Many diseases of the immune system such as autoimmune diseases, lymphomas and leukemias are caused by defects in the process of B cell maturation and activation. Various signals contribute to the tightly regulated B cell response, among which are crosslinking of BCR, T-cell help, TLR stimulation and the presence of cytokines and other ligands for membrane receptors, such as prostaglandins, in the B cell microenvironment. The identification of novel molecules that participate in the complex B cell signaling network will contribute to a better understanding of B cell responses and give rise to novel molecular targets for drug discovery and development.
In the present work we present two novel molecular targets for the modulation of B cell responses: serine proteases and the prostaglandin receptor EP4. In the first part of our research we tested if a series of inhibitors of serine proteases synthesized at the Faculty of Pharmacy were able to induce apoptotic cell death in human and murine B lymphoma cell lines. We have shown that compounds built on an azaphenylalanine scaffold are severely cytotoxic for B-cell lymphoma cells and they provoke biochemical and morphological changes typical of apoptosis, such as DEVDase activation, loss of mitochondrial membrane potential, nuclear degradation and genomic DNA fragmentation. Most of the inhibitors proved to be selective for thrombin, with inhibition constants (Ki) in the nanomolar range. However, they could also inhibit at least one additional serine protease (trypsin, chymotrypsin and/or coagulation factor X) with Ki values in the nanomolar or low micromolar range. These serine protease inhibitors constitute novel apoptosis inducing compounds in B-cell lymphoma cells. In the second part of our work we focused on the prostaglandin receptor EP4, which is the principal molecule conveying the growth-suppressive effect of prostaglandin E2. Our results suggest that an agonist of the EP4 receptor modulates the effects mediated by the receptors BCR and TLR4. We have shown that both receptors up-regulate the expression of EP4, which stimulation can in turn act as a positive feedback loop increasing the inhibitory effect of BCR crosslinking and as a negative feedback loop suppressing the proliferatory effect of TLR4 signaling. In WEHI-231 cells we demonstrated that this effect is at least in part mediated by the inhibition of activation of the transcription factor NFκB. Non-cytotoxic concentrations of the irreversible inhibitor of serine proteases TPCK were also able to inhibit NFκB activation. However, the combination of low concentrations of TPCK and an EP4 agonist (PGE1-OH) proved to be lethal for WEHI-231 cells, causing cell death. We suggest that this effect is caused by a synergistic inhibition of NFκB activation.