Search

The interaction of signal regulatory protein α (SIRPα) on macrophages with CD47 on cancer cells is thought to prevent antibody (Ab)-dependent cellular phagocytosis (ADCP) of the latter cells by the former. Blockade of the CD47-SIRPα interaction by Abs to CD47 or to SIRPα, in combination with tumor-targeting Abs such as rituximab, thus inhibits tumor formation by promoting macrophage-mediated ADCP of cancer cells. Here we show that monotherapy with a monoclonal Ab (mAb) to SIRPα that also recognizes SIRPβ1 inhibited tumor formation by bladder and mammary cancer cells in mice, with this inhibitory effect being largely dependent on macrophages. The mAb to SIRPα promoted polarization of tumor-infiltrating macrophages toward an antitumorigenic phenotype, resulting in the killing and phagocytosis of cancer cells by the macrophages. Ablation of SIRPα in mice did not prevent the inhibitory effect of the anti-SIRPα mAb on tumor formation or its promotion of the cancer cell-killing activity of macrophages, however. Moreover, knockdown of SIRPβ1 in macrophages attenuated the stimulatory effect of the anti-SIRPα mAb on the killing of cancer cells, whereas an mAb specific for SIRPβ1 mimicked the effect of the anti-SIRPα mAb. Our results thus suggest that monotherapy with Abs to SIRPα/SIRPβ1 induces antitumorigenic macrophages and thereby inhibits tumor growth and that SIRPβ1 is a potential target for cancer immunotherapy., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Tumor-associated macrophages (TAM) have been classified into an immunostimulatory M1 subset against microbes and malignancies, and an immunoregulatory M2 subset that secretes immunosuppressive cytokines in order to repair tissues damaged by malignancies. The infiltration of M2 in the tumor microenvironment is known to facilitate immunosuppression and tumor-promoting properties. In the present study, we investigated the phagocytic potential of these macrophage subsets in oral squamous cell carcinoma (OSCC) in relation to the expression of CD47, the 'don't eat me' signal against macrophages. The macrophage subsets M1 (induced by GM-CSF and IFN-γ) and M2 (induced by M-CSF and IL-10) were derived from the CD14(+) cells of healthy donors. Phagocytosis of the CFSE-labeled CD47(+) cell line HSC-3 by M1/M2 was assessed using flow cytometry and suppressed by an anti-CD47 neutralizing antibody or CD47 siRNA. Furthermore, CD68(+) and CD163(+) macrophage subset counts infiltrating tumor tissue and the expression of CD47 on cancer cells were examined immunohistochemically in 74 cases of OSCC, and their relationships with clinicopathological parameters or prognoses were determined. The phagocytic potential of M1 was similar to that of M2 in vitro. Phagocytosis by M1 increased in a CD47-dependent manner by the neutralizing antibody and siRNA, but did not in M2. An immunohistochemical (IHC) analysis revealed that the expression of CD47 did not correlate with macrophage subsets in peritumoral tissue or with any clinicopathological parameters; however, the stronger expression of CD47 by cancer cells and larger number of total macrophages/M2 were independently related to shorter survivals. Our results suggest that the expression of CD47 by cancer cells is related to evasion from phagocytosis, particularly that by M1 in vitro. IHC results indicate that various mechanisms are involved in the engulfing potential of TAM subsets in vivo.

Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein-tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow-derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2(flox/flox);Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Signal regulatory protein α (SIRPα), also known as SHPS-1/BIT/ CD172a, is an immunoglobulin superfamily protein that binds to the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region. CD47, another immunoglobulin superfamily protein, is a ligand for SIRPα, with the two proteins constituting a cell-cell communication system (the CD47-SIRPα signalling system). SIRPα is particularly abundant in the myeloid-lineage hematopoietic cells such as macrophages or dendritic cells (DCs), whereas CD47 is expressed ubiquitously. Interaction of CD47 (on red blood cells) with SIRPα (on macrophages) is thought to prevent the phagocytosis by the latter cells of the former cells, determining the lifespan of red blood cells. Recent studies further indicate that this signalling system plays important roles in engraftment of hematopoietic stem cells as well as in tumour immune surveillance through regulation of the phagocytic activity of macrophages. In the immune system, the CD47-SIRPα interaction is also important for the development of a subset of CD11c(+)DCs as well as organization of secondary lymphoid organs. Finally, the CD47-SIRPα signalling system likely regulates bone homeostasis by osteoclast development. Newly emerged functions of the CD47-SIRPα signalling system thus provide multiple therapeutic strategies for cancer, autoimmune diseases and bone disorders., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

SHPS-1 is a transmembrane protein predominantly expressed in macrophages. The possible role of SHPS-1 in regulation of Toll-like receptor (TLR)-dependent production of proinflammatory cytokines by macrophages has remained unknown, however. We now show that expression either of a mutant version of mouse SHPS-1 (SHPS-1-4F) in which the four tyrosine phosphorylation sites in the cytoplasmic region are replaced by phenylalanine or of a chimeric protein comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1-4F (CD8-4F) markedly promoted the production of tumor necrosis factor-alpha (TNF-alpha) or interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [poly(I : C)] in RAW264.7 macrophages. In contrast, expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region did not promote such responses. Expression of SHPS-1-4F promoted the LPS- or poly(I : C)-induced activation of NF-kappaB. LPS and poly(I : C) each induced the tyrosine phosphorylation of SHPS-1 through a Src family kinase and the association of SHPS-1 with SHP-1 and SHP-2. These results suggest that LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with SHP-1 and SHP-2 in a manner dependent on a Src family kinase. SHPS-1 then negatively regulates TLR4- or TLR3-dependent cytokine production through inhibition of NF-kappaB-dependent signaling.

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 Fc gammaR-mediated phagocytosis of mouse RBCs or SRBCs. Forced expression of another mutant form (SHPS-1-deltaCyto) 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-deltaCyto, in RAW264.7 cells enhanced Fc gammaR-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.

The interaction of CD47 on red blood cells (RBCs) with SHPS-1 on macrophages is implicated to prevent the phagocytosis of the former cells by the latter cells. Indeed, the rate of clearance of transfused CD47-deficient (CD47(-/-)) RBCs from the bloodstream of wild-type mice was markedly increased compared with wild-type RBCs. Conversely, the rate of clearance of transfused wild-type RBCs was markedly increased in mice that expressed a mutant form of SHPS-1 lacking most of the cytoplasmic region of the protein. However, we here found that the clearance of CD47(-/-) RBCs in SHPS-1 mutant mice was minimal. In addition, the phagocytosis of CD47(-/-) RBCs by splenic macrophages from SHPS-1 mutant mice was markedly reduced compared with wild-type macrophages. These results thus suggest an additional role for CD47 on RBCs in the negative regulation of phagocytosis by macrophages and in determination of the life span of circulating RBCs.

The lifespan of circulating red blood cells (RBCs) produced in bone marrow is determined by their elimination through phagocytosis by splenic macrophages. The mechanism by which RBC elimination is regulated has remained unclear, however. The surface glycoprotein SHPS-1, a member of the immunoglobulin superfamily, is abundant in macrophages. We have now examined the regulation of RBC turnover with the use of mice that express a mutant form of SHPS-1 lacking most of its cytoplasmic region. The mutant mice manifested mild anemia as well as splenomegaly characterized by expansion of the red pulp. The numbers of erythroid precursor cells in the spleen and of circulating reticulocytes were also increased in the mutant mice. In contrast, the half-life of circulating RBCs was reduced in these animals, and the rate of clearance of injected opsonized RBCs from the peripheral circulation was increased in association with their incorporation into splenic macrophages. Phagocytosis of opsonized RBCs by splenic macrophages from mutant mice in vitro was also increased compared with that observed with wild-type macrophages. These results suggest that SHPS-1 negatively regulates the phagocytosis of RBCs by splenic macrophages, thereby determining both the lifespan of individual RBCs and the number of circulating erythrocytes.

Mononuclear phagocytes such as dendritic cells (DCs) and macrophages in the lamina propria (LP) are thought to be important for both induction of inflammatory responses and maintenance of immunologic tolerance in the mammalian intestine. The molecular mechanisms by which these cells regulate intestinal immunity have remained poorly understood, however. Signal regulatory protein α (SIRPα) is a transmembrane protein that is specifically expressed in DCs, macrophages and neutrophils. Here, we show that SIRPα is abundant in CD11c CD11b LP cells of the mouse intestine. Whereas SIRPα did not appear to be important for the steady-state homeostasis of mucosal immunity in the intestine, the flagellin-stimulated production of IL-17 or interferon (IFN)-γ by LP cells of SIRPα mutant (MT) mice that lack the cytoplasmic region of the protein was markedly decreased compared with that observed with wild-type cells. Moreover, the flagellin-induced production of IL-6 by LP cells from SIRPα MT mice was also greatly reduced. SIRPα MT mice were also resistant to the development of colitis induced by IL-10 deficiency. Our data thus suggest that SIRPα expressed on CD11c LP cells is important for the production of IL-17 or IFN-γ in the LP as well as for the development of colitis induced by IL-10 deficiency. [ABSTRACT FROM AUTHOR]

SIRPβ (signal-regulatory protein β) is a transmembrane protein that is expressed in hematopoietic cells but whose functions are unknown. We have now cloned mouse SIRPβ cDNA and have shown that the gene is expressed in various tissues in addition to cells of the macrophage lineage. Engagement of SIRPβ by specific monoclonal antibodies promoted Fcγ receptor-dependent or -independent phagocytosis in mouse peritoneal macrophages. It also induced marked activation of MAPK and the upstream kinase MEK but weak activation of Akt. MEK inhibitors markedly blocked the promotion of phagocytosis by SIRPβ, whereas an inhibitor of phosphoinositide 3-kinase partly blocked such response. In addition, inhibitors of myosin light chain kinase or of myosin ATPase blocked the promotion of phagocytosis by SIRPβ. Furthermore, SIRPβ induced the formation of filopodia and lamellipodia in macrophages as well as the translocation of activated MAPK to these structures. It also elicited tyrosine phosphorylation of DAP12, Syk, and SLP-76, and a Syk inhibitor blocked the promotion of phagocytosis and activation of MAPK by SIRPβ. Our results suggest that engagement of SIRPβ promotes phagocytosis in macrophages by inducing the tyrosine phosphorylation of DAP12, Syk, and SLP-76 and the subsequent activation of a MEK-MAPK-myosin light chain kinase cascade. [ABSTRACT FROM AUTHOR]

Abstract: Signal regulatory protein α (SIRPα) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Wild-type (WT) C57BL/6 mice are known to be resistant to Leishmania major infection. We here found that C57BL/6 mice that express a mutant version of SIRPα lacking most of the cytoplasmic region manifested increased susceptibility to L. major infection, characterized by the marked infiltration of inflammatory cells in the infected lesions. The numbers of the parasites in footpads, draining lymph nodes and spleens were also markedly increased in the infected SIRPα mutant mice, compared with those for the infected WT mice. In addition, soluble leishmanial antigen-induced production of IFN-γ by splenocytes of the infected SIRPα mutant mice was markedly reduced. By contrast, the ability of macrophages of SIRPα mutant mice to produce nitric oxide in response to IFN-γ was almost equivalent to that of macrophages from WT mice. These results suggest that SIRPα is indispensable for protective immunity against L. major by the induction of Th1 response. [ABSTRACT FROM AUTHOR]

Signal regulatory protein (SIRP)α, also known as SHPS-1 or SIRPA, is a transmembrane protein that binds to the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is predominantly expressed in neurons, dendritic cells and macrophages. CD47, a widely expressed transmembrane protein, is a ligand for SIRPα, with the two proteins constituting a cell–cell communication system. The interaction of SIRPα with CD47 is important for the regulation of migration and phagocytosis. Recent studies have implicated the CD47–SIRPα signalling pathway in immune homeostasis and in regulation of neuronal networks. Advances in the structural and functional analyses of the CD47–SIRPα signalling pathway now provide exciting hints of the therapeutic benefits of manipulating this signalling system in autoimmune diseases and neurological disorders. [Copyright &y& Elsevier]