SIRPα Mutant Mice Research Articles

Lack of SIRPα phosphorylation and concomitantly reduced SHP-2–PI3K–Akt2 signaling decrease osteoblast differentiation

Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on intricate cellular signaling pathways, including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) have both been suggested to regulate bone cell differentiation. Here we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. This reduced osteoblastic differentiation potential in SIRPα-mutant stromal cells was associated with a significantly reduced expression of Runx2, osterix, osteocalcin, and alkaline phosphatase mRNA, as well as a reduced phosphorylation of SHP-2 and Akt2, as compared with that in wild-type stromal cells. Addition of a PI3K-inhibitor to wild-type stromal cells could mimic the impaired osteoblastogenesis seen in SIRPα-mutant cells. In conclusion, our data suggest that SIRPα signaling through SHP-2–PI3K–Akt2 strongly influences osteoblast differentiation from bone marrow stromal cells.

Non-Hematopoietic and Hematopoietic SIRPα Signaling Differently Regulates Murine B Cell Maturation in Bone Marrow and Spleen.

B lymphocyte development occurs in the bone marrow, while final differentiation and maturation can occur in both the bone marrow and the spleen. Here we provide evidence that signal regulatory protein α (SIRPα), an Ig-superfamily ITIM-receptor expressed by myeloid but not by lymphoid cells, is involved in regulating B cell maturation. Lack of SIRPα signaling in adult SIRPα-mutant mice resulted in a reduced maturation of B cells in the bone marrow, evident by reduced numbers of semi-mature IgD+IgMhi follicular type-II (F-II) and mature IgD+IgMlo follicular type-I (F-I) B cells, as well as reduced blood B cell numbers. In addition, lack of SIRPα signaling also impaired follicular B cell maturation in the spleen. Maturing BM or splenic B cells of SIRPα-mutant mice were found to express higher levels of the pro-apoptotic protein BIM and apoptosis was increased among these B cells. Bone marrow reconstitution experiments revealed that the B cell maturation defect in bone marrow and blood was due to lack of SIRPα signaling in non-hematopoietic cells, while hematopoietic SIRPα signaling was important for follicular B cell maturation in the spleen. Adding on to our previous findings of a stromal cell defect in SIRPα-mutant mice was the finding that gene expression of receptor activator of nuclear factor-ĸB ligand (RANKL) was significantly lower in cultured bone marrow stromal cells of SIRPα mutant mice. These data suggest a novel and opposite contribution of SIRPα signaling within non-hematopoietic and hematopoietic cells, respectively, to maintain B cell maturation and to prevent apoptosis in the bone marrow and spleen of adult mice.

Regulation of Phagocyte Migration by Signal Regulatory Protein-Alpha Signaling.

Signaling through the inhibitory receptor signal regulatory protein-alpha (SIRPα) controls effector functions in phagocytes. However, there are also indications that interactions between SIRPα and its ligand CD47 are involved in phagocyte transendothelial migration. We have investigated the involvement of SIRPα signaling in phagocyte migration in vitro and in vivo using mice that lack the SIRPα cytoplasmic tail. During thioglycolate-induced peritonitis in SIRPα mutant mice, both neutrophil and macrophage influx were found to occur, but to be significantly delayed. SIRPα signaling appeared to be essential for an optimal transendothelial migration and chemotaxis, and for the amoeboid type of phagocyte migration in 3-dimensional environments. These findings demonstrate, for the first time, that SIRPα signaling can directly control phagocyte migration, and this may contribute to the impaired inflammatory phenotype that has been observed in the absence of SIRPα signaling.

Signal regulatory protein alpha (SIRPα) regulates the homeostasis of CD103+CD11b+DCs in the intestinal lamina propria

Signal regulatory protein alpha (SIRPα/CD172a) is a conserved transmembrane protein thought to play an inhibitory role in immune function by binding the ubiquitous ligand CD47. SIRPα expression has been used to identify dendritic cell subsets across species and here we examined its expression and function on intestinal DCs in mice. Normal mucosa contains four subsets of DCs based on their expression of CD103 and CD11b and three of these express SIRPα. However, loss of SIRPα signaling in mice leads to a selective reduction in the CD103+CD11b+ subset of DCs in the small intestine, colon, and among migratory DCs in the mesenteric lymph node. In parallel, these mice have reduced numbers of TH17 cells in steady-state intestinal mucosa, and a defective TH17 response to Citrobacter infection. Identical results were obtained in CD47KO mice. DC precursors from SIRPα mutant mice had an enhanced ability to generate CD103+CD11b+ DCs in vivo, but CD103+CD11b+ DCs from mutant mice were more prone to die by apoptosis. These data show a previously unappreciated and crucial role for SIRPα in the homeostasis of CD103+CD11b+ DCs in the intestine, as well as providing further evidence that this subset of DCs is critical for the development of mucosal TH17 responses.

SIRPα signaling regulates podocyte structure and function

Signal-regulatory protein-α (SIRPα) is a transmembrane protein that contains tyrosine phosphorylation sites in its cytoplasmic region; two tyrosine phosphatases, SHP-1 and SHP-2, bind to these sites in a phosphorylation-dependent manner and transduce multiple intracellular signals. Recently, SIRPα was identified as one of the major tyrosine-phosphorylated proteins in the glomeruli and found to be expressed in podocytes. In the present study, we examined the role of SIRPα expression in podocytes using knockin mice (C57BL/6 background) expressing mutant SIRPα that lacks a cytoplasmic region (SIRPα-mutant mice). Light microscopic examination revealed no apparent morphological abnormalities in the kidneys of the SIRPα-mutant mice. On the other hand, electron microscopic examination revealed abnormal podocytes with irregular major processes and wider and flattened foot processes in the SIRPα-mutant mice compared with their wild-type counterparts. Significantly impaired renal functions and slight albuminuria were demonstrated in the SIRPα-mutant mice. In addition, adriamycin injection induced massive albuminuria together with focal glomerulosclerosis in the SIRPα-mutant mice, while their wild-type counterparts were resistant to adriamycin-induced nephropathy. These data demonstrate that SIRPα is involved in the regulation of podocyte structure and function as a filtration barrier under both physiological and pathological conditions.

SIRPα Controls the Activity of the Phagocyte NADPH Oxidase by Restricting the Expression of gp91phox

The phagocyte NADPH oxidase mediates oxidative microbial killing in granulocytes and macrophages. However, because the reactive oxygen species produced by the NADPH oxidase can also be toxic to the host, it is essential to control its activity. Little is known about the endogenous mechanism(s) that limits NADPH oxidase activity. Here, we demonstrate that the myeloid-inhibitory receptor SIRPα acts asanegative regulator of the phagocyte NADPH oxidase. Phagocytes isolated from SIRPα mutant mice were shown to have an enhanced respiratory burst. Furthermore, overexpression of SIRPα in human myeloid cells prevented respiratory burst activation. The inhibitory effect required interactions between SIRPα and its natural ligand, CD47, as wellas signaling through the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. Suppression of the respiratory burst by SIRPα was caused by a selective repression of gp91(phox) expression, the catalytic component of the phagocyte NADPH oxidase complex. Thus, SIRPα can limit gp91(phox) expression during myeloid development, thereby controlling the magnitude of the respiratory burst in phagocytes.

Signal regulatory protein α regulates the homeostasis of T lymphocytes in the spleen.

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell-cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4(+) T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin β receptor, as well as the in vivo response to lymphotoxin β receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.

Regulation by SIRPα of dendritic cell homeostasis in lymphoid tissues

AbstractThe molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11chigh DCs (conventional DCs, or cDCs), in particular, that of CD8−CD4+ (CD4+) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRPα that lacks the cytoplasmic region. We also found that SIRPα is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4+ cDCs. Differentiation of bone marrow cells from SIRPα mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4+ cDCs was markedly reduced in SIRPα mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4+ cDCs and CD8−CD4−(double negative) cDCs in the spleen. SIRPα as well as its ligand, CD47, are thus important for the homeostasis of CD4+ cDCs or double negative cDCs in lymphoid tissues.

Role of SIRPα in regulation of mucosal immunity in the intestine

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.

Essential roles of SIRPα in homeostatic regulation of skin dendritic cells

Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily protein that is predominantly expressed in dendritic cells (DCs). Its cytoplasmic region binds SHP-1 or SHP-2 protein tyrosine phosphatases, while its extracellular region interacts with CD47, another immunoglobulin superfamily protein, constituting cell-cell signaling. SIRPα was previously shown to be important for development of contact hypersensitivity, likely as a result of its positive regulation of the priming by DCs of CD4 + T cells. However, the mechanism by which SIRPα regulates DC functions remains unknown. Here we found that the number of I-A + cells, which represent migratory DCs such as Langerhans cells (LCs) or dermal DCs from the skin, in the peripheral lymph nodes (LNs) was markedly decreased in mice expressing a mutant form of SIRPα that lacks the cytoplasmic region compared with that of wild-type (WT) mice. In addition, an increase of fluorescein isothiocyanate (FITC)-bearing I-A + cells in the draining lymph nodes (LNs) after skin-painting with FITC was markedly blunted in SIRPα mutant mice. However, migratory ability, as well as expression of CCR7, of bone marrow-derived DCs prepared from SIRPα mutant mice were not impaired. By contrast, the number of I-A + LCs in the epidermis of SIRPα mutant mice was markedly decreased compared with that of WT mice. In addition, the mRNA expression of transforming growth factor-β receptor II in LCs of SIRPα mutant mice was markedly decreased compared with that of WT mice. These results suggest that SIRPα is important for homeostasis of LCs in the skin, as well as of migratory DCs in the LNs, but unlikely for migration of these cells from the skin to draining LNs.

Requirement of SIRPα for protective immunity against Leishmania major

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.