However, because α 4 integrin has additional roles to leukocyte recruitment, including T cell and effector cell activation, the mechanism of action of α 4 integrin blockade in EAE is not yet clear. Indeed, treatment with anti-α 4 integrin Abs has some protective effects in EAE and can partially reduce the numbers of inflammatory cells in the CNS of diseased animals ( 4, 5, 6). These studies have primarily focused on a role of α 4 integrin. Until very recently, in vivo investigations of EAE have had to rely on histological observations and clinical outcome following blockade of various adhesion molecules to infer the mechanisms of leukocyte recruitment to the CNS. Firm adhesion occurs when integrins are induced to bind their endothelial ligands with high affinity. The tethering and rolling steps are mediated by the selectins, as well as α 4 integrins. If appropriate signals are encountered, leukocytes then firmly adhere to the vessel wall before transmigrating into the surrounding tissue ( 1, 2, 3). Circulating leukocytes first tether to and then roll on endothelial cells expressing adhesion molecules. The current paradigm of recruitment has largely been worked out in vitro and in a few easily accessible tissues in vivo (mesentery, cremaster, and skin) in which intravital microscopy can be performed. The mechanisms of leukocyte recruitment to the CNS are not well understood. In conclusion, we describe increased leukocyte trafficking in the brains of EAE mice with important overlapping roles for both P-selectin and α 4 integrin in mediating leukocyte-endothelial cell interactions. A very similar reduction was seen when mice were treated with α 4 integrin-blocking Ab. In the absence of rolling (with P-selectin Ab), a 70% reduction in adhesion was noted. Interestingly, however, α 4 integrin-mediated rolling appeared to be entirely dependent on P-selectin as anti-P-selectin alone was able to completely block all leukocyte rolling. Addition of anti-P-selectin to α 4 integrin Ab-treated mice blocked all remaining rolling at each time point. The importance of α 4 integrin increased with time as anti-α 4 integrin blocked ∼20, 50, and 60% of leukocyte rolling 2 days before disease onset, 5 days and 2 wk postonset of symptoms, respectively, and 85% of rolling 5 wk postsymptoms. Expression was highest before symptom onset and decreased over the next 2 wk. Consistent with a role for P-selectin in recruitment to the CNS, P-selectin protein was detected in the brains and spinal cords of EAE mice. Rolling and adhesion remained elevated for 2 wk and returned to near normal levels by 5 wk postsymptom onset. In contrast, both rolling and adhesion was observed in brain postcapillary venules before onset of physical symptoms of EAE. No leukocyte rolling and very little adhesion was observed in healthy control mice. We used intravital microscopy to investigate the mechanisms of this recruitment. Experimental autoimmune encephalomyelitis (EAE) is mediated by inflammatory cells recruited from the circulation to the CNS.
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