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Background Inflammatory cell infiltration and synovial activation are important processes in rheumatoid arthritis. Chemotactic gradients of various chemokines are responsible for cell attraction and possibly for their activation. We have previously detected strong expression of chemokine receptor CXCR3 in the rheumatoid joint by immunostaining. Objectives Characterisation of the cells expressing CXCR3 in RA synovial membrane. Methods Synovial tissue samples were obtained from RA patients undergoing either synovectomy or a total joint replacement. Cells were released by digestion with collagenase, DNAse and briefly with hyaluronidase. A three colour fluorescence analysis was performed with FITC conjugated anti-CXCR3 mouse MAb (R&D) and with a panel of phycoerythrin (PE) conjugated MAb (anti-CD3, CD4, CD8, CD19, CD55, CD31, CD68, CD14 and CD45). Live cells were identified by propidium iodide. PBCs were stained using the same protocol. Results As expected, a proportion of CD3+ and CD4+ blood and synovial cells was CXCR3 positive. In addition, CXCR3 was also seen in synovial cells positive for CD55, CD14, CD8 and to a less extent CD31. However, in contrast to the surface staining of cells from peripheral blood, CXCR3 staining was identified only intracellularly in synovial cells. No CXCR3 staining could be detected on the surface of any type of viable synovial cell, including CD3 positive lymphocytes. Conclusion Flow cytometry identifies synovial cells that display intracellular CXCR3 staining. These cells are comprised of T lymphocytes, macrophages, possibly synovial fibroblasts and endothelial cell populations. The intracellular presence of CXCR3 suggests a possible internalisation of this molecule, which may be a consequence of ligand binding. The significance of this phenomenon and of CXCR3 expression in cell types other than leukocytes remains to be determined. This work was supported by a grant NI/6459 from IGA MZ CR.

Background Rheumatoid arthritis (RA) is characterised by a complex infiltration of many cell types including T and B lymphocytes, plasma cells, folicular dendritic cells, macrophages and other cells into synovial tissue of inflamed joints. Presence of B and plasma cells results in a local synthesis of multiple pathologic immunoglobulins contributing to the chronic joint inflammatory processes. Objectives To analyse mRNA from individual B and plasma cells isolated from synovium and peripheral blood of patients with RA, in order to investigate a clonality, VH and VL pairing and a molecular structure of produced antibodies. Methods RA synovial tissue after synovectomy was enzymatically digested. Single synovial B/plasma cells were sorted using immunofluorescent staining with anti-CD19, anti-IgM and anti-CD38. RA peripheral B cells were isolated in the same way without enzyme treatment. cDNA library from each single B and plasma cell was generated. Nested polymerase chain reaction was performed to analyse VH and VL gene usage. VH, DH and JH or VL and JL gene segments were assigned and somatic mutations determined by comparison to germline sequences on the V BASE/Genbank database. As a control peripheral B-lymphocytes from healthy donor were screened. Results Analysis of RA synovial mRNA revealed prevalence of C gamma rearranged transcripts that contained rearranged VH1, VH2, VH3, VH4, VH5 and VH6 genes. Utilisation of VH segments was similar between RA patients and normal subjects, but the accumulation of somatic mutations was elevated in RA synovial and peripheral B cells. Preferential utilisation of a limited number of VH and DH gene segments was found. There was an increased frequency of kappa light chains containing unusually long CDR3 when compared to normal peripheral B cells. mRNAs resulting from transcription of non-productive rearrangements were also detected as well as transcripts corresponding to isotype-switching. Conclusion Our findings are consistent with hypothesis that B cell response in RA synovium is probably antigen driven and oligoclonal. The project was supported by grant VS96129 from Czech Ministry of Education, Youth and Sport in the Czech Republic.

Inflammatory cell infiltration in synovium is a characteristic feature of rheumatoid arthritis (RA). Chemotactic gradients of various chemokines are responsible for cell attraction and possibly for their activation. Th1 cells are the predominant T cells in the synovium and have been shown to express high levels of chemokine receptors CXCR3 and CCR5.

Inflammatory cell infiltration and synovial activation are important processes in rheumatoid arthritis. Chemotactic gradients of various chemokines are responsible for cell attraction and possibly for their activation. We have previously detected strong expression of chemokine receptor CXCR3 in the rheumatoid joint by immunostaining.

Synovial tissue in rheumatoid arthritis displays a complex infiltration of many cell types like T and B lymphocytes, plasma cells, folicular dendritic cells, macrophages etc. Presence of B and plasma cells results in secretion of large amounts of multiple pathologic autoantibodies.