Following allergen exposure, eosinophils (Eos) and basophils (Basos) but not neutrophils (PMNs) are preferentially recruited to the lung in a Th2 environment supported by dendritic cells. The sequence of events is likely to include the release of histamine from resident mast cells within min of allergen binding. The presence of histamine is expected to lead within min to an upregulation of P-selectin on vascular endothelial cells that can serve in the initial Eo and Baso adherence. Within min to hrs, allergen specific Th2 cytokines IL4, IL-5, and IL-13 are generated by mast cells and T cells. IL4 and IL-13 regulate the expression of adhesion molecules on the endothelium. IL-5 enables VLA-4 dependent adhesion of Eos and Basos while the asthma specific chemokines that bind to CCR3 enable VLA-4 and LFA-1 dependent firm adhesion and transmigration. As a consequence Eos and Basos accumulate in bronchial mucosa and mucous cell metaplasia can arise later from bronchiolar inflammation. These considerations provide a temporal and mechanistic framework for analysis of Eo and Baso recruitment following allergen. We have shown that Eos use their P-selectin glycoprotein ligand (PSGL-1) to attach to CHO cells expressing low levels of P-selectin. At the low site density of P-selectin of endothelial cells, Eos attach preferentially as compared to PMNs in accord with observations that Eos but not PMNs use PSGL-1 to adhere to venules in the lung.
The first aim will focus on understanding the mechanism by which Eos and Basos but not PMNs preferentially use their PSGL-1 to engage P-selectin. VLA-4 plays a unique role in the recruitment of Eos and Basos because it can be regulated in distinct conformations that contribute both to cell capture and firm adherence. We have developed several novel real-time assays to examine how IL-5 and chemokine exposure alters the adhesive activity and affinity of VLA-4 on Eos.
The second aim i s to understand the relationship between the affinity of VLA-4 to the rolling and firm attachment of Basos and Eos. In animal models of allergic asthma, mAbs to P-selectin, VLA-4 and also LFA- 1 block the accumulation of Eos. Because LFA- 1 contributes to specific recruitment in so far as activated by cell specific stimuli, it must work in combination with P-selectin and VLA-4 to enable efficient transmigration.
The third aim will evaluate how the adhesion molecules work in combination in the recruitment. Taken together, the work will provide a novel understanding of the attachment of PSGL-1 to P-selectin, the use of VLA-4, and the coordination of the adhesion pathways in asthma.
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