Signaling through Fc gamma RIII is required for optimal T helper type (Th)2 responses and Th2-mediated airway inflammation

Abstract

Although inhibitory Fc gamma receptors have been demonstrated to promote mucosal tolerance, the role of activating Fc gamma receptors in modulating T helper type (Th)2-dependent inflammatory responses characteristic of asthma and allergies remains unclear. Here, we demonstrate that signaling via activating Fc gamma receptors in conjunction with Toll-like receptor 4 stimulation modulated cytokine production from bone marrow-derived dendritic cells (DCs) and augmented their ability to promote Th2 responses. Ligation of the low affinity receptor Fc gamma RIII was specifically required for the enhanced Th2 responses, as Fc gamma RIII(-/-) DCs failed to augment Th2-mediated airway inflammation in vivo or induce Th2 differentiation in vitro. Further, Fc gamma RIII(-/-) mice had impaired Th2 cytokine production and exhibited reduced airway inflammation, whereas no defect was found in Fc gamma RI(-/-) mice. The augmentation of Th2 immunity was regulated by interleukin 10 production from the DCs but was distinct and independent of the well-established role of Fc gamma RIII in augmenting antigen presentation. Thus, our studies reveal a novel and specific role for Fc gamma RIII signaling in the regulation of Th cell responses and suggest that in addition to immunoglobulin (Ig)E, antigen-specific IgG also contributes to the pathogenesis of Th2-mediated diseases such as asthma and allergies.

Figure 1.

Ligation of Fcγ receptors modulates TLR-4–mediated cytokine production from DCs. (A) BMDCs generated from BALB/c (filled bars) or C.C3-Tlr4^Lps-d (TLR-4^−/−) (open bars) mice were loaded with immune complexes produced by incubation of OVA with anti-OVA sera (OVA-IC) or OVA mixed with an aliquot of antisera in which IgG had been depleted (OVA-IgG^depl) to account for potential nonspecific effects from the antisera (***, P < 0.001; n.d., not detected). (B) BALB/c (filled bars) or TLR-4^−/− (open bars) BMDCs were cultured on plates coated with the indicated concentration of anti-FcγRIIb/RIII (2.4G2) in the presence of 10 ng/ml LPS. After 24 h of culture, cytokine production from the BMDCs was estimated. Fcγ receptor–mediated modulation of cytokine production was significantly reduced in the TLR-4^−/− BMDCs as compared with BALB/c BMDCs (P < 0.005 by two-way ANOVA analysis). The data shown in A are representative of three independent experiments, and those presented in B are representative of two independent experiments.

Figure 2.

Ligation of Fcγ receptors on the priming DCs augments Th2-mediated airway inflammation and cytokine production in vivo. BMDCs generated from B6 mice were cultured on a nonspecific control rat IgG (open bars) or anti-FcγRIIb/RIII (2.4G2) (filled bars) –coated plates along with OVA. After 24 h, these DCs were harvested and instilled i.t. into naive B6 recipient mice. After sensitization, the recipient mice were challenged with soluble OVA i.t. daily for 3 d starting on day 7, and BAL was performed 24 h after last challenge. (A) Airway inflammation was assessed by determining the cellular composition of the BAL fluid (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The error bars indicate variation between individual mice. (B) For each group, the BAL cells were pooled and cytokine production of the CD4⁺ T cells was determined by intracellular staining. (C) Pooled lung cells were restimulated in vitro with OVA for 48 h, and cytokine production was assessed by ELISPOT analysis (**, P < 0.01; ***, P < 0.001). The error bars indicate the SEM between replicate stimulations in different wells. The data represented in this figure were obtained from three independent experiments, and 9–10 mice per group were analyzed.

Figure 3.

Modulation of cytokine production by 2.4G2 requires expression of activating Fcγ receptors on the DCs. (A) BMDCs generated from B6 or Fcγ receptor–deficient mice were stimulated with 10 ng/ml LPS along with a control IgG or anti-FcγRIIb/RIII (2.4G2). The amount of cytokines present in culture supernatants was determined by ELISA. The amount of cytokine produced upon 2.4G2 treatment was normalized to the amount produced upon treatment with control IgG (**, P < 0.01; ***, P < 0.001). (B) The fold increase in IL-10 production from DCs stimulated with 2.4G2 in the presence of the indicated concentrations of ERK inhibitor (U0126), p38 inhibitor (SB202190), and PI3K inhibitor (Ly294002) was determined. The data shown in this figure are representative of two independent experiments. The error bars indicate variation between replicate stimulations in different wells (P < 0.0001 by one-way ANOVA analysis).

Figure 4.

Immune complexes up-regulate Th2 responses by cross-linking FcγRIII on DCs. BMDCs generated from B6 or FcγRIII^−/− mice were loaded with immune complexes produced by incubation of OVA with anti-OVA sera (OVA-IC; filled bars) or OVA mixed with an aliquot of antisera in which IgG had been depleted (OVA-IgG^depl; open bars). These DCs were instilled i.t. into naive B6 recipient mice challenged and analyzed as in Fig. 2. (A) Airway inflammation was assessed by determining the cellular composition of the BAL fluid (**, P < 0.01; ***, P < 0.001). (B) LN cells were restimulated in vitro with OVA for 48 h, and the amount of cytokines in culture supernatants was determined by ELISA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The data represented in this figure were obtained from two independent experiments. At least seven mice per group were analyzed.

Figure 5.

BMDCs from FcγRIII^−/− mice do not have a defect in antigen uptake, processing, or presentation. (A) To measure antigen uptake and processing, DQ-OVA (▪) or DQ OVA-ICs (▴) were loaded onto BMDCs generated from B6 (solid lines) or FcγRIII^−/− (dashed lines) mice. The cells were incubated at 37°C, and the amount of OVA processed into peptides was measured at the indicated time points as an increase in FITC fluorescence. Error bars represent variation observed between independent experiments. Statistical data presented were obtained using a two-way ANOVA analysis. The bottom panel depicts a representative histogram of the data obtained at 60 min. The shaded histograms represent baseline 60 min. (B) CFSE-dyed OTII TCR transgenic T cells were cultured for 96 h with B6 or FcγRIII^−/− DCs loaded with OVA (open bars) or OVA-IC (filled bars). The extent of proliferation was assessed as a dilution of the CFSE dye (P < 0.0001 by two-way ANOVA analysis).

Figure 6.

T cells exhibit reduced Th2 differentiation upon stimulation with FcγRIII^−/− BMDCs. (A) OTII T cells were cultured with OVA-IgG^Depl (open bars) or OVA-IC (filled bars) –loaded B6 or FcγRIII^−/− DCs for 7 d and restimulated with anti-CD3 for 48 h. The amount of cytokines in culture supernatants was determined by ELISA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). OTII TCR transgenic T cells were stimulated for 7 d with (B) B6 BMDCs or (C) FcγRIII^−/− DCs that were left untreated (Iso) or treated with anti-FcRIIb/RIII (2.4G2) and loaded with the agonist OVA_(323–339) peptide. T cells were subsequently restimulated for 48 h as in A, and the amount of cytokine production was determined by ELISA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The data shown are representative of two independent experiments. The error bars indicate SEM between replicate stimulations in different wells.

Figure 7.

IL-10 production from DCs plays a central role in the up-regulation of Th2 differentiation by immune complexes. (A) BMDCs generated from B6 or IL-10^−/− mice were loaded with immune complexes (filled bars) or control OVA-IgG^Depl (open bars). After 24 h of culture, cytokine production from the BMDCs was measured by ELISA. Th2 differentiation status of OTII T cells stimulated with (B) IL-10^−/− or (C) MCP-1^−/− DCs treated with immune complexes was assessed by analyzing cytokine production from the T cells as described in Fig. 6 A (**, P < 0.01; ***, P < 0.001). (D) Control OVA-IgG^Depl (open bars) or OVA-IC (filled bars) –loaded B6 or IL-10^−/− DCs were instilled into naive B6 recipients that were subsequently challenged as described in Fig. 2. Cytokine production from cells in the draining LNs was determined by ELISA. (E) Quantitative analysis (top) and a representative flow cytometry plot (bottom) of cytokine production from cells in the BAL as determined by intracellular staining. Data in the top panel are normalized to the amounts produced by recipients sensitized with OVA-IgG^Depl—loaded B6 DCs. (F) Analysis of cellular composition of the BAL fluid. These data are representative of three independent experiments. The error bars indicate SEM between (A–C) replicate stimulations in different wells or (D–F) variation between individual mice. At least eight mice per group were analyzed (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.d., not detected).

Figure 8.

FcγRIII^−/− mice exhibit a decrease in Th2-mediated airway inflammation and airway hyperresponsiveness. (A) FcRγ^−/− (n = 9), FcγRIII^−/− (n = 8), FcγRI^−/− (n = 8), and B6 mice were sensitized with i.p. injection of inactivated S. mansoni eggs on day 0. These mice were challenged with i.t. administration of SEA on day 7. On day 11, the mice were killed and the cellular composition of the BAL fluid was examined by flow cytometry (*, P < 0.05; **, P < 0.01). (B) Lung tissues from sensitized and challenged B6 and FcγRIII^−/− mice were fixed in 4% formalin and embedded in paraffin. The sections were stained with PAS or hematoxylin and eosin as noted. (C) Amount of IgE and SEA-specific IgG₁ in the sera of sensitized and challenged mice was estimated by ELISA (**, P < 0.01; P = 0.0012 obtained by two-way ANOVA analysis). (D) Goblet cell metaplasia, perivascular, and peribronchial inflammation were scored as described in Materials and methods. Four mice per group were analyzed. (E) Rrs to methacholine was measured at day 4 after challenge (P < 0.0053 by two-way ANOVA analysis). Data are representative of three independent experiments.

Figure 9.

In vivo Th2 differentiation and cytokine production are impaired in FcγRIII^−/− mice. B6 and FcγRIII^−/− mice were sensitized and challenged with S. mansoni as described in Fig. 8. (A) Cytokine production of the effector cells in the lungs was determined by intracellular staining as described in Materials and methods. (B) Quantitative analysis of data presented in A (*, P < 0.05). (C) LN cells were restimulated in vitro with SEA for 48 h, and the amount of cytokines in culture supernatants was determined by ELISA (**, P < 0.01; ***, P < 0.001). The data represented in this figure were obtained from two independent experiments.