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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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Effect of SHIP or SHIPΔP′ase expression on CR3-mediated phagocytosis in COS cells and macrophages. (A) COS cells transfected with the αM and β2 subunits of CR3 and coexpressing the indicated constructs were challenged with EC3bi for 30 min at 37°C in the presence or absence of 100 nM WM. The phagocytosis index of COS cells expressing CR3 alone, in the absence of WM (controls) was 651 ± 108. Phagocytosis was determined as the percentage of control. Data represent mean ± SEM, n = 3. Differences in phagocytosis between cells either incubated in WM or expressing wild-type SHIP, and controls were significant (P < 0.05). (B) RAW LR5 cells expressing the indicated constructs were challenged with EC3bi and 100 nM PMA and incubated in the absence or presence of 100 nM WM for 30 min at 37°C. The phagocytosis index of nonexpressing cells in the absence of WM (controls) was 55 ± 10. Phagocytosis was determined as the percentage of controls. Data represent mean ± SEM, n = 5. Differences in phagocytosis between cells either incubated in WM, or expressing wild-type SHIP or SHIPΔP′ase, and controls were significant (P < 0.05).
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Figure 5: Effect of SHIP or SHIPΔP′ase expression on CR3-mediated phagocytosis in COS cells and macrophages. (A) COS cells transfected with the αM and β2 subunits of CR3 and coexpressing the indicated constructs were challenged with EC3bi for 30 min at 37°C in the presence or absence of 100 nM WM. The phagocytosis index of COS cells expressing CR3 alone, in the absence of WM (controls) was 651 ± 108. Phagocytosis was determined as the percentage of control. Data represent mean ± SEM, n = 3. Differences in phagocytosis between cells either incubated in WM or expressing wild-type SHIP, and controls were significant (P < 0.05). (B) RAW LR5 cells expressing the indicated constructs were challenged with EC3bi and 100 nM PMA and incubated in the absence or presence of 100 nM WM for 30 min at 37°C. The phagocytosis index of nonexpressing cells in the absence of WM (controls) was 55 ± 10. Phagocytosis was determined as the percentage of controls. Data represent mean ± SEM, n = 5. Differences in phagocytosis between cells either incubated in WM, or expressing wild-type SHIP or SHIPΔP′ase, and controls were significant (P < 0.05).

Mentions: The above results are somewhat surprising in light of the data in human neutrophils and T cells, in which PI 3-kinase has been shown to be required for β2 integrin–mediated adhesion 212238. To confirm a role for PI 3-kinase and SHIP in CR3-mediated outside-in rather than inside-out signaling, we transfected COS cells with the αM and β2 subunits of CR3, alone and together, and assessed the binding and ingestion of EC3bi. Only cells that expressed both subunits demonstrated binding of EC3bi (data not shown). CR3-expressing COS cells demonstrated phagocytosis of EC3bi in the absence of PMA, and the addition of PMA did not result in enhancement of phagocytosis (data not shown), indicating that CR3 was constitutively and maximally activated. In addition, CR3-expressing COS cells, but not nonexpressing controls, demonstrated marked staining with mAb CBRM1/5, an antibody that recognizes an activation-dependent epitope on CR3 (26, and data not shown). Addition of WM led to an 87% inhibition of phagocytosis of EC3bi in transfected COS (Fig. 5 A) but did not inhibit particle binding or staining with mAb CBRM1/5 (data not shown). Coexpression of wild-type SHIP, but not catalytically inactive SHIP, also led to a decrease in phagocytosis, further implicating the lipid product of PI 3-kinase, PIP3, in CR3-mediating outside-in signaling and phagocytosis. Similarly, in macrophages overexpressing SHIP, there was a significant decrease in phagocytosis of EC3bi (Fig. 5 B); however, in contrast to COS cells, which lack endogenous SHIP expression (data not shown), expression of a catalytically inactive allele of SHIP in macrophages led to enhanced phagocytosis of EC3bi (compare Fig. 5a and Fig. b), consistent with a suppressive role for endogenously expressed SHIP in these cells. Collectively, these data suggest that one or more isoforms of PI 3-kinase is required for CR3-mediated phagocytosis in macrophages and COS cells, that this requirement is not due to the enhancement of receptor affinity or avidity (i.e., inside-out signaling), and that endogenously expressed SHIP downregulates CR3-mediated phagocytosis 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)

Effect of SHIP or SHIPΔP′ase expression on CR3-mediated phagocytosis in COS cells and macrophages. (A) COS cells transfected with the αM and β2 subunits of CR3 and coexpressing the indicated constructs were challenged with EC3bi for 30 min at 37°C in the presence or absence of 100 nM WM. The phagocytosis index of COS cells expressing CR3 alone, in the absence of WM (controls) was 651 ± 108. Phagocytosis was determined as the percentage of control. Data represent mean ± SEM, n = 3. Differences in phagocytosis between cells either incubated in WM or expressing wild-type SHIP, and controls were significant (P < 0.05). (B) RAW LR5 cells expressing the indicated constructs were challenged with EC3bi and 100 nM PMA and incubated in the absence or presence of 100 nM WM for 30 min at 37°C. The phagocytosis index of nonexpressing cells in the absence of WM (controls) was 55 ± 10. Phagocytosis was determined as the percentage of controls. Data represent mean ± SEM, n = 5. Differences in phagocytosis between cells either incubated in WM, or expressing wild-type SHIP or SHIPΔP′ase, and controls were significant (P < 0.05).
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Figure 5: Effect of SHIP or SHIPΔP′ase expression on CR3-mediated phagocytosis in COS cells and macrophages. (A) COS cells transfected with the αM and β2 subunits of CR3 and coexpressing the indicated constructs were challenged with EC3bi for 30 min at 37°C in the presence or absence of 100 nM WM. The phagocytosis index of COS cells expressing CR3 alone, in the absence of WM (controls) was 651 ± 108. Phagocytosis was determined as the percentage of control. Data represent mean ± SEM, n = 3. Differences in phagocytosis between cells either incubated in WM or expressing wild-type SHIP, and controls were significant (P < 0.05). (B) RAW LR5 cells expressing the indicated constructs were challenged with EC3bi and 100 nM PMA and incubated in the absence or presence of 100 nM WM for 30 min at 37°C. The phagocytosis index of nonexpressing cells in the absence of WM (controls) was 55 ± 10. Phagocytosis was determined as the percentage of controls. Data represent mean ± SEM, n = 5. Differences in phagocytosis between cells either incubated in WM, or expressing wild-type SHIP or SHIPΔP′ase, and controls were significant (P < 0.05).
Mentions: The above results are somewhat surprising in light of the data in human neutrophils and T cells, in which PI 3-kinase has been shown to be required for β2 integrin–mediated adhesion 212238. To confirm a role for PI 3-kinase and SHIP in CR3-mediated outside-in rather than inside-out signaling, we transfected COS cells with the αM and β2 subunits of CR3, alone and together, and assessed the binding and ingestion of EC3bi. Only cells that expressed both subunits demonstrated binding of EC3bi (data not shown). CR3-expressing COS cells demonstrated phagocytosis of EC3bi in the absence of PMA, and the addition of PMA did not result in enhancement of phagocytosis (data not shown), indicating that CR3 was constitutively and maximally activated. In addition, CR3-expressing COS cells, but not nonexpressing controls, demonstrated marked staining with mAb CBRM1/5, an antibody that recognizes an activation-dependent epitope on CR3 (26, and data not shown). Addition of WM led to an 87% inhibition of phagocytosis of EC3bi in transfected COS (Fig. 5 A) but did not inhibit particle binding or staining with mAb CBRM1/5 (data not shown). Coexpression of wild-type SHIP, but not catalytically inactive SHIP, also led to a decrease in phagocytosis, further implicating the lipid product of PI 3-kinase, PIP3, in CR3-mediating outside-in signaling and phagocytosis. Similarly, in macrophages overexpressing SHIP, there was a significant decrease in phagocytosis of EC3bi (Fig. 5 B); however, in contrast to COS cells, which lack endogenous SHIP expression (data not shown), expression of a catalytically inactive allele of SHIP in macrophages led to enhanced phagocytosis of EC3bi (compare Fig. 5a and Fig. b), consistent with a suppressive role for endogenously expressed SHIP in these cells. Collectively, these data suggest that one or more isoforms of PI 3-kinase is required for CR3-mediated phagocytosis in macrophages and COS cells, that this requirement is not due to the enhancement of receptor affinity or avidity (i.e., inside-out signaling), and that endogenously expressed SHIP downregulates CR3-mediated phagocytosis 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