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A regulatory role for Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) in phagocytosis mediated by Fc gamma receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18).

Cox D, Dale BM, Kashiwada M, Helgason CD, Greenberg S - J. Exp. Med. (2001)

Bottom Line: To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; alpha(M)beta(2))-dependent phagocytosis.In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis.We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating beta(2) integrin outside-in signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

ABSTRACT
The Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) is recruited to immunoreceptor tyrosine-based inhibition motif (ITIM)-containing proteins, thereby suppressing phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. The role of SHIP in phagocytosis, a PI 3-kinase-dependent pathway, is unknown. Overexpression of SHIP in macrophages led to an inhibition of phagocytosis mediated by receptors for the Fc portion of IgG (Fc gamma Rs). In contrast, macrophages expressing catalytically inactive SHIP or lacking SHIP expression demonstrated enhanced phagocytosis. To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; alpha(M)beta(2))-dependent phagocytosis. Macrophages overexpressing SHIP demonstrated impaired CR3-mediated phagocytosis, whereas macrophages expressing catalytically inactive SHIP demonstrated enhanced phagocytosis. CR3-mediated phagocytosis in macrophages derived from SHIP(-/-) mice was up to 2.5 times as efficient as that observed in macrophages derived from littermate controls. SHIP was localized to Fc gamma R- and CR3-containing phagocytic cups and was recruited to the cytoskeleton upon clustering of CR3. In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis. We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating beta(2) integrin outside-in signaling.

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SHIP, but not c-fms, localizes to CR3-mediated phagocytic cups. Adherent thio-macrophages obtained from SHIP+/+ mice were incubated with EC3bi, as described in Materials and Methods, for 12 min at 37°C. After fixation, cells were stained for the presence of SHIP or c-fms (negative control), and C3bi as described in Materials and Methods. (A and D) Phase–contrast; (B) anti-SHIP; (E) anti–c-fms; (C and F) anti-C3. Bar, 10 μm.
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Figure 6: SHIP, but not c-fms, localizes to CR3-mediated phagocytic cups. Adherent thio-macrophages obtained from SHIP+/+ mice were incubated with EC3bi, as described in Materials and Methods, for 12 min at 37°C. After fixation, cells were stained for the presence of SHIP or c-fms (negative control), and C3bi as described in Materials and Methods. (A and D) Phase–contrast; (B) anti-SHIP; (E) anti–c-fms; (C and F) anti-C3. Bar, 10 μm.

Mentions: To determine whether CR3 ligation results in an alteration in the distribution of SHIP, we performed indirect immunofluorescence in cells undergoing CR3-mediated phagocytosis. SHIP, but not the receptor for CSF-1, c-fms, localized to EC3bi-containing phagosomes (Fig. 6). The specificity of SHIP localization is evident from a lack of circumferential staining around phagosomes for another protein expressed in macrophages, c-fms (Fig. 6 E), and absence of SHIP staining in macrophages derived from SHIP−/− mice (data not shown). To confirm that CR3 ligation is sufficient to induce SHIP redistribution, we determined the cytoskeletal localization of SHIP and the αM subunit of CR3 after CR3 ligation. Both SHIP and αM, but not CD14, demonstrated enhanced cytoskeletal incorporation after mAb-induced clustering of CR3 (Fig. 7). In addition, there was a 2.2 ± 0.4–fold (mean ± SEM, n = 3) increase in the phosphotyrosyl content of SHIP after the clustering of CR3 using mAb M1/70 followed by a cross-linking antibody. In contrast, the phosphotyrosyl content of SHIP in macrophages incubated with an IgG2b control mAb followed by a cross-linking antibody was 0.8 ± 0.3–fold (mean ± SEM, n = 3) of unstimulated cells. Therefore, engagement of CR3 is sufficient to mediate an alteration in the localization of SHIP in macrophages.


A regulatory role for Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) in phagocytosis mediated by Fc gamma receptors and complement receptor 3 (alpha(M)beta(2); CD11b/CD18).

Cox D, Dale BM, Kashiwada M, Helgason CD, Greenberg S - J. Exp. Med. (2001)

SHIP, but not c-fms, localizes to CR3-mediated phagocytic cups. Adherent thio-macrophages obtained from SHIP+/+ mice were incubated with EC3bi, as described in Materials and Methods, for 12 min at 37°C. After fixation, cells were stained for the presence of SHIP or c-fms (negative control), and C3bi as described in Materials and Methods. (A and D) Phase–contrast; (B) anti-SHIP; (E) anti–c-fms; (C and F) anti-C3. Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 6: SHIP, but not c-fms, localizes to CR3-mediated phagocytic cups. Adherent thio-macrophages obtained from SHIP+/+ mice were incubated with EC3bi, as described in Materials and Methods, for 12 min at 37°C. After fixation, cells were stained for the presence of SHIP or c-fms (negative control), and C3bi as described in Materials and Methods. (A and D) Phase–contrast; (B) anti-SHIP; (E) anti–c-fms; (C and F) anti-C3. Bar, 10 μm.
Mentions: To determine whether CR3 ligation results in an alteration in the distribution of SHIP, we performed indirect immunofluorescence in cells undergoing CR3-mediated phagocytosis. SHIP, but not the receptor for CSF-1, c-fms, localized to EC3bi-containing phagosomes (Fig. 6). The specificity of SHIP localization is evident from a lack of circumferential staining around phagosomes for another protein expressed in macrophages, c-fms (Fig. 6 E), and absence of SHIP staining in macrophages derived from SHIP−/− mice (data not shown). To confirm that CR3 ligation is sufficient to induce SHIP redistribution, we determined the cytoskeletal localization of SHIP and the αM subunit of CR3 after CR3 ligation. Both SHIP and αM, but not CD14, demonstrated enhanced cytoskeletal incorporation after mAb-induced clustering of CR3 (Fig. 7). In addition, there was a 2.2 ± 0.4–fold (mean ± SEM, n = 3) increase in the phosphotyrosyl content of SHIP after the clustering of CR3 using mAb M1/70 followed by a cross-linking antibody. In contrast, the phosphotyrosyl content of SHIP in macrophages incubated with an IgG2b control mAb followed by a cross-linking antibody was 0.8 ± 0.3–fold (mean ± SEM, n = 3) of unstimulated cells. Therefore, engagement of CR3 is sufficient to mediate an alteration in the localization of SHIP in macrophages.

Bottom Line: To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; alpha(M)beta(2))-dependent phagocytosis.In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis.We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating beta(2) integrin outside-in signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.

ABSTRACT
The Src homology 2 domain-containing inositol 5'-phosphatase (SHIP) is recruited to immunoreceptor tyrosine-based inhibition motif (ITIM)-containing proteins, thereby suppressing phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathways. The role of SHIP in phagocytosis, a PI 3-kinase-dependent pathway, is unknown. Overexpression of SHIP in macrophages led to an inhibition of phagocytosis mediated by receptors for the Fc portion of IgG (Fc gamma Rs). In contrast, macrophages expressing catalytically inactive SHIP or lacking SHIP expression demonstrated enhanced phagocytosis. To determine whether SHIP regulates phagocytosis mediated by receptors that are not known to recruit ITIMs, we determined the effect of SHIP expression on complement receptor 3 (CR3; CD11b/CD18; alpha(M)beta(2))-dependent phagocytosis. Macrophages overexpressing SHIP demonstrated impaired CR3-mediated phagocytosis, whereas macrophages expressing catalytically inactive SHIP demonstrated enhanced phagocytosis. CR3-mediated phagocytosis in macrophages derived from SHIP(-/-) mice was up to 2.5 times as efficient as that observed in macrophages derived from littermate controls. SHIP was localized to Fc gamma R- and CR3-containing phagocytic cups and was recruited to the cytoskeleton upon clustering of CR3. In a transfected COS cell model of activation-independent CR3-mediated phagocytosis, catalytically active but not inactive SHIP also inhibited phagocytosis. We conclude that PI 3-kinase(s) and SHIP regulate multiple forms of phagocytosis and that endogenous SHIP plays a role in modulating beta(2) integrin outside-in signaling.

Show MeSH
Related in: MedlinePlus