Scatiil. J. Inmmnot. 36, 733-743, 1992
Antigen-Activated T Cells Inhibit Cartilage Proteoglycan Synthesis Independently of T-Cell Proliferation B. WILBR[NK*§. M. H O L E W I J N t . J. W. J. BIJLSMA*, J. L. A. M. VAN ROY*, R. VAN DER ZEEf, C. J. P. BOOGf. W. DEN O T T E R i & W. VAN EDENf Departments of *Rhcumatology and ^Pathology, University Hospital, and tinstitute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht. The Netherlands
Wilbrink B. Holwijn M. Bijisma JWJ. Van Roy JLAM. Van der Zee R, Boog CJP, Den Otter W, Van Eden W. Antigen-Activaled T Cells Inhibit Cartilage Proteoglycan Synthesis Independently of T-Cell Proliferation. Scand J Immunol 1992:36:733 43 Previously we have shown that blood mononuclear cells (MNC) obtained from patients with rheumatoid arthrilis (RA) have the capacity to induce depletion of proleoglycans (PG) in human cartilage cxplants. This was observed especially after stimulating MNC wilh mycobacterial antigens, rather ihan with the miiogen C\mcanavali!i A (Con A). We have now co-cultured cartilage explants in the presence o! T-cell clone A2b obtained from ihe rat model of adjuvanl arthritis (A A). We show ihat inhibiiion of the cartilage PG synthesis is a consequence ol'anligenspeciltc T-ccll aclivation and that it is mediated by a humoral factor. This seems to be a cytokine rather than an enzyme. Moreover, at the level of polyclonally responding Tcells. inhibition ofPG synthesis due to T-ccll activation by mycobacterial antigens was shown to depend on prior mycobacterial immunization. Arthrilogcnic T-cell clone A2b also showed PG synthesis inhibiiory effects when co-cultured with cartilage alone. The inhibitory activity was shown to be unrelated lo Ihe degree of T-cell proli feral ion. We conclude that anligen-specilic T-cell activation may be one of the initiating events leading to cartilage damage in arthritic processes. The measurement of T-cell-mediated PG synthesis inhibition may be a more sensitive and relevant assay lor the detection of pathogenic T cells than T-cell proliferation. Profes.sor Dr J. W. J. Bijtsnui. Departmeni of Rttctimatologv. F non-essential amino acids. Antigens and aniihodies. The 65-kDa recomhinant protein [10], its epiiope with amino acid sequence 18018i< (nonapeptidc) asdelined by the arthritogenic T-ccll clone A2b [lO], and BP [20] were prepared as described. Monoclonal antibodies against the iji T-cell receptor (R73) [21]. a T helper lymphocyte marker (W3.25) [22], MHC class II B locus (0X6) [2.1]. or a B lymphocyte marker (0X33) were added (50/il/wcll), R73wasakind gift of Dr T. Hiinig. Munchen. Germany, The OX antibodies and W3/25 were kind gifts of the late Dr A. F, Williams, Oxford, tJK, Proliferation cissux for T-cell ciomw. Proliferative responses were measured for 18 h by the incorporation or['H]-lhymidini; (7,4 kBq well) in cells (2 x lO-'/wcll) cultured in restimulation medium (seeabove) for 4days in triplicate. Culture was performed in 96-well flatbottomed microtitre trays, in the presence of irradiated (3000 Rad) syngeneic ihymocytes (IO''/well) as accessory cells and various amounts of antigen with or without monoclonal antibodies. To test the blocking ability ofthe antibodies the cells were preincubated for 2 h before antigens were added. Cells were harvested on libreglass (iUers and incorporation was measured wilh a liquid scintillation counter. The mean cpm oTcultures in the presence of antigen (either or not wilh aniibodiesj divided by the mean cpm of the control cultures, i.e. in the absence ofantigen, is used as the stimulation mdex (SI), T-cell elone culiure supernalanls. Supernatants were harvested from parallel cultures after 48 h. One hundred //I of supernatant per well was harvested and transferred into wells of round-bottomed trays. To test the heat stability ofthe efl'ector molecules, the supernatants were subjected to 60 C for 30 min. Size exclusion HPLC was used to determine the molecular weight of the active component ofthe supernatant. Three ml of supernatant was lyophilized and redissolved In 200 /il PBS and fractionated on a TSK G2000SW column (Toya Soda, Japan) using PBS as elucnt at 1 ml/min. Fractions of I ml were collected and tested in a 50";, dilution on cartilage (100/il/well). Calibration of the column was done with protein standards (bovine pancreatic ribonuclease A. Mr= 13,7k and bovine serum albumin. Mr = 6}
Antigen-Activated T Cells Inhibit Cartilage Proteoglycan Synthesis Independently of T-Cell Proliferation B. WILBR[NK*§. M. H O L E W I J N t . J. W. J. BIJLSMA*, J. L. A. M. VAN ROY*, R. VAN DER ZEEf, C. J. P. BOOGf. W. DEN O T T E R i & W. VAN EDENf Departments of *Rhcumatology and ^Pathology, University Hospital, and tinstitute of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht. The Netherlands
Wilbrink B. Holwijn M. Bijisma JWJ. Van Roy JLAM. Van der Zee R, Boog CJP, Den Otter W, Van Eden W. Antigen-Activaled T Cells Inhibit Cartilage Proteoglycan Synthesis Independently of T-Cell Proliferation. Scand J Immunol 1992:36:733 43 Previously we have shown that blood mononuclear cells (MNC) obtained from patients with rheumatoid arthrilis (RA) have the capacity to induce depletion of proleoglycans (PG) in human cartilage cxplants. This was observed especially after stimulating MNC wilh mycobacterial antigens, rather ihan with the miiogen C\mcanavali!i A (Con A). We have now co-cultured cartilage explants in the presence o! T-cell clone A2b obtained from ihe rat model of adjuvanl arthritis (A A). We show ihat inhibiiion of the cartilage PG synthesis is a consequence ol'anligenspeciltc T-ccll aclivation and that it is mediated by a humoral factor. This seems to be a cytokine rather than an enzyme. Moreover, at the level of polyclonally responding Tcells. inhibition ofPG synthesis due to T-ccll activation by mycobacterial antigens was shown to depend on prior mycobacterial immunization. Arthrilogcnic T-cell clone A2b also showed PG synthesis inhibiiory effects when co-cultured with cartilage alone. The inhibitory activity was shown to be unrelated lo Ihe degree of T-cell proli feral ion. We conclude that anligen-specilic T-cell activation may be one of the initiating events leading to cartilage damage in arthritic processes. The measurement of T-cell-mediated PG synthesis inhibition may be a more sensitive and relevant assay lor the detection of pathogenic T cells than T-cell proliferation. Profes.sor Dr J. W. J. Bijtsnui. Departmeni of Rttctimatologv. F non-essential amino acids. Antigens and aniihodies. The 65-kDa recomhinant protein [10], its epiiope with amino acid sequence 18018i< (nonapeptidc) asdelined by the arthritogenic T-ccll clone A2b [lO], and BP [20] were prepared as described. Monoclonal antibodies against the iji T-cell receptor (R73) [21]. a T helper lymphocyte marker (W3.25) [22], MHC class II B locus (0X6) [2.1]. or a B lymphocyte marker (0X33) were added (50/il/wcll), R73wasakind gift of Dr T. Hiinig. Munchen. Germany, The OX antibodies and W3/25 were kind gifts of the late Dr A. F, Williams, Oxford, tJK, Proliferation cissux for T-cell ciomw. Proliferative responses were measured for 18 h by the incorporation or['H]-lhymidini; (7,4 kBq well) in cells (2 x lO-'/wcll) cultured in restimulation medium (seeabove) for 4days in triplicate. Culture was performed in 96-well flatbottomed microtitre trays, in the presence of irradiated (3000 Rad) syngeneic ihymocytes (IO''/well) as accessory cells and various amounts of antigen with or without monoclonal antibodies. To test the blocking ability ofthe antibodies the cells were preincubated for 2 h before antigens were added. Cells were harvested on libreglass (iUers and incorporation was measured wilh a liquid scintillation counter. The mean cpm oTcultures in the presence of antigen (either or not wilh aniibodiesj divided by the mean cpm of the control cultures, i.e. in the absence ofantigen, is used as the stimulation mdex (SI), T-cell elone culiure supernalanls. Supernatants were harvested from parallel cultures after 48 h. One hundred //I of supernatant per well was harvested and transferred into wells of round-bottomed trays. To test the heat stability ofthe efl'ector molecules, the supernatants were subjected to 60 C for 30 min. Size exclusion HPLC was used to determine the molecular weight of the active component ofthe supernatant. Three ml of supernatant was lyophilized and redissolved In 200 /il PBS and fractionated on a TSK G2000SW column (Toya Soda, Japan) using PBS as elucnt at 1 ml/min. Fractions of I ml were collected and tested in a 50";, dilution on cartilage (100/il/well). Calibration of the column was done with protein standards (bovine pancreatic ribonuclease A. Mr= 13,7k and bovine serum albumin. Mr = 6}
