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Autoimmune arthritis can be induced in rats by immunization with cartilage-specific types II and XI collagen. Type XI collagen is composed of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains, in which alpha 3(XI) is essentially identical to alpha 1(II) of type II, and alpha 1(XI) and alpha 2(XI) are similar to alpha 1(V) and alpha 2(V) of type V collagen. To characterize the immune response to type XI collagen, Lewis rats were injected with bovine type XI collagen, and the cellular and humoral responses were compared with those of rats injected with types V and II collagen. Arthritis, IgG deposits in cartilage, and joint destruction were seen in rats immunized with types XI and II collagen. Type XI elicited strong cellular responses to rat types XI, V, and II; conversely, types II and V collagen elicited strong responses to rat type XI. Antitype XI Abs reacted with rat type XI, moderately with rat type V, but poorly with rat type II. Direct and inhibition ELISA showed that cross-reactions between types XI and V collagen resulted from recognition of determinants shared by their respective alpha 2(XI) and alpha 1(V) chains. Abs eluted from joints of rats immunized with type XI collagen, however, reacted only with native rat type XI collagen. These data demonstrate that type XI collagen induces diverse populations of Abs differing in collagen-type specificity, and suggest that only those Abs to native rat type XI collagen are central to the pathogenesis of type XI collagen-induced arthritis.

Chick type II collagen (CII), a protein commonly found in joint cartilage, induces an autoimmune arthritis when administered to susceptible strains of mice. A cyanogen bromide fragment of CII, CB11, contains the requisite epitopes critical for inducing collagen-induced arthritis. If administered as a tolerogen, however, before immunization, CB11 prevents the onset of disease. Therefore, delineation of structural elements of CB11 that can regulate autoreactive T cells became the goal of this study. To delineate the structural elements of CB11 antigenic to T cells, 14 peptides containing overlapping sequences of CB11 were generated. Mononuclear cells from CII-immunized DBA/1 mice were cultured with these peptides and the resulting supernatants examined for the production of IFN-gamma. Two peptides, CII 181-209 and CII 245-270, generated the greatest responses. The ability of these two peptides to regulate arthritis was tested by administering them to neonatal DBA/1 mice as tolerogens before immunization with CII. Both peptides suppressed the incidence of arthritis whereas no other peptide used as a tolerogen significantly altered the course of the disease. T cells from four arthritis-resistant murine strains did not recognize either peptide when immunized with CII, whereas cells from the disease-susceptible B10.Q mice responded well to both. Thus, the coincidence of T cell responses to CII 181-209 and CII 245-270 in CIA-susceptible mice and the lack of response in disease-resistant strains or CII-tolerized mice identify these two peptides as containing important T cell epitopes that regulate CIA.

The immunization of genetically susceptible strains of mice with type II collagen (CII) elicits a collagen-induced arthritis that resembles rheumatoid arthritis. Our laboratory previously identified a region of CII, CII-245-270, that contains a T cell epitope that is prominent in the immune response to CII. Residues critical to the I-Aq-restricted presentation of this determinant have been characterized. To produce synthetic peptides with the potential of disrupting I-Aq-restricted Ag presentation, synthetic analogue peptides were developed that contain site-directed substitutions in critical positions. One analogue peptide was found to be an efficient competitor for binding to I-Aq and to be capable of inhibiting T cell responses in vitro. When DBA/1 mice were coimmunized with CII and the analogue peptide, the incidence and severity of arthritis were greatly reduced, concordant with the humoral immune responses to CII.

A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.

To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.

Increased collagen deposition is a common feature of granulomatous and nongranulomatous inflammation associated with certain types of cell-mediated immune reactions in vivo. In the present study, we found that normal human peripheral blood mononuclear leukocytes cultured in vitro and stimulated by antigens or T cell mitogens release a 100 to 170 K m.w., heat-labile, trypsin-sensitive protein that stimulates dermal fibroblasts to produce increased quantities of type I and III collagens. Our data suggest that this collagen production protein is of T lymphocyte origin and that it preferentially stimulates production of collagen. We also observed that human mononuclear leukocytes release a different effector molecule with an m.w. of 55 K that inhibits fibroblast collagen production. Mononuclear leukocytes in culture are capable of releasing both the stimulator and the inhibitor of collagen production. The relative amounts of each of these factors elaborated by mononuclear leukocytes in culture appear to be influenced by several variables, such as cell density, type of stimulant used, and the duration of the culture period. These observations suggest that collagen production by fibroblasts in close proximity to sites in vivo where cell-mediated immune reactions are occurring might be regulated by both of these effector molecules.

The NH2-terminal sequence of type 1 M protein was determined by automated Edman degradation of purified polypeptide fragments extracted from whole streptococci by limited digestion with pepsin. Three polypeptide fragments were purified by slab gel electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide followed by electroelution. The purified fragments migrated as 28-, 25-, and 23.5-kDa fragments, respectively. Each of the fragments inhibited opsonization of a diluted antiserum prepared in rabbits by immunization with whole type 1 streptococci. The amino-terminal sequences of the peptide fragments were confirmed by comparison with the primary structure predicted from the nucleotide sequence of the type 1 M protein structural gene. The 28-kDa fragment contained the NH2-terminal asparagine residue of the processed type 1 M protein, whereas the NH2-terminal sequences of the 25- and 23.5-kDa peptides began at residues 27 and 36, respectively. A seven-residue periodicity with respect to polar and nonpolar residues was observed beginning at residue 22 and, therefore, the secondary structural potential of type 1 M protein is similar to that reported for other M proteins. In contrast to the other M proteins, however, identical repeats were rare, the longest sequence identity consisting of a three-amino acid acid sequence Lys-Asp-Leu at positions 30-32 repeated once at positions 65-67. A 23-residue synthetic peptide of the amino-terminus of the type 1 M protein evoked opsonic antibodies against type 1 streptococci. These results indicate that the NH2-terminal region of type 1 M protein retains the secondary structural characteristics of other M serotypes. Moreover, it contains epitopes that evoke protective immune responses. Our studies may have bearing in the development of safe and effective vaccines against group A streptococcal infections.

The protective immunogenicity of chemically synthesized copies of the NH2-terminal region of type 6 streptococcal M protein was investigated. Four overlapping peptides were synthesized by copying residues 1-20, 10-20, 12-31, and 22-31. Rabbit antisera raised against whole cells of type 6 streptococci reacted at high dilutions (1/12,800 to 1/51,200) with S-M6(1-20) and S-M6(10-20), and at low dilutions (1/100-1/800) with S-M6(12-31) and S-M6(22-31), indicating that the NH2-terminal region of type 6 M protein bears immunodominant epitopes. When covalently linked to tetanus toxoid and emulsified in complete Freund's adjuvant, the synthetic peptides S-M6(1-20), S-M6(10-20), and S-M6(12-31), but not S-M6(22-31), evoked type-specific opsonic antibodies against type 6 streptococci. Although the immune sera reacted in low dilutions by enzyme linked immunoabsorbent assay (ELISA) with the heterologous M protein polypeptides pep M5, pep M19, and pep M24, they failed to opsonize the streptococci from which these M protein polypeptides were derived. Each of the immune sera reacted in high dilution by ELISA with the respective immunizing peptides. All except those against S-M6(22-31) also reacted with pep M6. None of the immune sera reacted with human cardiac tissue by immunofluorescence or with muscle myosin by ELISA. The pattern of the inhibition of opsonization by each of the synthetic peptides of each of the immune sera indicates the presence of at least three protective epitopes in the NH2-terminal region of type 6 M protein. Our results indicate that the NH2-terminal region of type 6 M protein contains both protective and nonprotective epitopes, and chemically synthesized copies of this region lack cardiac tissue cross-reactive epitopes. These studies hold promise for the development of safe and effective vaccines against group A streptococci, especially against the strains giving rise to rheumatic fever and rheumatic heart disease.