LIMITING DILUTION ANALYSIS OF THE FREQUENCY OF AUTOREACTIVE LYMPH-NODE CELLS ISOLATED FROM MICE WITH ANTIGEN-INDUCED ARTHRITIS

Antigen-induced arthritis (AIC) in mice occurs after the single inject ion of methylated bovine serum albumin (mBSA) into the knee joint of a nimals preimmunized with the same antigen in complete Freund's adjuvan t. A short acute reaction is followed by a chronic inflammation which shows similar histological features to human rheumatoid arthritis. The mechanisms leading to the chronicity of arthritis are not yet clear. Previous data suggest that autoimmune responses to cartilage component s contribute to the persistence of arthritis. In the present study we estimate the frequency of autoreactive cells in the draining lymph nod es of arthritic mice by means of limiting dilution analysis. Graded co ncentrations of lymph node cells were stimulated for 7 days with type II collagen (CII) or cartilage proteoglycans (PG) in the presence of i rradiated syngeneic feeder cells, and the frequency of responding cell s was calculated. In the draining lymph nodes of immunized mice withou t arthritis induction, the frequency of CII reactive lymph node cells ranged from 1/41,182 to 1/57,424 whereas only 1 out of 3 experiments r evealed a detectable frequency of PG reactive cells (1/136,128). For c omparison, no CII or PG reactive cells were detected in the lymph node s of normal mice. Intra-articular challenge of immunized mice with mBS A resulted in chronic destructive arthritis and caused a significant i ncrease in the frequencies of CII and PG reactive lymph node cells (1/ 12,776 to 1/24,611 and 1/79,964 to 1/93,075, respectively). When using incomplete Freund's adjuvant for immunization, a comparable acute art hritis developed after the intra-articular injection of antigen, but t he transition into the chronic stage was missing. The frequencies of a utoreactive cells in the lymph nodes of these animals were below the l evel of detection and were judged to be below 1/150,000. The results s uggest that autoimmune reactions against cartilage constituents might contribute to the perpetuation of joint inflammation, and that the myc obacteria in the complete Freund's adjuvant play an essential role in this process. (C) 1996 Academic Press Limited