Integrin/Chemokine receptor interactions in the pathogenesis of experimental autoimmune encephalomyelitis

Abstract

Excessive infiltration of leukocytes and the elaboration of inflammatory cytokines are believed to be responsible for the observed damage to neurons and oligodendrocytes during multiple sclerosis (MS). Blocking adhesion molecules or preventing the effects of chemotactic mediators such as chemokines can be exploited to prevent immune cell recruitment to inflamed tissues. An anti-α4 integrin antibody (anti-VLA-4mAb/natalizumab (Tysabri®)) has been used as a treatment for MS and reduces leukocyte influx into the brain. In patients, anti-VLA-4 reduces relapses and disability progression. However, its mechanism of action in the brain is not completely understood. The anti-VLA-4mAb was demonstrated to mobilize hematopoietic progenitor cells. Interestingly, the chemokine SDF-1/CXCL12 and its receptor CXCR4 are also key factors regulating the migration of hematopoietic stem cells. Moreover, studies have revealed a crosstalk between SDF-1/CXCR4 and VLA-4 signaling in regulating cell migration. In this study, we address the effects of anti-VLA-4 on chemokine signaling in the brain during MS. We assessed the ability of anti-VLA-4 to regulate Experimental Autoimmune Encephalomyelitis (EAE) and chemokine/receptor signaling. Preclinical administration of anti-VLA-4 delayed clinical signs of EAE. We found that anti-VLA-4 treatment reduced chemokine expression. In order to further explore the interaction of anti-VLA-4 with chemokine/receptor signaling we used dual color transgenic mice. After EAE induction, the expression of both SDF-1/CXCL12 and CXCR4 receptor was upregulated, treatment with anti-VLA-4 inhibited this effect. The effects of anti-VLA-4 on chemokine signaling in the CNS may be of importance when considering its mechanism of action and understanding the pathogenesis of EAE.

Figure 1

A: Preclinical administration of anti-VLA-4 Ab effectively delays the clinical signs of EAE. By day 11 after priming, mice treated with the control antibody showed clinical signs of EAE (n=12, mean clinical score of 1.2) whereas none of the anti-VLA-4-treated mice were showing any signs of the disease (n=12). B: Treatment with anti-VLA-4 Ab at the peak of acute phase does not interfere with clinical signs of EAE. (Arrows show the beginning of the treatment). Both groups followed a similar pattern of disease severity. Arrows show the start of the treatment.

Figure 2

Effect of anti-VLA-4 Ab treatment on SDF-1/CXCR4 expression in EAE mice. In order to examine the expression of SDF-1/CXCL12 and CXCR4 after anti-VLA-4 Ab treatment in EAE, SDF-1-RFP/CXCR4-EGFP dual transgenic mice were used. Preclinical treatment with anti-VLA-4 Ab reduces the expression of CXCR4 (green) in the posterior part of the subventricular zone (SVZ) (B), corpus callosum (cc) (D) and cerebellum (F). SDF-1/CXCL12 (red) is downregulated in the SVZ and cc (B and F respectively). Panels A, C, and E show the expression of CXCR4 and SDF-1/CXCL12 in EAE mice injected with the control antibody.

Figure 3

Characterization of SDF-1/CXCL12 and CXCR4 expressing cells in SDF-1RFP/CXCR4-EGFP EAE mouse brain. CXCR4 is expressed by cells exhibiting the morphology of migrating progenitors in the posterior part of the SVZ (A). Immunostaining using an anti-Olig1 antibody shows that these CXCR4-expressing cells (panel A, green) colocalize with Olig1 (panel B, red). Arrowheads in panel C show the colocalization of CXCR4 and Olig1. CXCR4 is also upregulated in CD68 expressing macrophages in EAE mouse brain (BV: blood vessel). Arrowheads show the colocalization of CXCR4 and CD68 expression (D). SDF-1/CXCL12 expression is mainly confined to GFAP positive astrocytes (arrowheads in E) and IBA1 positive microglia (arrowheads in F).

Figure 4

Treatment of anti-VLA-4 Ab reduces the expression of SDF-1/CXCL12 in EAE brain. In order to quantify the expression of SDF-1/CXCL12 after treatment with anti-VLA-4 Ab, ELISA was performed. SDF-1/CXCL12 expression is lower in EAE brains treated with anti-VLA-4 preclinically (A) or at the peak of the acute phase (B).