The immune response to normal joint proteins probably plays a critical role in the joint destruction of rheumatoid arthritis. Many patients have detectable immunity to various connective tissue components and arthritis can be induced in rodents by immunizing them either with collagen or proteoglycans isolated from cartilage. The discovery that the antigen fragments that bind to MHC molecules and are recognized by T cells can be represented by, linear synthetic peptides has greatly facilitated the elucidation of the exact nature of antigen recognition. Localizing determinants of connective tissue with the capacity to generate immunogenic responses may give us a better understanding of the way in which arthritis is initiated and regulated.
The aims of this proposal include identifying determinants of type II collagen (CII) which are immunogenic. Two approaches will be taken to identify the structural requirements of antigens mediating both suppression and induction of arthritis. various peptides will be generated by a combination of cyanogen bromide and proteolytic cleavage of type II collagen and will be purified. Synthetic peptides (23-25 amino acid residues long) representing discrete regions of the molecule will be prepared. Collagen fragments and synthetic peptides will be used to tolerize mice prior to immunization to CII and observed for the subsequent suppression of arthritis. Fragments will also be emulsified with CFA and used to immunize DBA/1 mice which will subsequently be observed for the development of arthritis. Then T cell lines and clones reactive with determinants of CII will be isolated from spleens and lymph nodes of immunized and tolerized mice and tested for the ability to either induce tolerance and subsequent suppression of arthritis, induce arthritis itself, or alter established disease. Finally we will determine the active site of each immunogenic peptide using overlapping peptides and peptides having single amino acid substitutions and testing these peptides for activity using in vitro T cells proliferative assays and in vivo assays to determine whether they elicit the same immune response as the parent peptide. Elucidating the mechanisms by which tolerance to autoantigens can be induced and maintained has important implication for therapy of all autoimmune diseases.
