ABSTRACT
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-1alpha (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.
Subject(s)
Microglia/chemistry , Microglia/immunology , Receptors, Chemokine/genetics , Receptors, Chemokine/immunology , Animals , Calcium/metabolism , Calcium Signaling/immunology , Cells, Cultured , Chemokine CCL2/pharmacology , Chemokine CCL3 , Chemokine CCL4 , Chemokine CCL5/pharmacology , Cytosol/metabolism , DNA Primers , Enzyme Inhibitors/pharmacology , Gene Expression/drug effects , Gene Expression/immunology , Lipopolysaccharides/pharmacology , Macrophage Inflammatory Proteins/pharmacology , Microglia/cytology , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Receptors, CCR1 , Receptors, CCR2 , Receptors, CCR5/genetics , Receptors, CCR5/immunology , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Ryanodine/pharmacology , Thapsigargin/pharmacology , Virulence Factors, Bordetella/pharmacologyABSTRACT
Macrophage inflammatory protein is a member of the C-C subfamily of chemokines, which exhibits, in addition to proinflammatory activities, a potent endogenous pyrogen activity. In this study, we analysed the time-course of expression and cellular source of macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta, in inflammation of the rat brain associated with ischemia and endotoxemia. Using in situ hybridization histochemistry, we observed that intravenously injected bacterial lipopolysaccharide induced a transient expression of macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta messenger RNAs throughout the brain, with maximal expression 8-12 h after lipopolysaccharide treatment. We also revealed an early increase in macrophage inflammatory protein-1alpha and macrophage inflammatory protein-1beta messenger RNA levels, after permanent and transient middle cerebral artery occlusion, starting as early as 1 h after the occlusion and reaching a peak of expression 8-16 h after middle cerebral artery occlusion. The induction of macrophage inflammatory protein-1 messenger RNA was clearly stronger in the transient than in the permanent middle cerebral artery-occluded rat brains, showing that the reperfusion process influences the extent of the chemokine response after middle cerebral artery occlusion. In situ hybridization combined with immunohistochemistry for glial fibrillary acidic protein, a specific marker for astrocytes, excluded astrocytes as the cellular source of macrophage inflammatory protein-1 messenger RNAs after both middle cerebral artery ischemia and lipopolysaccharide treatment. Using immunohistochemistry, macrophage inflammatory protein-1alpha protein expression was shown to be induced in a time-dependent manner after lipopolysaccharide treatment and middle cerebral artery occlusion. Macrophage inflammatory protein-1alpha immunopositive cells co-localized with cells stained with OX-42 antibody, a microglia/macrophage marker. These results indicate that macrophage inflammatory protein-1 is implicated in the inflammatory reaction of the brain in response to ischemia or infection, and might modulate the host defence febrile response to a pathogenic stimulus.
Subject(s)
Brain Ischemia/metabolism , Brain/metabolism , Lipopolysaccharides/pharmacology , Macrophage Inflammatory Proteins/metabolism , Animals , Chemokine CCL4 , Immunohistochemistry , In Situ Hybridization , Injections, Intravenous , Macrophage Inflammatory Proteins/genetics , Male , RNA, Messenger/metabolism , Rats , Rats, Inbred SHR , Tissue Distribution/physiologyABSTRACT
Increasing evidence indicates a key role of chemoattractant cytokines in the accumulation of leukocytes in the central nervous system (CNS) during the course of inflammatory processes. Monocyte chemoattractant protein (MCP-1/JE), a member of the beta-chemokine (C-C chemokine) family, functions as a potent chemoattractant and activator for monocytes. We have investigated the induction of MCP-1 mRNA using in situ hybridization histochemistry (ISH) and characterized its cellular source by combination of ISH and immunocytochemistry in ischemic rat brains as well as in brains of endotoxin-treated rats. Our results show that 6 h-2 d after middle cerebral artery occlusion (MCAO), MCP-1 mRNA is present in astrocytes surrounding the ischemic tissue (penumbra). At later time points (after 4 d), MCP-1 mRNA is found in macrophages and reactive microglia in the infarcted tissue. Peripheral administration of the bacterial lipopolysaccharide (LPS) induced MCP-1 mRNA throughout the brain in a time-dependent manner (1 h-1 d, peak of expression 6-8 h) and was found in astrocytes. In summary, we have found expression of MCP-1 in (a) astrocytes and to a lesser extent in macrophages/reactive microglia after MCA-occlusion and in (b) astrocytes after peripheral administration of LPS. These findings support that MCP-1 is involved in the CNS response to acute trauma or infection and thus may play a key role in inflammatory processes of the brain.