FcγRI is required for TGFβ2-treated macrophage-induced tolerance

Abstract

Macrophages treated with TGFβ2 (TGFβ2-Mϕ) and antigen are highly tolerogenic in vivo, and induce antigen-specific and long-lasting tolerance in both naïve and primed mice via induction of suppressor/regulatory T cells. In this study, we examined the molecular pathways, including the requirements for Smad-dependent signaling, that are involved in the induction and function of tolerogenic TGFβ2-Mϕ. Treatment of murine macrophages with TGFβ2 induced translocation of Smad2/3 to the nucleus, and impairment of Smad3-, but not Smad2-, dependent signaling inhibited the tolerogenic function of a TGFβ2-treated murine macrophage cell line. Gene expression in murine macrophages treated with TGFβ2 was evaluated by microarray analysis. The FcγRI gene was one of a number of immune-related genes differentially expressed in TGFβ2-Mϕ, and appeared to be critical for tolerance in this system, since TGFβ2-Mϕ from FcγRI deficient mice were unable to induce tolerance. The role that FcγRI plays in TGFβ2-Mϕ-mediated tolerance is currently unclear. The results of this study provide important information about the factors that are critical for the induction of TGFβ2-Mϕ-mediated tolerance, and a better understanding of these mechanisms could lead to the development of more effective tolerance-inducing strategies for the treatment of autoimmune/inflammatory diseases.

Keywords: ACAID; ANOVA; APC; Antigen presenting cells; CFA; DN; DTH; Delayed type hypersensitivity; FcγR; LPS; Macrophages; Mice; Mϕ; Mϕ59; OVA; PEC; TGFβ; Tolerance; WT; analysis of variance; anterior chamber-associated immune deviation; antigen presenting cells; complete Freund's adjuvant; delayed type hypersensitivity; dominant negative; i.v.; intravenous; lipopolysaccharide; macrophage; macrophage hybridoma cell line; ovalbumin; peritoneal exudates cells; s.c.; subcutaneous; transforming growth factor β; wild-type.

Figure 1

Flow cytometric analysis of thioglycolate-elicited peritoneal exudate cells (PEC). Mice were injected with thioglycolate i.p. and 4 days later, PEC were harvested and cultured overnight with TGFβ2 and soluble protein antigen (LPS-free OVA). After culture, adherent cells were harvested, labeled with anti-F4/80 and anti-I-A antibodies then analyzed by flow cytometry.

Figure 2

TGFβ2 induces translocation of Smad2/3 from cytosol to nucleus in primary macrophages (PEC). Control Mφ (PBS-treated) and TGFβ2-Mφ (TGFβ-treated) were analyzed for the presence of Smad2/3 in cytosolic and nuclear extracts by western blot (A). The relative nuclear/cytosol Smad2/3 ratios are displayed as mean±SEM. Statistical differences were analyzed using a Student’s t test. * p<0.02 compared to the control (B). PECs were labeled with anti-Smad2/3 and Alexa488-anti-IgG and DRAQ5, then analyzed by confocal microscopy (C). (n=3)

Figure 3

TGFβ2 induces translocation of Smad2/3 from cytosol to nucleus in a murine macrophage hybridoma cell line (Mφ59). Control Mφ59 and TGFβ2-Mφ59 were analyzed for the presence of Smad2/3 in cytosolic and nuclear extracts by western blot (A). The relative nuclear/cytosol Smad2/3 ratios are displayed as mean±SEM. Statistical differences were analyzed using a Student’s t test. * p<0.02 compared to the control (B). Mφ59 cells were labeled with anti-Smad2/3 and Alexa488-anti-IgG and DRAQ5, then analyzed by confocal microscopy (C). (n=3)

Figure 4

Smad3-dependent TGFβ signaling is required for TGFβ2-Mφ-induced tolerance in vivo. Mφ59 cells untransfected (TGFβ Control) or stably transfected with either Smad2-DN, Smad3-DN or Smad2 and Smad3 (Smad2/3) DN (dominant negative) constructs were treated with TGFβ and OVA overnight, then injected i.v. into naïve mice (n=5 mice/group). After seven days, mice were immunized with OVA and CFA, then ear-challenged 10–14 days later. Ear swelling was measured after 24 hrs. Statistical differences were analyzed using ANOVA and the Tukey-Kramer multiple comparisons test. * p<0.05 compared to the positive control.

Figure 5

FcγRI expression by TGFβ2-Mφ is required for tolerance induction. mRNA extracted from Control Mφ or TGFβ2-Mφ (primary cells) was reverse transcribed and subjected to quantitative real-time PCR using FcγRI primers (A). TGFβ2-Mφ from either wild-type (WT) or FcγRI-deficient (FcγRI^−/−) mice were injected i.v. into naïve WT mice (n=5 mice/group). After seven days, mice were immunized with OVA and CFA, then ear-challenged 10–14 days later. Ear swelling was measured after 24 hrs (B). Statistical differences were analyzed using ANOVA and the Tukey-Kramer multiple comparisons test. * p<0.05 compared to positive control.