Studies on mononuclear cell adhesion to mucosal endothelial cells in rheumatoid arthritis.

The aim of the first part of the thesis was to develop and validate an in vitro adherence assay involving porcine mononuclear cells (MCs) and porcine endothelium, present within gut and lymph node. Factors involved in MC / endothelium interactions were determined. In summary we found that cell adhesion in our assay system was temperature, Ca2+ and Mn2+ sensitive, required metabolic activity, was inhibited by the phosphorylated monosaccharide galactose 6-phosphate, and unaffected by the presence of mucus. These findings reflected certain aspects of in vivo cell adhesion, present within the in vitro assay used. The adhesion characteristics of porcine Peyer' s patch (PP), peripheral blood (PB), and lymph node (LN) MCs to porcine gut and lymph node endothelium was examined and used as an guiding model for the future study of human MCs adherence. It was found that PP MCs adhered significantly better to gut endothelium than to LN endothelium and similarly LN MCs adhered significantly better to LN endothelium than to gut endothelium. PP MCs exhibited significantly greater levels of adherence to gut endothelium as compared to PB and LN MCs. The second part of the thesis was to apply the assay in order to study the adherence of MCs from patients with rheumatoid arthritis (RA) and to compare results with those obtained from porcine MCs. In summary we found that RA synovial fluid (SF) MCs exhibited adherence characteristics in keeping with those of PP MCs, with the adherence of RA SF MCs to gut endothelium significantly greater than to lymph node, and also significantly greater than RA synovial membrane (SM), PB and control PB MCs adherence to gut endothelium. This pattern of adhesion was maintained when adherence to human gut endothelium was also examined. The possible adhesion molecules involved were also studied by inhibition experiments using various monoclonal antibodies. The third part of the thesis, studied the in vitro effects of corticosteroid (methylprednisolone) treatment on cell adhesion, using porcine MCs. We then proceeded to study the effects of in vivo methylprednisolone (MP), given intravenously to RA patients. The effects on ex-vivo adherence to porcine gut endothelium showed on average a 58% inhibition of adherence of PB MCs, 24 hours following MP therapy. The question of whether this was a quantitative or qualitative effect was addressed by the immunophenotyping of circulating and adherent PB MC subsets. We observed no significant effect on the majority of circulating PB MC subset populations, except for the CD3+, CD4+ and CD45RA+ subsets, no significant effect on adherent subsets except for the HLA-DR+ subset was observed and additionally no significant effect upon the circulating levels of soluble VCAM-1 was observed. In conclusion, this thesis has shown a reliable and reproducible in vitro cell adhesion assay, RA SF MCs which share adhesion characteristics with porcine PP MCs and differ from porcine LN and RA SM MCs. In vivo corticosteroid treatment in RA patients which significantly reduces adhesion and strengthens the likely relevance of the assay in understanding adhesion properties and likely origin of MCs involved in the rheumatoid process.