Tethering of Apoptotic Cells to Phagocytes through Binding of CD47 to Src Homology 2 Domain-Bearing Protein Tyrosine Phosphatase Substrate-1

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein β (Sirpβ1 and Sirpβ4) molecules were identified as differentially expressed in CD8− cDC. Genomic sequence analysis revealed a third Sirpβ member localized in the same gene cluster. These Sirpβ genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpβ1 and β2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpβ genes were expressed by CD8− cDC, but not by CD8+ cDC or plasmacytoid pre-DC. The related Sirpα gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpα and Sirpβ1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpβ1. Cross-linking of Sirpβ1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpβ1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8− cDC.

Section: Discussionmentioning

confidence: 99%

Signal Regulatory Protein Molecules Are Differentially Expressed by CD8− Dendritic Cells

Lahoud

Proietto

Gartlan

et al. 2006

“…The mechanism by which SHPS-1-4F facilitates the attachment of RBCs to RAW264.7 cells remains unclear, however. The binding of CD47 on apoptotic cells to SHPS-1 on macrophages is implicated in the tethering of the former cells to the latter (24). Indeed, the attachment of IgG-opsonized CD47 Ϫ/Ϫ RBCs to either parental RAW264.7 cells or m4F13 cells was markedly reduced compared with that of WT RBCs, suggesting that the interaction between CD47 (on RBCs) and SHPS-1 (on RAW264.7 cells) also contributes to the tethering of the former cells to the latter.…”

Section: Discussionmentioning

confidence: 99%

“…A mouse mAb to rat SHPS-1 (9F10) was generated with a rat SHPS-1 fusion protein (the extracellular region of rat SHPS-1 fused to the Fc region of human IgG) as Ag, as described previously (23). Hamster mAbs to mouse SHPS-1 (15D9 and FG2) (24) were provided by S. Nagata (Osaka University, Osaka, Japan). Rabbit polyclonal Abs to SHPS-1 (anti-SHPS-1 Cyto) were obtained from ProSci.…”

Section: Abs and Reagentsmentioning

confidence: 99%

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

Ikeda

Okazawa

Ohnishi

et al. 2006

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.

“…Its interaction with SIRP-␣ prevents the elimination and clearance of RBC and mononuclear cells by macrophages (18,19). By contrast, CD47 expression on apoptotic cells is absolutely required for their engulfment by APCs, suggesting that CD47/SIRP-␣ interaction works as a tethering step in phagocytosis (20). Finally, CD47/SIRP-␣ interaction modulates neutrophil transendothelial and transepithelial migration (21).…”

Section: Endritic Cells (Dc)mentioning

confidence: 99%

Semimature Stage: A Checkpoint in a Dendritic Cell Maturation Program That Allows for Functional Reversion after Signal-Regulatory Protein-α Ligation and Maturation Signals

Braun

Galibert

Nakajima

et al. 2006

CD47 on live cells actively engages signal-regulatory protein-α (SIRP-α) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-α ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-α allowed the generation of mature migratory DCs not producing IL-12, IFN-γ-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1β, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-α-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-α expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-α pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.