Mutational Analysis of the Mechanism of Negative Regulation by Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate-1 of Phagocytosis in Macrophages

Abstract

Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) is a transmembrane protein predominantly expressed in macrophages. The binding of CD47 on RBCs to SHPS-1 on macrophages is implicated in inhibition of phagocytosis of the former cells by the latter. We have now shown that forced expression in mouse RAW264.7 macrophages of a mutant version (SHPS-1-4F) of mouse SHPS-1, in which four tyrosine phosphorylation sites are replaced by phenylalanine, markedly promoted FcγR-mediated phagocytosis of mouse RBCs or SRBCs. Forced expression of another mutant form (SHPS-1-ΔCyto) of mouse SHPS-1, which lacks most of the cytoplasmic region, did not promote such phagocytosis. Similarly, forced expression of a rat version of SHPS-1-4F, but not that of rat wild-type SHPS-1 or SHPS-1-ΔCyto, in RAW264.7 cells enhanced FcγR-mediated phagocytosis of RBCs. Tyrosine phosphorylation of endogenous SHPS-1 as well as its association with Src homology 2 domain-containing protein tyrosine phosphatase-1 were not markedly inhibited by expression of SHPS-1-4F. Furthermore, the attachment of IgG-opsonized RBCs to RAW264.7 cells was markedly increased by expression of SHPS-1-4F, and this effect did not appear to be mediated by the interaction between CD47 and SHPS-1. These data suggest that inhibition by SHPS-1 of phagocytosis in macrophages is mediated, at least in part, in a manner independent of the transinteraction between CD47 and SHPS-1. In addition, the cytoplasmic region as well as tyrosine phosphorylation sites in this region of SHPS-1 appear indispensable for this inhibitory action of SHPS-1. Moreover, SHPS-1 may regulate the attachment of RBCs to macrophages by an as yet unidentified mechanism.

‘Clustering’ SIRPα into the Plasma Membrane Lipid Microdomains Is Required for Activated Monocytes and Macrophages to Mediate Effective Cell Surface Interactions with CD47

“…Studies have shown that SIRPα and CD47-mediated cell surface interactions play a critical role in monocyte chemotactic transmigration and macrophage recognition of phagocytic targets. Inhibition of the SIRPα-CD47 interaction delays monocyte transmigration [6] and also perturbs macrophages to elicit intracellular signaling, which normally restrains phagocytosis of the surrounding cells [2,17-22,34]. Consistently, our cell adhesion assays showed that monocytes that had been activated and had transmigrated through the endothelial monolayers or that had differentiated into macrophages displayed significant direct binding to CD47.…”

“…It has been suggested that the extracellular binding of SIRPα to CD47 triggers tyrosine phosphorylation in its cytoplasmic ITIMs, resulting in the association of the SH2 domain-containing protein tyrosine phosphatase (SHP-1 or 2), which consequently initiates negative signaling events leading to the inhibition of cell function [11-16]. Studies of macrophage phagocytosis indicate that ligation of macrophage SIRPα by CD47 expressed on the encountered cells prohibits macrophage phagocytosis, whereas failure of SIRPα engagement by CD47, or deficiency of SIRPα ITIM-mediated signaling, promotes macrophage engulfment of the cell [2,8,17-22]. It has also been suggested that increased CD47 expression on cancer cells serves as a crucial mechanism for the evasion of immune clearance [23,24].…”

Loss of Cell Surface CD47 Clustering Formation and Binding Avidity to SIRPα Facilitate Apoptotic Cell Clearance by Macrophages

“…Accumulating evidence has shown that ligation of macrophage SIRPα by CD47 expressed on the encountered cells prohibits macrophage phagocytosis, whereas failure of SIRPα engagement by CD47, or deficiency of SIRPα ITIM-mediated signaling, promotes macrophage engulfment of the host cell (1, 3, 42–45). Through over-expressing CD47 on the surface, certain types of cancer cells can escape from macrophage-mediated immune clearance (46, 47).…”

Cd47

“…40,41 This signal depends on the docking of SHP-1 phosphatase to phosphorylated ITIMS in the SIRP␣ cytoplasmic domain leading to SHP-1 activation and presumably dephosphorylation of one or more key components of the phagocytic machinery. 42,43 A function of this mechanism is suggested by the report that phagocytosis of xenograft cells is augmented by the species incompatibility of donor CD47 with host SIRP␣. 44 …”