However, it is still unclear whether CXCR2 intervenes during adhesion and/or transmigration. remain undetermined. Methods CXCR2 ligand expression was examined in the CNS of mice suffering from EAE or exposed to bacterial toxins by quantitative RT-PCR and em in situ /em hybridization. CXCL1 expression was analyzed in IL-6-treated endothelial cell cultures Rabbit Polyclonal to FCRL5 by quantitative RT-PCR and eCF506 ELISA. Granulocytes were counted in the brain vasculature after treatment with a neutralizing anti-CXCL1 antibody using stereological techniques. Results CXCL1 was the most highly expressed ligand of the granulocyte receptor CXCR2 in the CNS of mice subjected to EAE or infused with lipopolysaccharide (LPS) or pertussis toxin (PTX), the latter being commonly used to induce EAE. IL-6 upregulated CXCL1 expression in brain endothelial cells by acting transcriptionally and mediated the stimulatory effect of PTX on CXCL1 expression. The anti-CXCL1 antibody reduced granulocyte adhesion to brain capillaries in the three conditions under study. Importantly, it attenuated EAE severity when given daily for a week during the effector phase of the disease. Conclusions This study identifies CXCL1 not only as a key regulator of granulocyte recruitment into the CNS, but also as a new potential target for the treatment of neuroinflammatory diseases such as multiple sclerosis. strong class=”kwd-title” Keywords: Neuroinflammation, Neuroimmunity, Chemotaxis, Myeloid cells, Polymorphonuclear cells, Neutrophils, Cerebral endothelium, Autoimmunity. Background Myelin-reactive CD4+ T lymphocytes play a critical role in the pathogenesis of multiple sclerosis and its animal model, EAE [1]. A basic principle in immunology states that these cells eCF506 do not act alone, but rather in concert with different populations of myeloid phagocytes, which activate them by presenting antigens and producing proinflammatory molecules, and which execute effector functions. The phagocytes best known to be involved in EAE are monocyte-derived CD11c+ dendritic cells [2-5] and Ly6C+ macrophages originating from either microglia or monocytes [2,4,6,7]. In addition, mounting evidence suggests the involvement of a third population of phagocytes, namely granulocytes. Indeed, it has been reported that granulocytes massively infiltrate the CNS of EAE mice [8-17], and that EAE is markedly attenuated in mice either treated with antibodies against the granulocyte proteins CXCR2 and Ly6G, treated with a small molecule antagonist of CXCR2, or genetically manipulated to suppress CXCR2 [12,18-20]. Therefore, granulocytes and the extracellular signaling pathways that control them represent novel potential therapeutic targets for multiple sclerosis. We have uncovered a population of rod-shaped granulocytes that patrol the CNS vasculature by crawling on the luminal endothelial surface [17,21,22]. These cells are recruited in greater number in mice suffering from EAE or exposed to bacterial products such as LPS and PTX [17,21]. According to the classical model, the mechanism underlying this recruitment includes the following steps: 1) proinflammatory cytokines induce the expression of chemokines and adhesion molecules on the endothelial surface; 2) these chemokines activate granulocytes by promoting the conversion of integrins from a low-affinity to eCF506 a high-affinity state; and 3) the latter integrins allow the firm attachment of granulocytes to endothelial adhesion molecules [23]. So far, we have gathered evidence that PTX induces ICAM1 expression in brain capillaries indirectly through IL-6, and that granulocytes bind to these vessels through interaction between Mac1 (integrin M2) and intercellular adhesion molecule 1 (ICAM1) [17]. In contrast, granulocyte adhesion in response to LPS is independent of eCF506 IL-6, but dependent on TNF and IL-1 [17,21]. An important question that remains to be addressed is what chemokine(s) control granulocyte adhesion in the cerebral microvasculature. The G-protein-coupled receptor CXCR2 is crucial for granulocyte infiltration into the brain parenchyma, as demonstrated using CXCR2-knockout mice exposed to LPS or em Staphylococcus aureus /em [24,25]. However, it is still unclear whether CXCR2 intervenes during adhesion and/or transmigration. CXCR2 binds to chemokines of the CXC family containing the glutamate-leucine-arginine (ELR) motif (i.e., CXCL1, CXCL2, CXCL3, CXCL5, and CXCL7 in mouse and human, in addition to CXCL6 and CXCL8 in human only) [26]. This family also comprises mouse CXCL15, which does not appear to bind CXCR2 [27]. Both CXCL1 and CXCL2 are upregulated in the CNS of mice suffering from endotoxemia [24] or EAE [13,14,16,28-30]. Such information is not available for the other CXCR2 ligands, leading to question the relative importance of these molecules in granulocyte recruitment into the CNS. The primary objective of the present study was to identify the main CXCR2 ligand responsible for granulocyte adhesion to brain capillaries during EAE and after exposure to bacterial toxins, especially PTX, which is commonly used as an adjuvant to induce EAE [31], but whose mechanism of action is still not fully understood. The second objective was to examine whether the effect of PTX on CXCL1 expression is mediated by IL-6, a cytokine essential for EAE development [32-36] and previously eCF506 identified as mediating the effect of PTX on endothelial ICAM1 expression.

However, it is still unclear whether CXCR2 intervenes during adhesion and/or transmigration