SHPS-1 promotes the survival of circulating erythrocytes through inhibition of phagocytosis by splenic macrophages

Abstract

IntroductionThe lifespan of circulating red blood cells (RBCs) is approximately 120 and 40 days in humans and mice, respectively, and is determined by their production in bone marrow (BM) and their clearance from the peripheral circulation, predominantly in the spleen and liver. [1][2][3] The production of RBCs is controlled by the primary erythropoietic regulator erythropoietin, 2,4 whereas clearance of old RBCs by the spleen is achieved mostly as a result of their specific recognition and phagocytosis by splenic macrophages. 5,6 The precise molecular mechanism by which splenic macrophages recognize senescent RBCs for phagocytosis is largely unknown, however. [1][2][3] Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), 7,8 also known as signal-regulatory protein ␣, 9 brain immunoglobulin (Ig)-like molecule with tyrosinebased activation motifs, 10 and p84 neural adhesion molecule, 11 is a transmembrane protein that is especially abundant in macrophages. [12][13][14] The putative extracellular region of SHPS-1 comprises 3 Ig-like domains, and its cytoplasmic region contains 4 tyrosine phosphorylation sites that mediate the binding of Src homology 2 domain-containing protein tyrosine phosphatases designated SHP-1 and SHP-2. 7,9 Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors and cytokines as well as by integrinmediated cell adhesion to extracellular matrix proteins. 7,9,[15][16][17][18] SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli. In macrophages, tyrosine-phosphorylated SHPS-1 binds SHP-1, 12,19,20 which is implicated in negative regulation of the functions of a variety of hematopoietic cells. [21][22][23] The complex of SHPS-1 and SHP-1 is thus thought to regulate macrophage functions in a negative manner.CD47 is a ligand for the extracellular region of SHPS-1. 24,25 This protein, which was originally identified in association with ␣v␤3 integrin, is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, 5 putative membrane-spanning segments, and a short cytoplasmic tail. 26 CD47 and SHPS-1 constitute a cell-cell communication system (the CD47-SHPS-1 system) that plays important roles in a variety of cellular processes including cell migration, 27,28 adhesion of B cells, 29 and T-cell activation. 14,30 In addition, the CD47-SHPS-1 system is implicated in negative regulation of phagocytosis by macrophages. CD47 is highly expressed on the surface of RBCs, where it associates with the Rh protein complex instead of with integrins. 31 The rate of clearance of CD47-deficient RBCs from the bloodstream was found to be markedly increased compared with that of wild-type (WT) cells. 6,32 Furthermore, the phagocytosis of CD47-deficient RBCs by splenic or BM-derived macrophages was greatly enhanced Supported by a Grant-in-Aid for Scientific Research on Priority Areas Cancer; a Grant-in-Aid for Scientific Research (B); a Grant-in-Aid for Young Scie...

Cd47-Sirpα interaction and IL-10 constrain inflammation-induced macrophage phagocytosis of healthy self-cells

“…Resolution of at least two puzzles remains paramount: first, why macrophages do not attack self in mice deficient of Cd47 or Sirpα? Despite that the Cd47-Sirpα-mediated inhibition is missing, mice deficient of Cd47 (26), Sirpα (established in this study), or the Sirpα cytoplasmic domain (27) are relatively healthy, manifesting no or only minor phenotypes (9,18,28) that suggest macrophage phagocytosis toward self-cells. Adoptive transfer of Cd47-null RBCs into these mutant mice also failed to induce rapid elimination (1) (also shown in this study).…”

“…conjunction with therapeutic anticancer antibodies (4,9,(16)(17)(18)(19)(20)(21). Conversely, the lack of a compatible CD47 to ligate the recipient SIRPα is considered to be associated with tissue rejection in xenotransplantation (22)(23)(24)(25).…”

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

“…Furthermore, the phagocytosis of CD47-deficient RBCs by either splenic or bone marrow-derived macrophages was markedly enhanced in an in vitro assay (6,7). Conversely, we recently showed that the rate of clearance of transfused RBCs from the bloodstream was markedly increased in mice that express a mutant SHPS-1 protein lacking most of the cytoplasmic region, compared with that apparent for WT mice (9). In addition, phagocytosis of opsonized RBCs by peritoneal macrophages (PEMs) derived from the SHPS-1 mutant mice was enhanced compared with that apparent with WT PEMs (8).…”

“…The surface expression of SHPS-1 on RAW264.7 cells was examined by flow cytometry, as described (8,9). In brief, cells were detached from culture dishes by treatment with 0.01% EDTA and then washed with icecold PBS.…”

“…Phagocytosis was assayed in vitro, as previously described (8,9). In brief, opsonized or nonopsonized normal mouse RBCs or RBCs from CD47-deficient mice (6, 7), or IgG-opsonized SRBCs, suspended in 200 l of RPMI 1640, were overlaid on RAW264.7 cells that had been plated at a density of 1.25 ϫ 10 5 cells/well in 24-well plates.…”