Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

Abstract

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.

Cutting Edge: The Wiskott-Aldrich Syndrome Protein Is Required for Efficient Phagocytosis of Apoptotic Cells

“…Therefore, in the absence of WASp, other related molecules such as SCARs/WAVEs presumably contribute to the recruitment of the Arp2/3 complex and subsequent actin polymerization, as suggested for CR3-mediated phagocytosis (13). Consistent with this, we have observed that Rac, which can activate SCARs/WAVEs, as well as Cdc42, is required for apoptotic cell uptake (18,24).…”

“…Cdc42 is known to activate WASp, and indeed we have observed that Cdc42 is required for uptake of apoptotic cells (18). It is likely that interaction between WASp and the Arp2/3 complex contributes significantly to the actin polymerization necessary for this process to occur (19).…”

“…Apoptotic cell uptake by normal macrophages is accompanied by polymerization of actin and the formation of an actin cup (18). To determine the role of WASp in this process, we examined its localization during formation of the actin cup around apoptotic Baf-3 cells.…”

A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

“…These data suggest that lamellipodial actin spreading is required for the formation of CD48iGFP APC extensions. Cdc42 regulates lamellipodial actin spreading (37) as well as phagocytosis (38) and macropinocytosis (44). When we blocked lamellipodial actin spreading with 100 nM dominant-negative Cdc42 as a protein transduction reagent (Cdc42dn) (37), only 31% of the T cell/APC couples showed CD48iGFP APC extensions as opposed to 69% with buffer only ( p Ͻ 0.001).…”

State of the Art. Apoptosis and Cell Homeostasis in Chronic Obstructive Pulmonary Disease

“…In addition, apoptotic cell uptake is significantly regulated by the balance between Rac-1, which is required, and RhoA, which is inhibitory (30)(31)(32), and this balance can be seen both in vivo and in vitro, and across different cell types. This raises the intriguing possibility that an intrinsic, or induced, imbalance between these low-molecular-weight GTPases in potential efferocytes may contribute to the impaired clearance, and indeed to the potential for some smokers to develop COPD.…”