Human type II collagen is processed in lysosomal compartments of macrophages for presentation of the glycosylated arthritogenic epitope hCII_259–273to CD4 T cells in HLA-DR1 transgenic mice

Background

Post-translational modification of human type II collagen (hCII) in the form of hydroxylation of Pro and Lys residues and glycosylation of some hydroxylated Lys residues has been shown to correlate with hCII arthritogenicity in susceptible strains of mice [1, 2]. At the epitope level, O-linked glycosylation of Lys264 located within the arthritogenic region hCII_259–273 has been implicated in the creation of neoepitopes recognized by arthritogenic T cells [3]. Macrophages and to lesser extent primed B cells have been implicated in processing hCII for presentation of hCII_259–273 epitope to specific T cells [4, 5], whereas Langerhans dendritic cells are unable to process CII [6]. Macrophages may thus play a pivotal role in activation of autoreactive T cells during collagen-induced arthritis. However, no information is available on the mechanisms of antigen processing of the glycosylated arthritogenic epitope, although it is likely to be crucial for an understanding of the activation of autoimmune T cells in rheumatoid arthritis.

Objective

We investigated the mechanisms of intracellular processing of hCII for presentation of the glycosylated epitope hCII_259–273 to CD4 T cells in macrophages from HLA-DR1-transgenic mice.

Methods

HLA-DRB*0101 C57BL/6J0-0 transgenic mice (designated HLA-DR1-tg) were developed by backcrossing HLA-DRB*0101⁺Aq⁺ mice onto a MHC class II-deficient background. T-cell hybridomas specific for the glycosylated and non-glycosylated hCII_259–273 epitope were developed to study antigen presentation of the glycosylated epitope by bone marrow macrophages used as antigen-presenting cells. Subcellular fractions of macrophages were used as a source of enzyme activity to digest hCII at pH 4.5 in the presence and absence of enzyme inhibitors to localize stages of hCII degradation to particular endosomal/lysosomal compartments and to identify the families of enzymes involved.

Results

HLA-DR1-tg mice lacking mouse MHC class II were susceptible to collagen-induced arthritis. Macrophages from DR1-tg mice processed intact hCII for presentation of the glycosylated epitope hCII_259–274 to T-cell hybridomas. T-cell hybridomas specific for the glycosylated peptide did not cross-react with the non-glycosylated peptide. Intracellular processing of hCII for presentation of the glycosylated epitope was prevented by inhibitors of serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteinases or agents that raise endosomal pH, suggesting a requirement for extensive lysosomal processing. Lysosome-enriched subcellular fractions of macrophages were identified as the main organelles involved in processing and presentation of the glycosylated epitope from hCII, as these compartments contained: proteolytic enzymes of the serine-proteinase and cysteine-proteinase families that could generate the glycosylated hCII epitope; the glycosylated hCII epitope itself generated by intracellular processing of hCII; peptide-receptive HLA-DR1 molecules; and complexes of HLA-DR1 molecules with the glycosylated and non-glycosylated hCII_259–274 epitopes.

Conclusion

We showed stringent conditions for intracellular lysosomal processing of hCII for presentation of the arthritogenic glycosylated epitope by HLA-DR1 molecules to CD4 T cells, which may explain the lack of tolerance to glycosylated collagen and induction of arthritis in HLA-DR1-tg mice.