthesis of anti-EA at similar 2 WEEKS AFTER INTRAARTICULAR CHALLENGE Fig 5. 14C-amino acid incorporation into anti-EA in 21 hour cultures of synovium. lymph node and spleen cells expressed as percent of counts/min incorporated into Ig. = Antigen-injected joints; 0 = Saline-injected joints; Nl = Spleen cell suspension and El = Lymph node cell suspension. Immune rabbits were injected IA with 2.5 rng EA or saline solution 2. 4 and 6 weeks previously. Saline-injected animals were challenged with an equivalent dose of antigen intraperitoneally. ovium excised from the joints (Figure 4), fourfold to eightfold greater mean incorporation was observed in the antigen-injected joints compared with the saline-injected joints. It is again apparent that inflamed synovial tissues continued to synthesize Ig very actively 6 weeks after induction of the arthritis. Specific antibody synthesis by synovium and lymphoid cells. T h e radioactive amino acid incorporation into anti-EA antibody, ex50 r Immunized Side I ; Nonimmunized I Side I ! a W 30 kg a .-c 20 E \ \ - : ; lo s* 0 1 Synovium 1 I Pop1I t eal 332 t Axlllary Synovium 1 t Fig 6. In vitro 14C-amino acid incorporation into anti-EA by synoviurn, spleen and lymph node cells expressed as percent of counts/min incorporated into Ig. W = Antigen-injected joints; F 3 = Lymph node cell suspensions and = Spleen cell suspensions. Two rabbits were immunized unilaterally with EA in CFA and subsequently challenged in both knees with 2.5 rng EA in saline solution. The tissues were obtained 4 weeks after the I A injection. Spleen Pop1 Iteal Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) ANTIGEN-INDUCED ARTHRITIS. I Table 1. Sum of Histologic Grades of Synovitis in Right and Left Knee after the lntraarticular Injection of EA* Duration of arthritis (wk) 2 4 6 No. of animals 8 10 4 Average grade 7.8 7.3 4.5 Range 6-11 2-10 3-6 "2.5 m g injected into each joint levels. These findings indicate that, although injection of antigen with CFA is a prerequisite for the prolonged synthesis of anti-EA by the local lymph nodes, the occurrence of synovitis and its persistence is dependent on systemic immunity, as well as the intraarticular deposition of inducing antigen. It appears to be independent of the immune response in the adjacent lymph nodes. Correlation of serum antibody level with severity of arthritis. In an attempt to correlate the initial serum antibody response with the severity of the arthritis, sera obtained just before intraarticular challenge with antigen were assayed for anti-EA titers by the ammonium sulphate precipitation technic, using radioactive EA. These were plotted against the sum of the histology grades assigned to the right and left knees of each rabbit. Since the histologic examination showed a decrease in average severity of arthritis 6 weeks after induction (Table 1 ) and the anti-EA titers were all determined immediately prior to intraarticular challenge, a linear relationship between these parameters would not be expected. Therefore, only the 18 rabbits with arthritis of 2 and 4 weeks duration were included. When the values at these time intervals were correlated with serum antibody levels at the time of indyction of arthritis, a positive correlation ( r = 0.66) and a statistically significant straight-line relationship ( P < 0.005) were obtained (Figure 7). The fate of '251-labeledantigen. T w o groups of rabbits immunized with either BSA (experimental) or EA (control) were challenged in both knees 3 weeks later by the IA injection of the respective inducing antigen. T h e experimental group received 2.5 mg I2jI-BSA containing 15 x lo6 counts/min in the right knee and the control group received 2.5 mg EA plus 15 x 106 counts/min of '251-BSA. In addition, 2.5 mg 1251-BSA(28 x lo6 counts/min) was injected into both knees of two normal, nonimmune rabbits. Using the external probe, the percent of initially injected radioactivity retained in the right knees was measured and plotted against time. In Figure 8 are seen the elimination patterns of Iz5I-BSA in these groups of rabbits. T h e early patterns are similar in the normal, nonimmune and control group with an EA induced synovitis. There is an initial rapid decrease in radioactivity, lasting for about 5 days, which probably represents equilibration with the rest of the body. T h e experimental group, preimmunized and injected locally with BSA, showed a more rapid initial decrease, reaching approximately 0.25% of the initial radioactivity by 5 days. At this time, the normal I0 ?? 2l L I 100 I 200 I 300 I 400 I 500 ANTIGEN BINDING CAPACITY (pgN1 Fig7. Positive correlation (r = 0.66; P < 0.005) of antigen-binding capacity of sera (ABC-33) of individual rabbits prior to IA challenge with antigen and subsequent severity of arthritis at 2 and 4 weeks assessed by the added histology grades from both knees. Lining layer hyperplasia and mononuclear cell infiltration each graded 0 to4. Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) 333 COOKE AND JASlN 100.0 r Fig 8. Elimination curves of IA lZ51-BSA in immune arthritis and control rabbits obtained with an external probe. Experimental rabbits (BSA-immune) -A-: arthritis was induced with 2.5 m g BSA bilaterally. The r i ht knees were injected w i t h 1'51-BSA (15 x lo6 counts/min). Control arthritic rabarthritis bits (EA-immune) ---Or--: was induced with 2.5m g EA bilaterally. I n addition. the right knees were i n j e c t e d w i t h (15 x lo6 counts/min) lZ51-BSA. Normal rabbits (nonimmune) - 0 - injected only with 2.5 ~TI~"~I-BSA (28 x 1o6counts/min). I 0.001; 2 3 4- WEEKS and control rabbits knees retained four times this amount. T h e difference presumably represents immune elimination in the rabbits preimmunized to BSA. After the initial 5-day period, the unimmunized and control animals with EA-induced arthritis then eliminated I2jI-BSA at a steady and comparable rate. Between 10 to 14 days however, a n abrupt change in slope was seen which coincides with the development of the expected primary immune response to BSA. This response decreased the remaining articu- lar antigen to lower levels than those remaining in the experimental group, and was followed by a very slow, constant rate of elimination. A similar slow component in the experimental group began immediately after the initial rapid phase. Sufficient radioactivity remained in the experimental group to allow computation of the half-life of the remaining intraarticular antigen. T h i s was estimated to be 20 days or more. A reciprocal, but otherwise identical, experiment was carried out in EA preimmunized Table 2. Retention of lz5I-BSA in Joints of Normal or Preimmunized Rabbits lz5I-BSA in iected Immunizing antigen BSA EA BSA EA - Weeks after lntraarticular intraarticular Counts/min antigen injection x 106 BSA EA BSA EA BSA 2 2 4 4 4 15 15 15 15 28 lZ51-BSAretained Cou nts/min 32.930" 12.120" 24.600 4,310 9.200-t Countshnin (BSA) 010 0.220 0.094 0.165 0.029 0.033 Countshnin (EA) 2.3 - 5.7 - *Average counts/min for the right knees of two rabbits ?Average counts/min for both knees of two rabbits 334 Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) ANTIGEN-INDUCED ARTHRITIS. I Table 3. Retention of 1251-EA in Arthritic Joints of Immunized Rabbits Induced Previously by EA or BSA. Both Groups Received lz5I-EA* in the Right Knees I mrnunizing antigen Inducing antigen EA BSA EA BSA EA BSA EA BSA Duration of arthritis (wks) 2 2 4 4 1251-EAretained Counts/min (EA) Cou nts/mi n t Counts/min (BSA) 24,350 4,970 21,250 2.900 4.9 7.1 ”30 x 106counts/min injected tAverage counts/min of the right knees of two rabbits animals by injecting 30 x lo6 counts/min of I2jI-EA into knees of rabbits with EA (experimental) and BSA (control) arthritis. T h e elimination patterns of 1251-EA by external monitoring were similar to the first experiment. Selective retention of radioactive antigen. Selective retention of radioactive antigen in rabbit knees with a synovitis induced by the same antigen was confirmed when the rabbits were sacrificed, the knee joints removed and their radioactivity determined. After injection of radioactive BSA (Table 2), the joints of rabbits immunized to BSA retained 2.3 and 5.7 times as much antigen as the control rabbits with arthritis induced by EA and injected with 12’I-BSA. T h e normal, nonimmune rabbits sacrificed 4 weeks after the IA injection retained 0.03% of the initial dose, a level similar to that measured in control arthritic joints. T h e results using l2jI-EA are seen in Table 3. Again, marked retention of radioactivity is evident in the joints of rabbits immune to EA; 4.9 and 7.1 times as much 1251-EAwere retained at 2 and 4 weeks than in the BSA inducedjoints, which also had been injected with 12’I-EA. DISCUSSION In this investigation, a number of aspects of an immunologically-induced, chronic inflammatory reaction have been studied in the antigen-induced a r t h r i t i s of D u m o n d e a n d Glynn (1,4). T h e arthritis was induced by in- jection of EA or BSA into the knees of rabbits previously immunized with either antigen in CFA. T h e experiments were designed to quantitate immunoglobulin and specific antibody synthesis concurrently in synovium, lymph nodes and spleen. These were measured by immune precipitation of 14C-amino acid-labeled immunoglobulin and anti-EA synthesized in in vitro cultures of these tissues. T h e method utilized has been previously described from this laboratory (6, 12). T h e synovitis induced was associated with an intense and prolonged synthesis of immunoglobulin for as long as 8 weeks after induction, the longest interval tested. This finding was consistent with the prominent infiltration of the synovial m e m b r a n e w i t h lymphocytes a n d plasma cells. In the EA-injected joints, anti-EA comprised 30 to 40% of the total Ig synthesized throughout the 6-week observation period. Anti-EA synthesis in these joints made up a much larger fraction of the total Ig synthesized than observed in control joints and spleen. Prolonged synthesis of large amounts of specific antibody, directed at the locally injected antigen, provided indirect evidence for the persistence of the antigen. Control cultures of synovium from immunized animals which were not challenged intraarticularly, synthesized small but reproducible amounts of Ig and, on occasion, small amounts of specific antibody. T h i s is consistent with the observation of Jasin and Ziff (12) who Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) 335 COOKE AND JASlN demonstrated synthesis of antibody to intravenously-injected BSA in t h e synovial membrane of an animal injected intraarticularly with the nonspecific mitogens streptolysin-S and phytohemagglutinin. Moreover, the synovium from saline-injected joints also demonstrated mild inflammatory changes as long as 6 weeks after IA injection. It is possible that the mild acute inflammatory response produced by the saline solution may have attracted antigen and immunocompetent cells to the joint, leading to the accumulation of cells synthesizing specific antibody to the intraperitoneally injected EA. In unilaterally primed rabbits, anti-EA synthesis in lymphoid tissue occurred only in lymph nodes from the immunized side, confirming previous work by Askonas and White (16). In contrast, synovial synthesis of anti-EA was similar on both immunized and unimmunized sides, indicating that the accumulation of antibody producing cells in the synovium was not influenced either by the site of immunization or the immune status of the adjacent lymph nodes and depended only on the systemic immunity of the animals. T h e level of the circulating antibody present at the time of intraarticular challenge appeared to influence the severity of the subsequent arthritis, since a positive correlation was found between pre-intraarticular challenge serum antibody levels and the degree of subsequent inflammation. It would appear from this that the severity of the synovitis was related to the magnitude of the humoral antibody response. Glynn (2) has emphasized a requirement for the presence of delayed hypersensitivity to the immunizing antigen for the development of the arthritis on the basis of a) the necessity for immunization with CFA and b) the correlation of the arthritis with a positive delayed skin reaction to the antigen. T h e present findings provide evidence that the humoral antibody response also plays a direct role. T h e finding of prolonged specific antibody synthesis in the inflamed synovial cultures supports the concept that a local immune re336 sponse is being maintained by persisting antigen. Webb, Ford and Glynn (17) have shown that systemic immunization of rabbits up to 4 weeks after intraarticular injection of antigen results in the appearance of a chronic synovitis in the antigen-injected joints. Studies in a subsequent paper (18), describing the retention of antigens in this model of arthritis and the site of their localization, indicate that persisting antigen may indeed be directly responsible for the chronicity of the arthritis. T h e fate of '251-labeled antigen in normal and arthritic rabbits was markedly influenced by the immune status of the animal at the time of joint challenge, since animals immune to the labeled antigen retained it in greater amounts than control rabbits with arthritis induced by another antigen or normal rabbits. Similar results were recently reported by Consden et a1 (4); intraarticular retention of EA was increased in preimmune rabbits. This suggests that intraarticular antigen interacts with antibody to form complexes which are retained in the joint. A similar mechanism has been found to operate in germinal centers of lymph nodes of immunized animals where antigen and antibody, presumably in the form of complexes, are r e t a i n e d f o r c o n s i d e r a b l e p e r i o d s of time (19-21). T h e role of antibody would also be compatible with the observed positive correlation between severity of arthritis and preexisting serum antibody levels. A similar pathogenesis may underlie the chronic arthritis produced in piglets by injection of live Mycoplasma hyorhinis (22). In this arthritis, inflammatory activity continues long after the disappearance of viable organisms, suggesting that antigen may be sequestered locally. In this arthritis, synovial fluid antibody titers were frequently higher than in serum, indicating that a local immune response to the Mycoplasma antigens was also present. Following IA injection of antigen, there was a rapid phase of immune elimination, thereafter, the remaining intraarticular antigen was eliminated at a very slow rate. In the immune Arthritis and Rheumatism, Vol. 15, No. 4 (July-August 1972) ANTIGEN-INDUCED ARTHRITIS. I rabbits, the half-life of the intraarticular antigen was estimated to be greater than 20 days. These findings provide support for the concept that the prolonged local immune response may be a consequence of the persistence of antigen. In another paper (18), evidence is provided that the antigen is retained in the form of immune complexes bound with complement components. This indicates a dual role for the intraarticular antigen: a) as a stimulus for the prolonged local antibody synthesis and b) as a source of antigen-antibody complexes mediating continuous inflammatory activity. REFERENCES 1. Dumonde DC, Glynn L E : The production of arthritis in rabbits by an immunological reaction to fibrin. Br J Exp Pathol43:373-383, 1962 2. Glynn LE: T h e chronicity of inflammation and its significance in rheumatoid arthritis. Ann Rheum Dis27:105-121,1968 3. Glynn L E : Aetiology of rheumatoid arthritis with regard to chronicity. Ann Kheum Dis (Suppl) 28:3-4. 1969 4. Consden R, Doble A, Glynn LE, et al: Production of a chronic arthritis with Ovalbumin. Its retention in rabbit knee joints. Ann Rheum Dis 30:307-3 IS, 197 1 5. Phillips J M , Kaklamanis P, Glynn L E : Experimental arthritis associated with autoimmunization to inflammatory exudates. Ann Rheum Dis 2s: 165-174, 1966 6. 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Cooke T D , Hurd ER, Ziff M, et al: T h e pathogenesis of chronic inflammation in experimental antigen-induced arthritis. 11. Preferential localization of antigen-antibody complexes to collagenous tissues. J Exp Med 135:323, 1972 19. Ada GL, Nossal GJV, Austin CM: Antigens in immunity. IV. Cellular localization of 12jl and l 3 I I labeled flagella in lymph nodes. Aust J Exp Biol Med Sci 42:311-330, 1964 20. White KG, French V, Stark J M : Germinal center formation and antigen localization in Malpighian bodies of the chicken spleen. Germinal Centers in Immune Responses. Edited by H. Cottier a n d W . O d a r t c h e n k o . New York, Springer Verlag Inc, 1967, pp 131-144 21. Sordat B, Sordat M, Hess ,MW, et al: Specific antibody within lymphoid germinal center cells of mice after primary immunization with horseradish peroxidase: a light and electron microscopicstudy. J Exp Med 131:77-91, I970 22. Barden JA, Decker J L : Mycoplasma hyorhinis swine arthritis. I. 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