Giachelli, Cecilia MLund, Susan Amanda2012-09-132013-09-142012-09-132012Lund_washington_0250E_10186.pdfhttp://hdl.handle.net/1773/20898Thesis (Ph.D.)--University of Washington, 2012Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases. Macrophages are central players in chronic inflammation and OPN orchestrates macrophage function at multiple levels by mediating adhesion, migration, and survival. However, the molecular mechanisms by which OPN regulates macrophage biology are not well understood. Insights into these mechanisms could allow for the creation of therapeutics designed to selectively target OPN function in inflammatory diseases. We explored the role of osteopontin in macrophage-mediated inflammation and defined the OPN functional domains and cell surface receptors mediating this process. OPN interacts with integrins via two main functional domains: the RGD motif that binds to alphaV-containing integrins and the SLAYGLR sequence that binds to integrins alpha4beta1 and alpha9beta1. In chemotaxis studies, we found that migration to OPN was mediated via integrins alpha4 and alpha9 in vitro. We also evaluated the relative contributions of the RGD and SLAYGLR domains of OPN to leukocyte accumulation in an in vivo model of acute inflammation. For these studies we created chimeric mice expressing mutated forms of OPN in macrophages. We found that the SLAYGLR domain of OPN mediates macrophage accumulation in response to thioglycollate-elicited peritonitis. Together, these data suggest that the SLAYGLR domain of OPN interacts with integrins alpha4 and alpha9 to regulate macrophage migration and accumulation. We also determined the effect of OPN on macrophage activation state. Contrary to previous reports, we show that OPN does not affect macrophage activation in terms of pro-inflammatory cytokine expression or cell surface receptor expression. We also found that primary macrophages from wild type and OPN-null mice did not differ in their ability to be polarized to either the M1 or M2 macrophage activation state. Finally, OPN could not induce NF-kB signaling in macrophages, further evidence that OPN does not regulate macrophage activation. Overall, these studies indicate that while OPN does not affect macrophage activation, it does promote macrophage migration and accumulation. Consequently, specifically targeting the interactions between the SLAYGLR domain of OPN and integrins alpha4 and alpha9 could be used therapeutically to reduce macrophage-mediated inflammation.application/pdfen-USCopyright is held by the individual authors.inflammation; macrophage; migration; osteopontinBiomedical engineeringBiologyImmunologyBioengineeringThesis