Neutralization of the Chemokine CXCL10 Reduces Inflammatory Cell Invasion and Demyelination and Improves Neurological Function in a Viral Model of Multiple Sclerosis

A, CXCR3 mRNA expression. CXCR3 mRNA expression in CD4⁺ and CD8⁺ T lymphocytes isolated from the CNS of mice treated with either anti-CXCL10 or NRS at 21 and 28 days p.i. CD4⁺ T lymphocytes obtained from mice treated with anti-CXCL10 display a reduced expression of CXCR3 mRNA transcripts when compared with control mice treated with NRS at 21 days p.i. In contrast, comparable levels of CXCR3 mRNA expression in CD8⁺ T lymphocytes are found in mice treated with anti-CXCL10 and NRS at 21 and 28 days p.i., supporting FACS data showing similar levels of CD8⁺ T lymphocyte infiltration into the CNS at all times examined. Corresponding to the return of inflammatory cells into the CNS at 28 days p.i., CD4⁺ T lymphocytes isolated from mice formerly treated with anti-CXCL10 display increased expression of CXCR3 mRNA when compared with those observed at 21 days p.i. Top panel, CXCR3 (111 bp); bottom panel, L32 (183 bp). The results presented represent enriched lymphocytes pooled (three mice per group) at each time point. B, CCL5 protein levels at 21 days p.i. CCL5 protein levels from brains of mice at 21 days p.i were determined by ELISA. Mice treated with anti-CXCL10 displayed significantly decreased levels of CCL5 when compared with either NRS- or anti-CXCL9-treated mice (21 days p.i.). ∗, p < 0.05 (anti-CXCL10: n = 3; anti-CXCL9: n = 3; and NRS: n = 3). C, IFN-γ mRNA levels at 21 days p.i. IFN-γ protein was not detected by ELISA; therefore, RT-PCR analysis was used to evaluate IFN-γ expression at day 21 p.i. Mice treated with anti-CXCL10 show a marked decrease in the levels of IFN-γ mRNA transcripts in the CNS when compared with NRS-treated mice. Top panel, IFN-γ (365 bp); bottom panel, L32 control (183 bp). Each lane represents an individual mouse. D, CCL5 and IFN-γ mRNA levels at 28 days p.i. RT-PCR analysis of CCL5 and IFN-γ expression within the CNS of MHV-infected mice at 28 days p.i. Total RNA was extracted from the brains of MHV-infected mice formerly treated with either anti-CXCL10 or NRS at 28 days p.i. and subjected to RT-PCR. Mice formerly treated with anti-CXCL10 and NRS show comparable levels of both CCL5 and IFN-γ mRNA transcripts in the CNS. Top panel, CCL5 (312 bp); middle panel, IFN-γ (365 bp); bottom panel, L32 control (183 bp). Each lane represents an individual mouse.

LFB staining of spinal cords. Demyelination is reduced in mice treated with anti-CXCL10 as compared with NRS- or anti-CXCL9-treated mice at day 21 p.i. Representative spinal cord sections from mice treated with anti-CXCL10 21 days p.i. (A), mice treated with anti-CXCL9 21 days p.i. (B), or mice treated with NRS 21 days p.i. (C). Sections were stained with LFB to assess the severity of demyelination. Numerous inflammatory foci and areas of myelin stripping are present within the white matter tracts of NRS- or anti-CXCL9-treated mice, whereas limited numbers of inflammatory cells are detected in white matter tracts of anti-CXCL10-treated mice, and only minimal damage is detected. (magnification, ×100, inset ×400).