Background Rheumatoid arthritis (RA) is the most common inflammatory arthritis. B and T lymphocytes play a central role in the pathophysiology of RA. RasGRP is a member of the CDC25 family of Ras guanyl nucleotide exchange factors. RasGRP1 is expressed in T and B cells whereas RasGRP3 is only expressed in B cells. In previous studies, we have shown that RasGRP3 expression level significantly decreased in Peripheral blood mononuclear cells (PBMC) from RA patients responders to adalimumab after 3 months, leading to the question of TNFα involvement in pathways including RasGRP1 and RasGRP3.
Objectives To study TNFα effects on RasGRP1 and RasGRP3 expression levels in vitro.
Methods We measured by qRT-PCR, RasGRP1 and RasGRP3 expression levels, i) in PBMC from 3 healthy controls (HC), ii) in negative selected B and T cells from PBMC isolated from 3 buffy coat. In each condition, cells were cultured with or without BCR or TCR stimulation for 4 days and TNFα was added for 24 or 48 hours. Immunofluorescence staining was performed to cheque the cell purity and B and T cells stimulation by flow cytometry. To test the functionals effects of RasGRP1 and RasGRP3 overexpression in T and B cells respectively, IL-2 production was measured by ELISA in T-cells, and Elk-1 expression level was measured by qRT-PCR in B cells before and after TNFα stimulation. In addition, TNFα effects on cell proliferation were evaluated by [3H] thymidine incorporation by the B and T cells.
Results In B cells, TNFα induced an increase of RasGRP1 (p < 0.001) and RasGRP3 (p < 0.001) expression levels in absence of BCR stimulation. In the same way, in T cells, TNFα induced an increase of RasGRP1 (p < 0.001) and RasGRP3 (p < 0.001) expression levels in absence of TCR stimulation. Furthermore, TNFα induced a significantly increase of IL-2 production (p < 0.05) in unstimulated T cells and of Elk-1 expression level (p < 0.01) in unstimulated B cells. However, TNFα have no effects on B and T cells proliferation.
Conclusions This study suggests the RasGRP1 and RasGRP3 regulation by TNFα, independently of B and T cells stimulation. The increasing of RasGRP3 and RasGRP1 in B and T cells specifically via TNFα binding on its receptors could promote the activation and proliferation of B and T cells by an independent antigen pathway. This second pathway could explain the maintenance of B and T cells activation.
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