Background/Purpose: B cells are recognized as key players in the pathogenesis of rheumatoid arthritis (AR) through the production of autoantibodies, the local production of proinflammatory soluble factors and, when acting as antigen-presenting cells, the regulation of T-cell functions. Experimental evidence suggests that B cells should migrate to and accumulate within the synovial microenvironment to exert their pathogenic action in RA. However, little is known about the driving force responsible for the recruitment of B cells in the rheumatoid synovium. Chemokines and their receptors expressed in leucocytes help to control the selective migration and activation of inflammatory cells in the inflamed synovium. Objective: to determine the chemokine or chemokines responsible for the recruitment of B cells in the rheumatoid synovium.

Methods: Surface expression levels of CD27, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CXCR2, CXCR4, CXCR5 and CXCR7 were assessed by double- or triple-staining flow cytometry analysis in CD20+ mononuclear cells isolated by Ficoll-gradient from the peripheral blood (PB) and synovial fluid (SF) of 15 RA patients. In a sample of patients, the total expression (intra- and extracellular) of specific chemokine receptors was analyzed by flow cytometry in CD20+ cells PB and SF permeabilized mononuclear cells from PB and SF. Transwell experiments were used to study the synergism and migration capabilities of negatively immunoselected PB B-cells from normal donors or in CD20+ mononuclear cells from PB and SF of RA patients in response to a single chemokine or a mix of two chemokines.

Results: B cells from the SF of RA patients showed a significant increase in the surface expression of CCR1 (2.1± 0.1-fold), CCR2 (2.4±0.2 fold), CCR4 (6±2 fold), CCR5 (2±0.1 fold) and CXCR4 (2.5±0.8-fold) with respect to PB. Remarkably, SF B cells expressed consistently lower amounts of CXCR5 (0.15±0.05 fold, p<0.01), CXCR7 (0.7±0.1 fold, p<0.05) and CCR6 (0.75±0.1 fold, p<0.05) with respect to PB. This differential pattern of chemokine receptor expression was not modified by the previous contact of B cells with antigen, as assessed by CD27 expression levels. Flow cytometry results showed a relative increase in the expression of CXCR5 and CCR6 in permeabilized CD20+ cells from SF compared to those from PB, which suggests that both receptors undergo cell internalization in B cells that migrate to the rheumatoid synovium. In Transwell experiments, MIP-3α and BCA-1, ligands of CCR6 and CXCR5, respectively, caused a significantly higher migration on B cells from PB than in those from the SF of RA patients. Together, the two chemokines synergistically increased B cells migration from PB but not from SF.

Conclusion: B cells present in the synovial microenvironment of patients with RA down-regulate the surface expression of CXCR5, CCR6 and CXCR7 via an internalization mechanism. Individually, BCA-1 and MIP-3α show higher chemoattractant effects in B cells from PB versus those from SF, while together they exert a synergistic effect in B cells from PB, but not from SF. These results suggest that BCA-1 and MIP-3a might play major roles in RA pathogenesis by acting synergistically in the accumulation of B lymphocytes within the inflamed synovium.