Characterization of the Effects of Cross-Linking of Macrophage CD44 Associated with Increased Phagocytosis of Apoptotic PMN

Simon P Hart,Adriano G Rossi,Ian Dransfield,Christopher Haslett

doi:10.1371/journal.pone.0033142

Abstract

Control of macrophage capacity for apoptotic cell clearance by soluble mediators such as cytokines, prostaglandins and lipoxins, serum proteins, and glucocorticoids may critically determine the rate at which inflammation resolves. Previous studies suggested that macrophage capacity for clearance of apoptotic neutrophils was profoundly altered following binding of CD44 antibodies. We have used a number of different approaches to further define the mechanism by which CD44 rapidly and specifically augment phagocytosis of apoptotic neutrophils. Use of Fab' fragments unequivocally demonstrated a requirement for cross-linking of macrophage surface CD44. The molecular mechanism of CD44-augmented phagocytosis was shown to be opsonin-independent and to be distinct from the Mer/protein S pathway induced by glucocorticoids and was not functional for clearance of apoptotic eosinophils. CD44-cross-linking also altered macrophage migration and induced cytoskeletal re-organisation together with phosphorylation of paxillin and activation of Rac2. Investigation of signal transduction pathways that might be critical for CD44 augmentation of phagocytosis revealed that Ca2+ signalling, PI-3 kinase pathways and altered cAMP signalling were not involved, but did implicate a key role for tyrosine phosphorylation events. Finally, although CD44 antibodies were able to augment phagocytosis of apoptotic neutrophils by murine peritoneal and bone marrow-derived macrophages, we did not observe a difference in the clearance of neutrophils following induction of peritonitis with thioglycollate in CD44-deficient animals. Together, these data demonstrate that CD44 cross-linking induces a serum opsonin-independent mechanism of macrophage phagocytosis of apoptotic neutrophils that is associated with reduced macrophage migration and cytoskeletal reorganisation.

Highlights

Development of novel, effective therapeutic strategies for treatment of inflammatory diseases requires an understanding the cellular and molecular mechanisms underlying development and progression of inflammation [1]
We previously reported that F(ab9)2 fragments of CD44 mAb were able to augment macrophage phagocytosis of apoptotic cells [24]
When bound 5A4 Fab9 fragments were cross-linked by addition of F(ab9)2 anti-mouse immunoglobulins, we observed a rapid increased in the proportion of macrophages capable of phagocytosis of apoptotic PMN, unequivocally demonstrating a requirement for cross-linking of CD44

Summary

Introduction

Development of novel, effective therapeutic strategies for treatment of inflammatory diseases requires an understanding the cellular and molecular mechanisms underlying development and progression of inflammation [1]. A critical event in the resolution of inflammatory responses is the clearance of recruited inflammatory granulocytes, via the co-ordinated induction of programmed cell death (apoptosis) and subsequent clearance of apoptotic cells by tissue phagocytes [8]. This mechanism has been elegantly confirmed in experimental models of inflammation, where acceleration of neutrophil apoptosis facilitates early resolution and reduction in tissue injury [9]. Multiple molecular mechanisms may be involved in the clearance of apoptotic cells by phagocytes [13], uptake of apoptotic cells suppresses toll-like receptor-driven production of pro-inflammatory mediators by macrophages and can induce release of IL-10 and TGF-b that have the potential to exert anti-inflammatory effects [14,15]

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Results

Conclusion