Ligation of antigen to immunoglobulin receptor BCR induces activation and proliferation of mature B cells and apoptosis of immature B cells. Stimulation of BCR activates signal pathways in cells that induce transcriptional reprogramming. One of the genes, whose expression is highly induced, is also Ptger4, that codes for prostaglandin receptor EP4. Endogenous ligand for this receptor, PGE2, is a well-known immunomodulator. Ligation of PGE2 on EP4 receptor activates cAMP-dependent and independent pathways that promote modulation of BCR induced phenotypic response of B lymphocyte. Activation of BCR induces interleukin 6 gene expression, which has CREB binding site in promotor region of the gene. We investigated the role of receptor EP4 in modulation of BCR induced interleukin 6 gene expression. The experimental work was based on pharmacological treatment of WEHI 231 cell line. We stimulated the cells with agonists, antagonists and inhibitors of signal pathway components. Measuring the interleukin 6 gene expression after stimulation of EP4 receptor and BCR helped us to determine how both receptors modulate the expression of the fore mentioned gene. Besides that we also measured the excretion of interleukin 6 to cell media with cytometric cytokine assay. Using the enzyme-linked immunoassay we measured the concentration of cAMP after ligation of EP4 receptor. The function of EP4/cAMP dependent signal pathways in modulation of BCR induced interleukin 6 gene expression was determined by using relevant inhibitors of adenylate cyclase and protein kinase A. In the last part of our research work we determined the effect of interleukin 6 on proliferation of WEHI 231 cell line using the MTS method.
The results clearly show that stimulation of WEHI 231 cells with PGE2 raises BCR induced interleukin 6 gene expression. Using specific EP4 receptor agonist and antagonist we proved, that PGE2 mediates its effects mainly through EP4 receptor. As ligation of EP4 receptor without simultaneous ligation of BCR does not induce interleukin 6 gene expression, we conclude, that ligation of EP4 receptor modulates BCR induced interleukin 6 gene expression. The stimulation of EP4 receptor raised intracellular production of cAMP. By inhibiting the enzyme adenylate cyclase and protein kinase A we managed to completely diminish the effects of EP4 receptor stimulation on BCR induced interleukin 6 gene expression. We summarize, that stimulation of EP4 receptor modulates BCR induced interleukin 6 gene expression via cAMP/PKA dependent signal pathway. Most probably this pathway leads to activation of CREB nuclear factor. Excreted interleukin 6 stimulates proliferation of WEHI 231 cells. Despite the rise in interleukin 6 gene expression after simultaneous stimulation of EP4 receptor and BCR (as we proved in the first part of our research) the expression of interleukin 6 receptor gene remains low. Even more, its expression reduces 4 hours after stimulation of EP4 receptor and BCR which could indicate a negative feedback loop.
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