FcgammaR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways

Abstract

Phagocytosis of IgG-coated particles via FcgammaR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcgammaR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcgammaR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1beta and IL-6 production in response to FcgammaR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1beta production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcgammaR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1beta production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways.

Figure 1.

SHIP downregulates FcγR-induced superoxide production. (A) BMMs derived from SHIP^+/+ and SHIP^–/– mice were activated with heat-aggregated IgG for the time points indicated in the figure. Generation of superoxide was measured using 10 μM fluorescent probe DHE. DHE fluorescence intensity is plotted in the graph. (B) BMMs derived from SHIP^+/+ and SHIP^–/– mice were activated with heat-aggregated IgG for 2 hours. Generation of superoxide was measured using fluorescent probe DHE. Data represent mean and SEM of 4 independent experiments. Data were analyzed by Student t test. *P < .05. (C) Protein-matched whole-cell lysates (WCLs) were analyzed by Western blotting with SHIP antibody (i). The same membrane was reprobed with actin antibody (ii). IB indicates immunoblot. (D) Mac-1 expression on the SHIP^+/+ and SHIP^–/– BMMs was analyzed by flow cytometry. For this, the cells were labeled with APC-labeled Mac-1 antibody in the presence of the anti-FcγRII/III monoclonal antibody (mAb) 2.4G2 (to block Fcγ receptors; - - -). Cells were also labeled with APC-labeled isotype control antibody (—).

Figure 2.

FcγR-induced Rac activation is enhanced in SHIP-deficient macrophages. (A) SHIP^+/+ and SHIP^–/– macrophages were activated by clustering Fcγ receptors for the indicated time points. GTP-bound Rac was captured with PAK-1 PBD beads as bait from protein-matched cell lysates and visualized by Western blotting with anti-Rac antibody. Unhydrolizable GTP and GDP analogs (obtained from Chemicon International) were used as positive and negative controls, respectively. (B) Fold induction of GTP Rac in the activated samples over resting (R). The graph represents mean and SEM of 3 independent experiments. Data were analyzed by Student t test (*P ≤ .05).

Figure 3.

Overexpression of wild-type SHIP downregulates FcγR-induced Rac activation. (A) Raw 264.7 cells were retrovirally infected using vector alone, wild-type SHIP, or catalytic-deficient D675A SHIP. GTP-bound Rac was measured in these transfectants after FcγR clustering. The bottom panel represents the band intensity of the Rac GTP. Values obtained from 3 independent experiments are represented as mean and SEM. (B) The stable transfectants were analyzed for SHIP expression by immunoprecipitating SHIP with rabbit polyclonal SHIP antibody and immunoblotting with SHIP antibody. The last lane is a control immunoprecipitate (IP) with normal rabbit serum. Data were analyzed by the Student t test (*P ≤ .05).

Figure 4.

SHIP downregulates FcγR-induced production of IL-1β and IL-6. BMMs obtained from SHIP^+/+ and SHIP^–/– mice were stimulated for the time points indicated in the figure with heat-aggregated IgG. (A) The levels of IL-1β and (B) IL-6 in lysates and supernatants were measured by ELISA. The graphs represent the mean and SEM of values obtained from 3 independent experiments. Data were analyzed by Student t test (*P ≤ .05).

Figure 5.

Differential requirement for the PI3K and Ras/MAPK pathways in FcγR-induced IL-1β and IL-6 production. BMMs were treated with Me₂SO (DMSO) or 10 μM LY294002 or 2.5 μM UO126 for 30 minutes at 37°C prior to stimulation with heat-aggregated IgG. (A) The levels of IL-1β and (B) IL-6 in cell lysates and supernatants were measured by ELISA. The graphs represent the mean and SEM of 3 independent experiments. Data were analyzed by Student t test. *P ≤ .05. (C) Protein-matched lysates from unstimulated and stimulated cells (stimulated for 7 minutes) were analyzed by Western blotting with phospho-Erk antibody. The bottom panel is a reprobe of the same membrane with anti-Erk antibody. (D) Parallel samples were probed with anti-phospho Serine Akt antibody, and the membrane was reprobed with anti-Akt antibody (bottom panel).

Figure 6.

SHIP downregulates FcγR-induced activation of Erk. (A) SHIP^+/+ and SHIP^–/– macrophages were activated by clustering Fcγ receptors for the indicated time points. Protein-matched lysates were probed with anti-phospho–ERK and reprobed with antibody against actin (bottom panel). The graph shown below is a quantitative estimate of Erk phosphorylation and represents the mean and SEM of values obtained from 3 independent experiments. Data were analyzed by Student t test. *P ≤ .05. □ indicates SHIP^+/+; ▴, SHIP^–/–. (B) Erk phosphorylation was likewise measured in stable transfectants overexpressing vector alone, wild-type SHIP, or catalytic-deficient D675A SHIP.

Figure 7.

SHIP downregulates FcγR-induced IL-1β and IL-6 production through the inhibition of PI3K and Ras/MAPK pathways. SHIP^+/+ and SHIP^–/– macrophages were treated with either Me₂SO (DMSO), 10 μM LY294002, or 2.5 μM UO126 for 30 minutes at 37°C prior to stimulation with heat-aggregated IgG. (A) The levels of IL-1β in cells treated with DMSO or UO126 (i) and IL-1β in cells treated with DMSO or LY294002 (ii) were measured by ELISA. The graphs represent the mean and SEM of values obtained from 3 independent experiments. Data were analyzed by Student t test. *P ≤ .05. (B) The levels of IL-6 in supernatants of cells treated with DMSO or UO126 (i) and cells treated with DMSO or LY294002 (ii) were measured by ELISA. The graphs represent the mean and SEM of values obtained from 3 independent experiments. Data were analyzed by Student t test. *P ≤ .05. (C) Protein-matched lysates were analyzed by Western blotting with phospho-Erk antibody (top panels). Parallel samples were probed with antiphospho serine Akt antibody (middle panels), and the membrane was reprobed with anti-Akt antibody (bottom panels). (D) Raw 264.7 stable transfectants overexpressing vector alone, wild-type SHIP, or catalytic-deficient D675A SHIP were stimulated with heat-aggregated IgG. Production of IL-1β and IL-6 was analyzed by measuring lysates and supernatants, respectively, by ELISA at 5 hours and 8 hours after stimulation. Data were analyzed by Student t test. *P ≤ .05.