Cytokines regulation of chemokine and chemokine receptor in relation to multiple sclerosis.

Expression of chemokines CXCL10 and CCL2 is elevated within inflammatory lesions in the central nervous system (CNS) of multiple sclerosis (MS) patients, particularly in astrocytes. These chemokines play a critical role in the recruitment of inflammatory cells into the CNS during inflammation. However, the cerebrospinal fluid of MS patients also shows high levels of CXCL10 at the time of relapse but by contrast CCL2 is decreased. In the present study, the mechanisms controlling the synthesis and release of these two chemokines in MS were assessed in vitro using primary human brain astrocytes isolated from MS and non-MS individuals. Pro-inflammatory cytokines (interleukin-1beta , tumour necrosis factor and interferon-gamma) increased the expression of both CCL2 and CXCL10 by astrocytes at the mRNA and protein level, as determined by real time PCR and enzyme linked immunosorbent assays (ELISA), respectively. CCL2 binding to astrocytes was then determined to evaluate any autocrine action on astrocytes in a single astrocyte preparation. CCL2 bound constitutively and following cytokine treatment. CCL2-binding was not the result of the interaction with its receptor since astrocytes did not express CCR2 on this astrocyte culture. CCR2-independent binding of CCL2 was confirmed by the absence of intracellular signalling, evidenced by the lack of calcium influx as well as of Erk and Akt phosphorylation, in CCL2-treated astrocytes. Even though astrocytes expressed CXCR3, similar negative results on calcium influx and downstream signalling pathways were observed for CXCL10. D6 chemokine decoy receptor expression was then assessed in vitro and in situ to further investigate the mechanism(s) of chemokine binding to astrocytes. Cultured astrocytes constitutively expressed the D6 decoy receptor at the mRNA and protein level, but levels were unchanged following cytokine treatment. D6 was expressed in situ in MS normal appearing white matter and in control brain tissue, at both the mRNA and protein level. D6 expression was detected on neurons and microglia but not astrocytes using imunohistochemical methods. Incubation of frozen brain sections with biotinylated CCL2 resulted in partial co-localisation with D6 staining.Altogether, these results suggest a role for astrocytes in regulating inflammation through synthesis and secretion of CCL2 and CXCL10. Subsequently, CCL2 binding to astrocytes, either by binding to D6 decoy receptor or by alternative mechanisms, may establish a chemokine gradient in the CNS, and direct the migration of leukocytes.