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A1271 - Platelet CCR3 Receptors Are Involved in the Recruitment and Migration of Platelets During Allergic Inflammation
Author Block: S. Shah, Y. R. Vasquez, C. P. Page, S. Pitchford; Sackler Institute of Pulmonary Pharmacology, King's College London, London, United Kingdom.
Rationale Platelets express CCR1, CCR3, CCR4 and CXCR4 chemokine receptors (Clemetson et al., 2000), and have been shown to undergo chemotaxis in response to these chemokines in vitro (Kraemer et al., 2010). In allergic inflammatory diseases such as asthma, platelets can migrate extravascularly into lungs in response to allergen (Pitchford et al., 2008). The mechanisms behind the migration of platelets from the vasculature to sites of allergic inflammation and the roles of chemokine receptors in this process are not fully understood. The aim of this study was to investigate the vascular interactions of platelets in allergic mice following allergen challenge using intravital microscopy of the mouse cremaster muscle (CM), in the presence of chemokine receptor antagonists. Methods Mice were sensitised intranasally with 25 μg house dust mite extract (HDM), 10 times over 2 weeks. The CCR3, CCR4 and CXCR4 receptor antagonists SB328437 (30 mg/kg), C-021 (30 mg/kg) and AMD3100 (10 mg/kg) respectively, were administered intraperitoneally at 30 minutes before 100 μg HDM challenge was administered subcutaneously in the scrotal sack. Anti-mouse CD49b PE antibody (0.1 mL intravenously) was injected to visualise platelets at 1 hour before the CM was prepared for intravital microscopy of post capillary venules, 24 hours after injection of chemokine receptor antagonists. Platelet rolling, adhesion, and extravascular presence were then enumerated via video analysis and immunohistochemical staining (IHC). N=8 per group, data presented as mean +/- SEM with one-way ANOVA and Dunnett’s post hoc statistical tests. Results The number of leukocytes present within the CM decreased in AMD3100 treated groups compared to positive controls (from 15.3±1.8 to 4.3±0.8 (leukocytes/30 μm2); P≤0.001), but were not affected by CCR3 or CCR4 antagonism. Fluorescently labelled platelets in SB328437 treated groups compared to positive controls decreased rolling (from 20.7±2.3 to 12.3±2.9 (rolling platelets/10 sec); P≤0.05) and adhesion (from 1.1±0.1 to 0.6±0.2 (platelets/30 μm2); P≤0.05) events in post capillary venules. There was also a decreased number of CD42b positive extravascular CM platelet staining events in SB328437 treated groups, as determined by IHC (from 12.4±1.4 to 8.9±0.7 (platelets/ mm2); P≤0.05). Neither CXCR4 nor CCR4 antagonism affected these parameters. Conclusions CXCR4 receptor antagonism reduced leukocyte, but not platelet recruitment, to the CM after allergen challenge, suggesting platelets accumulate independently of leukocytes. The localized platelet accumulation that occurs in response to allergen challenge was attenuated by CCR3 receptor antagonism, indicating the CCR3 receptor plays a role in platelet migration to sites of allergic inflammation.