The inositol phosphatase SHIP-2 down-regulates FcgammaR-mediated phagocytosis in murine macrophages independently of SHIP-1

Abstract

FcgammaR-mediated phagocytosis of IgG-coated particles is a complex process involving the activation of multiple signaling enzymes and is regulated by the inositol phosphatases PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP-1 (Src homology [SH2] domain-containing inositol phosphatase). In a recent study we have demonstrated that SHIP-2, an inositol phosphatase with high-level homology to SHIP-1, is involved in FcgammaR signaling. However, it is not known whether SHIP-2 plays a role in modulating phagocytosis. In this study we have analyzed the role of SHIP-2 in FcgammaR-mediated phagocytosis using independent cell models that allow for manipulation of SHIP-2 function without influencing the highly homologous SHIP-1. We present evidence that SHIP-2 translocates to the site of phagocytosis and down-regulates FcgammaR-mediated phagocytosis. Our data indicate that SHIP-2 must contain both the N-terminal SH2 domain and the C-terminal proline-rich domain to mediate its inhibitory effect. The effect of SHIP-2 is independent of SHIP-1, as overexpression of dominant-negative SHIP-2 in SHIP-1-deficient primary macrophages resulted in enhanced phagocytic efficiency. Likewise, specific knockdown of SHIP-2 expression using siRNA resulted in enhanced phagocytosis. Finally, analysis of the molecular mechanism of SHIP-2 down-regulation of phagocytosis revealed that SHIP-2 down-regulates upstream activation of Rac. Thus, we conclude that SHIP-2 is a novel negative regulator of FcgammaR-mediated phagocytosis independent of SHIP-1.

Figure 1.

SHIP-2 down-regulation of FcγR-mediated phagocytosis is dependent on an intact SH2 domain as well as the C-terminal proline-rich domain. Raw 264.7 cells were transiently transfected with empty vector alone, HA-SHIP-2 WT, HA-SHIP-2 ΔSH2, or HA-SHIP-2 ΔPRD. GFP was cotransfected as a marker for transfection. (A) IgG-coated SRBCs were added to the transfectants 24 hours after transfection. The samples were incubated for 1 hour at 37°C to allow phagocytosis to take place. Cells were then subjected to brief hypotonic lysis prior to fixation in paraformaldehyde to be viewed under a fluorescence microscope. Phagocytosis was measured by counting the total number of SRBCs ingested by 100 transfectants (GFP-positive) each time for a total of 3 readings per sample in each experiment. ^*P < .05 compared with cells transfected with empty vector alone. The graph represents the mean ± SD. (B) Whole cell lysates from the transfectants were incubated with anti-HA antibody and protein G-agarose beads overnight. The precipitated proteins were next separated on 10% SDS-PAGE and transferred to nitrocellulose membrane, which was Western blotted with anti-HA antibody. The same membrane was washed and reprobed with anti-SHIP-2 antibody. These results are representative of 3 independent experiments.

Figure 2.

Ectopic expression of SHIP-2 in SHIP-1^-/- BMMs. (A) BMMs from SHIP-1^+/+ and SHIP-1^-/- animals were tested for the expression of SHIP-1 and SHIP-2 by Western blotting. (B) Using the Nucleofector, BMMs were transfected with plasmids encoding GFP (left: immunofluorescence image; right: phase contrast image of the same field). Images were obtained using an inverted fluorescence microscope with ×40 magnification. (C) SHIP-1 knockout BMMs were transiently transfected with empty vector alone, wild-type SHIP-2 (SHIP-2 WT), or catalytically deficient SHIP-2 (SHIP-2 D608A). GFP was cotransfected as a marker for transfection. Whole cell lysates from the transfectants were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane, which was blotted with anti-SHIP-2 or anti-GFP antibody. These results are representative of 3 independent experiments.

Figure 3.

SHIP-2 knockdown by siRNA results in enhanced FcγR-function. (A) Raw 264.7 cells were transiently transfected with (1) control siRNA, (2) SHIP-2 siRNA1, and (3) SHIP-2 siRNA2. Cells were harvested 24 hours after transfection, and protein-matched lysates were analyzed by Western blotting with anti-SHIP-2 antibody (upper panel) and anti-SHIP-1 antibody (lower panel). The middle panel is a quantitative measurement of SHIP-2 band intensities. (B) Raw 264.7 cells were transiently transfected with control siRNA or SHIP-2 siRNA1. Twenty-four hours after transfection, the IgG-coated SRBCs were added. The samples were incubated for 1 hour at 37°C to study phagocytosis. Cells were then subjected to brief hypotonic lysis prior to fixation in paraformaldehyde to be viewed under a fluorescence microscope. Phagocytosis was measured by counting the total number of RBCs ingested by 100 transfectants. ^*P < .05. (C) Raw 264.7 cells transfected with control siRNA or SHIP-2 siRNA were incubated with IgG-coated SRBCs for 1 hour at 4°C to study binding. Cells were then subjected to brief washing prior to fixation in paraformaldehyde to be viewed under a fluorescence microscope. The binding activity was expressed as the total number of bound SRBCs on 100 rosetting Raw cells that each bound 3 or more SRBCs (binding index). Graphs represent means ± SD.

Figure 4.

SHIP-2 down-regulates Rac activation. Raw 264.7 cells were transiently transfected with either a nonspecific control siRNA or SHIP-2 siRNA. (A) Eight hours after transfection, cells were starved in incomplete RPMI media (ie, with no FBS) for 16 hours. After starvation, cells were stimulated with 2.4G2 followed by MAR (mouse anti-rat IgG) for the indicated time. Protein-matched cell lysates were incubated with GST-PAK1-PBD-agarose beads for 1 hour at 4°C. Active Rac bound to the beads was eluted and loaded on 12% SDS-PAGE (top panel). Equal volume of lysis buffer only and whole cell lysate were loaded as negative and positive controls (neg and WCL). The membranes were reprobed with anti-GST antibody (middle panel). Rac band intensities were quantitated and are presented as fold increase over the resting control siRNA-transfected sample (bottom panel). The graph represents means ± SD. (B) Whole cell lysates from the resting samples were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane, which was then probed with anti-SHIP-2 (top panel) or anti-SHIP-1 antibody (bottom panel).

Figure 5.

SHIP-2 down-regulates phagocytosis by suppressing upstream Rac activation. (A) Raw 264.7 cells were transiently transfected with constitutively active Rac (CA-Rac). Eight hours after transfection, cells were starved in incomplete RPMI media (ie, with no FBS) for 16 hours. After starvation, cells were stimulated with 2.4G2 followed by MAR (mouse anti-rat IgG) for 5 minutes to cluster FcγR. Protein-matched cell lysates were incubated with GST-PAK1-PBD-agarose beads for 1 hour at 4°C. Equal volume of lysis buffer was incubated with beads as a negative control (neg). Active Rac bound to the beads was eluted and loaded on 12% SDS-PAGE (top panel) and analyzed by Western blotting with anti-Rac antibody. The membrane was washed and reprobed with an anti-GST antibody (bottom panel). (B) Raw 264.7 cells were cotransfected with either a nonspecific control siRNA or SHIP-2 siRNA with CA-Rac. Twenty-four hours after transfection, whole cell lysates were made and separated on 12% SDS-PAGE and probed with an anti-Rac antibody. (C) Parallel samples were separated by 10% SDS-PAGE and probed with anti-SHIP-2 (top panel) or anti-SHIP-1 (bottom panel) antibody. (D) IgG-coated SRBCs were incubated with the transfectants for 1 hour at 37°C to assess phagocytic efficiency. Phagocytosis was measured by counting the total number of RBCs ingested by 100 phagocytosing cells. □ indicates control siRNA; ▪, SHIP-2 siRNA. The graph represents means ± SD.

Figure 6.

SHIP-2 is recruited to the site of phagocytosis. Raw 264.7 cells were grown on coverslips and either left untreated (resting) or incubated with IgG-coated SRBCs (red) for 5 minutes at 37°C. Cells were then fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. After blocking, cells were stained with a goat anti-SHIP-2 antibody followed by a Cy5-conjugated donkey anti-goat F(ab′)₂ fragment (yellow). F-actin was stained with FITC-phalloidin (green), and nuclei were stained with Hoechst (blue). Coverslips were mounted on slides and read using Zeiss 510 confocal microscope with ×63 magnification. SHIP-2 has a diffused cytoplasmic pattern in resting cells (top panel). SHIP-2 localizes to the site of phagocytosis (middle panel), where it colocalized with F-actin. Cells stained with normal goat IgG followed by Cy5-conjugated donkey anti-goat F(ab′)₂ fragment showed clean background (bottom panel).

Figure 7.

SH2 and PRD domains are both required for translocation of SHIP-2 to the site of phagocytosis. Raw 264.7 cells transfected with HA-tagged SHIP-2 constructs were grown on coverslips and either left untreated (resting) or incubated with IgG-coated SRBCs (red) for 5 minutes at 37°C. Cells were then fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. After blocking, cells were stained with a mouse anti-HA antibody followed by an Alexa Fluor 594-conjugated goat anti-mouse F(ab′)₂ fragment (yellow). Coverslips were mounted on slides and read using Zeiss 510 confocal microscope with ×63 magnification. (A) Empty vector-tranfected cells; (B) HA-SHIP-2 WT transfectants; (C) HA-SHIP-2 ΔSH2 transfectants; and (D) HA-SHIP-2 ΔPRD tranfectants.