Molecular biology of microglia cytokine and chemokine receptors and microglial activation

Activation of brain microglial cells can be subdivided into a number of stages. Early stages likely are proliferation and migration to sites of cell damage. These two stages have been studied exemplarily on the IL-3 receptor beta-subunit and on the CC-chemokine receptor 5 using molecular biological methods. First, IL-3 receptor beta-subunit cDNA has been cloned in full length from rat microglia. Since cultured microglia are already activated to some extent, mRNA of this subunit has been detected in the isolated cells, but was absent in normal rat brain. Lipopolysaccharide (LPS) increased this mRNA in the cultured cells and LPS injected into the circulation of rats induced the mRNA specifically in brain microglia as revealed by in situ hybridizations. Next, we obtained partial cDNAs of receptor-coupled protein tyrosine kinases JAK 1 and JAK 2. These mRNAs were present both in cultured microglia and in rat brain, but were not influenced by LPS. Finally, a full-length cDNA of the rat chemokine receptor 5 has been obtained by PCR methodology. Its mRNA was increased by administration of LPS both in cultured microglia and in vivo. It is expected, that further investigations on these receptors could help to develop improved strategies to combat chronic inflammatory events in the brain

Molecular biology of microglia cytokine and chemokine receptors and microglial activation

Activation of brain microglial cells can be subdivided into a number of stages. Early stages likely are proliferation and migration to sites of cell damage. These two stages have been studied exemplarily on the IL-3 receptor beta-subunit and on the CC-chemokine receptor 5 using molecular biological methods. First, IL-3 receptor beta-subunit cDNA has been cloned in full length from rat microglia. Since cultured microglia are already activated to some extent, mRNA of this subunit has been detected in the isolated cells, but was absent in normal rat brain. Lipopolysaccharide (LPS) increased this mRNA in the cultured cells and LPS injected into the circulation of rats induced the mRNA specifically in brain microglia as revealed by in situ hybridizations. Next, we obtained partial cDNAs of receptor-coupled protein tyrosine kinases JAK 1 and JAK 2. These mRNAs were present both in cultured microglia and in rat brain, but were not influenced by LPS. Finally, a full-length cDNA of the rat chemokine receptor 5 has been obtained by PCR methodology. Its mRNA was increased by administration of LPS both in cultured microglia and in vivo. It is expected, that further investigations on these receptors could help to develop improved strategies to combat chronic inflammatory events in the brain

Cloning of rat HIV-1-chemokine coreceptor CKR5 from microglia and upregulation of its mRNA in ischemic and endotoxinemic rat brain

Chemokine receptors play a crucial role in the recruitment of immune cells to sites of inflammation. Although chronic diseases of the brain are often accompanied by inflammatory events, there is presently no information about the occurrence and regulation of these receptors in the central nervous system (CNS), Moreover, one CC-chemokine receptor, CKR5, has recently been identified as coreceptor for HIV-1 entry into macrophages, HIV-1 target cells in brain are macrophage-related microglia, which suggests that they are infected by the same mechanism (He et al,,: Nature 385:645-649, 1997), Although rats are not susceptible to HIV-1 infection, they can be used to study chemokine receptor regulation in a variety of brain pathologies. After cloning CC-CKR5 and establishing reverse transcriptase polymerase chain reaction (RT-PCR) for its ligands macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and regulated on activation, normal T cell-expressed and secreted (RANTES), we studied expression of these four mRNAs in purified microglia and compared it with their expression in rat brain, Lipopolysaccharide (LPS)-treated microglia showed transiently increased mRNA levels of both CKR5 and its ligands, Similar data were obtained from brains of LPS-injected rats, In middle cerebral artery occluded (MCAO)-animals, RANTES mRNA was unaffected, whereas CKR5 mRNA showed a sustained rise until 96 hr after surgery, MIPs exogenously added to microglial cultures markedly reduced CKR5 mRNA expression, whereas RANTES did not, MIP mRNAs, in contrast to RANTES and CKR5 mRNAs, were undetectable in normal brain, RANTES appears to play a role distinct from MIPs in brain, In summary, upregulation of CC-chemokines and CKR5 in the CNS upon bacterial infection or in ischemia may impact on microglial activation stage and result in increased risk of HIV-1 infection, J, Neurosci, Res. 53:16-28, 1998 (C) 1998 Wiley Liss, Inc

Cultured rat microglia express functional beta-chemokine receptors

We have investigated the functional expression of the beta-chemokine receptors CCR1 to 5 in cultured rat microglia. RT-PCR analysis revealed constitutive expression of CCR1, CCR2 and CCR5 mRNA. The beta-chemokines MCP-1 (1-30 nM) as well as RANTES and MIP-1 alpha (100-1000 nM) evoked calcium transients in control and LPS-treated microglia. Whereas, the response to MCP-1 was dependent on extracellular calcium the response to RANTES was not. The effect of MCP-1 but not that of RANTES was inhibited by the calcium-induced calcium release inhibitor ryanodine. Calcium responses to MCP-1- and RANTES were observed in distinct populations of microglia. (C) 1999 Elsevier Science B.V. All rights reserved