Expression Of CCR7 And The Effect Of It With The Ligand CCL19 On Biological Function Of Mouse Microglial Cells

Chemokines deliver their activity by interacting with cell surface expressed chemokine receptors that belong to the family of seven transmembrane domain G protein-coupled rhodopsin-like receptors. CCR7 was identified as a gene induced by the Epstein-Barr virus (EBV), and is thought to be a mediator of EBV effects on B lymphocytes. Previous results told us that such receptor is expressed in various lymphoid tissues and activates B and T lymphocytes. Important functions of CCR7 have been shown to control the migration of memory T cells to inflamed tissues and stimulate dendritic cell maturation. Because of the importance of such receptor, many researchers have investigated CCR7 in many kinds of cells. However, no report has been made regarding the presence of CCR7, the receptor for CCL19 and CCL21 in microglia; instead, CCL21 is found to induce chemotactic behavior by interacting with CXCR3 in microglia. Moreover, recent studies have shown that it is highly unlikely that this signaling system plays a role in inflammation in the CNS.Experimental animals used in this study were gcd-10 wild type mouse. Our present results provide the first evidence that RT-PCR and Western blotting analysis showed CCR7 expression was detected in activated primary cultures of microglia, but not in the resting mocroglia. Primary cultures of microglia stimulated with LPS express CCR7 mRNA in time and dose-dependent way. CCR7 mRNA expression was also found in cultured microglia treated by cell debris. LPS, but not IFN-γinduced CCR7 expression in microglial cell. Similar to previous results, expression of CXCR3 mRNA was found in the cultured microglial cells.TGF-βM has known to be associated with production of various kinds of cytokines. TGF-β1 inhibits various kinds of cytokine mRNA expression. In present study, we found that LPS-induced expression CCR7 mRNA is almost suppressed by TGF-β1. However, LY294002, a PI3K inhibitor, has no effect on LPS-induced CCR7 mRNA expression in cultured microglia, suggesting that TGF-β1 shows its activity not through PI3K signal pathway. Previously, it has been reported that induction of CCR7 expression in thymocytes and RLm6 depends on activation of the MEK-ERK signaling pathway and an increase in intracellular Ca²⁺ levels. Here we examined the effect of U0126, an inhibitor of the extracellular signal-regulated kinase kinase (ERKK/MEK), on expression of CCR7 mRNA in activated microglia. Interestingly, however, CCR7 mRNA expression was up-regulated after adding the U0126. In the present study we also investigated other pathways such as p38, which is possibly responsible for the induction of CCR7 mRNA expression in the activated primary cultures of microglia. Our data indicated that CCR7 mRNA expression does not due to the activation of the ERKK/MEK or p38. Although the induction mechanism of CCR7 expression in activated microglia requires further investigation, the cell type, experimental condition-dependent effect and some other factors, such as NF-κB, the interplay of NF-κB with MAPKs or the interplay within the MAPKs, could be due to mechanism for the induction of CCR7 mRNA expression.Similar to previous results, TNF-αand iNOS mRNA expression were detected in LPS stimulated microglia.The findings that CCR7 mRNA is expressed in activated microglia led us to examine effects of its ligands. In the present study, we examined the effects of CCL19 on LPS-treated primary microglial cells culture. Percent of Cell death was almost 20% in LPS-treated cells. This percentage was consistently reduced to about 10% when the cells were cultured in the presence of 0.lμg/ml CCL19, implying that CCL19 reduced about 50% of dead cells. Our results clearly show that CCL19 is able to augment the function of phagocytosis for LPS-stimulated microglia. After adding the CCL19 in LPS-stimulated microglia, phagocytosis was augmented, indicating that the interaction between CCR7 and its ligand, CCL19 enhances phagocytosis of microglia. With the adding of CCL19 a transient increase in intracellular calcium was detected in the cultured microglia under activated condition but not under normal condition, indicating that level of Ca²⁺ influx can be elevated by CCL19 in activated microglia.In conclusion, CCR7 is barely expressed in microglia under normal conditions, but markedly up-regulated upon their activation. Indeed, its ligand CCL19 augmented phagocytosis in activated microglia, suggesting that under activated conditions the CCL19/CCR7 interaction becomes predominant, which may play a role in regulating chemokine receptors and cytokine production, cell survival, phagocytosis and transient increase in intracellular calcium.