Neutrophils, polymorphonuclear leukocytes representing the major class of blood leukocytes, move during infection or injury to sites of inflammation in a process mediated by surface interactions with endothelial cell adhesion molecules. Selective removal and turnover of neutrophils and other leukocytes is the key to inflammatory resolution, but little is known about the mechanisms involved. It is commonly thought that extravasated, activated neutrophils undergo apoptosis during inflammation, and that this leads to their phagocytosis and removal through recognition of surface exposed phosphatidylserine (PS). However, the precise contribution of apoptosis to this process, if any, is not clear. There is accumulating evidence that neutrophils entering inflamed tissues may be removed by phagocytosis in apoptosis-independent pathways. While in the process of identifying factors that contribute to leukocyte turnover independently of apoptosis, we discovered that endothelial cells express high levels of galectin-1 (Gal-1), a member of the large family of B-galactoside-binding lectins. Specific binding of Gal-1 to activated, but not resting, human neutrophils induces surface expression of PS and leads to recognition and phagocytosis by macrophages inde4pendently of apoptosis. We found that the signaling responses to Gal-1 binding were shared by several other recombinant galectins that may also be expressed by endothelial cells. Interestingly, Gal-1 did not signal PS exposure in activated lymphocytes. Moreover, we found in activated neutrophils that Gal-1 binding leading to surface exposure of PS elevates cytosolic Ca[2+] and engages Src kinases, phospholipase C-y, and possibly scramblase-like proteins. In addition, our data implicate an association of this pathway with specialized membrane microdomains (lipid rafts). Our findings suggest an alternative mechanism to apoptosis-dependent surface expression of PS, one that marks neutrophils for phagocytosis through galectin-dependent signaling. We hypothesize that galectins bind to surface glycoconjugates on activated neutrophils and leads to PS exposure. This has important implications for regulation of leukocyte turnover and the resolution of inflammation. We propose three specific aims to explore this novel hypothesis and the potential role(s) of galectins and their receptors in the signaling pathway of this process. It is important to note that these aims complement and support the aims of Projects 1 and 3 in this application.
Aim 1 We will isolate and characterize the leukocyte surface receptors for galectins in resting versus activated human neutrophils, galectin-responsive selected leukocyte cell lines, and leukocytes and endothelial cells from mice engineered to express altered glycans (Projects 1 and 3). Preliminary results indicate that N-glycosylated proteins are special ligands for galectins and may reside in lipid rafts.
Aim 2 We will investigate the mechanism of galectin-induced PS exposure, the role of Ca2+ mobilization and scramblase-like proteins, and the functions of membrane microdomains in this signaling pathway. Our preliminary evidence indicates that Gal-1 signaling in activated human neutrophils is propagated through Src kinase and PLC-y, and involves intra- and extracellular Ca[2+] mobilization.
Aim 3 We will explore the expression of galectins in human and murine ECs (Projects 1 and 3) and the mechanisms and factors that may cause secretion of active galectins in EC cells. Our preliminary studies show that human umbilical vein endothelial cells (HUVECs) constitutively synthesize Gal-1. ECs may express other galectins, but the mechanisms of galectin(s) release/export are not known. Our success in these studies should provide important insights into galectin functions in cell signaling and mechanisms regulating leukocyte turnover. These will be important in understanding inflammatory resolution and are likely to expedite development of new treatments for inflammation-related diseases.
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