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  • Immature and mature B cells adapt differently


    Immature and mature dihydroquercetin adapt differently to signal transduction via BCR [2]. This functional dichotomy is also regulated by noncytokine substances in the surroundings of mature and immature B lymphocytes, such as the prostanoid family of lipid mediators including PGD2, PGE2, PGF2α, PGI2 and thromboxane A2 [7]. Of the prostanoids, PGE2 has been shown to exhibit growth suppressing effects in immature mouse B lymphocytes [8], [9], [10]. In addition, when surface Ig on immature B cells are crosslinked, the E series prostaglandins promote B cell unresponsiveness [11]. Four G protein coupled receptors (GPCRs), designated EP1, 2, 3 and 4, which are often expressed in the same cell type, mediate the effects of PGE2 [12]. Recently we have identified Ptger4, coding for EP4 receptor, as a gene whose expression on mature and immature B lymphocytes is significantly upregulated after BCR-triggering [13]. In addition to its induction on BCR cross-linking, Ptger4 also appears as a promising candidate regulator of immature B cell response, because PGE2–EP4 signaling prevents BCR-induced proliferation of mature B lymphocytes [14]. Depending on the cell type, activation of the EP4 receptor by PGE2 leads to elevated levels of cAMP and in some cases to activation of cAMP independent pathways, including activation of PI3K/AKT [15], MEK/ERK1/2 [16], and modulation of NF-κB transcription factor [17]. No detailed insight into the signaling components and mechanism of the attenuating effect of PGE2/EP4 on immature B lymphocytes has been provided.
    Materials and methods
    Discussion While recognition of antigen by mature B cells leads to clonal expansion, self reactive immature B cells respond to BCR engagement by apoptosis [1]. The mechanism of immature and transitional immature B cell negative selection can be regulated by the microenvironment context in which the immature and transitional immature B cells encounter antigens. In the present study we have addressed the role of EP4 receptor as a potent modulator of the BCR induced response of dihydroquercetin immature B lymphocytes. BCR-triggered activation of B cells leads to transcriptional reprogramming that can be observed by a strong induction of immediate-early genes within minutes following mitogenic stimulation, preparing cells to respond quickly with proliferation of mature B lymphocytes or apoptosis of immature B lymphocytes. Our interest in the PGE2–EP4 signaling pathway was triggered by one of our previous studies confirming that the Ptger4 gene, coding for EP4 receptor, is up-regulated on BCR triggering in immature and mature primary murine B lymphocytes [13]. In the search of novel modulators of the BCR response, we showed that BCR receptor crosslinking on WEHI 231 cells up-regulates Ptger4 expression in WEHI 231 cells, whose mRNA expression kinetics appear similar to those of a wave of delayed-early genes. Since the functional outcome of PGE2 is dependent on PGE2 signal strength/EP receptor affinity and/or the maturation/activation status dependent expression of EP subclasses in B cells, it is important to stress that none of the other genes coding for PGE2 receptors showed altered expression following BCR triggering of immature B cells. Exogenous PGE2 has been previously reported to play an immunosuppressive role in B cell proliferation [11], [12]. Indeed we showed that exogenous PGE2 has growth inhibitory effects on WEHI 231 cells over a wide concentration range from 10μM to as low as 1nM. Using a selective EP4 receptor agonist and a specific EP4 receptor antagonist we confirmed that EP4 receptor is the only receptor transmitting the PGE2 delivered signals on immature WEHI 231 B cells. The observed action of PGE2 can be explained by specific upregulation of EP4 receptor on immature B cells, sensitizing the cells to PGE2 mediated inhibitory effects. Macrophages are a major source of prostaglandins in the germinal centre (GC) microenvironment, and it was proposed that these scavengers of apoptotic lymphocytes may use prostaglandin E2 to down-regulate the GC reaction [20]. We demonstrated that, while EP4 receptor stimulation by PGE2 or Pge1–OH leads to inhibition of proliferation and not apoptosis, the engagement of EP4 receptor strongly enhances caspase mediated, BCR induced apoptotic responses of WEHI 231 cells, indicating that upregulation of EP4 receptor and its activation plays an important role in the negative selection of autoreactive immature B cells. This notion is confirmed by the fact that mice treated with PGE2 have fewer B-lineage precursors in their bone marrow than untreated mice, which suggests a regulatory role of PGE2 in the elimination of self-reactive or defective immature B-cell precursors [21]. Based on our findings, EP4 receptor could play a pivotal role in the observed action of in vivo administered PGE2. This is consistent with results reported by Murn et al., since Ptger4 mice consistently harbored significantly increased numbers of mature B cells in the spleen compared with wild type controls [14].