CD4 cell-secreted, posttranslationally modified cytokine GIF suppresses Th2 responses by inhibiting the initiation of IL-4 production

Abstract

T helper 2 (Th2) cells are critical to the induction of IgE antibody and allergic inflammation, but how the pathological pathways are controlled in nonallergic individuals remains unclear. Here we report that glycosylation-inhibiting factor (GIF) suppresses Th2 effector generation. GIF is a cytokine encoded by the same gene that codes for macrophage migration inhibitory factor (MIF). GIF-deficient mice demonstrated enhanced T-dependent antibody formation especially of IgE isotype and allergic airway inflammation with the generation of regulatory T cells unaffected. GIF-deficient macrophages and dendritic cells revealed normal responsiveness to toll-like receptor (TLR) ligands. GIF undergoes a unique posttranslational modification, cysteinylation. The modified GIF, mainly secreted by activated T cells derived from CD4(+)CD25(-) cells, inhibited IL-4 production by the same cells whereas the unmodified GIF showed no effect. Bone marrow chimera experiment demonstrated that T cell-derived GIF suppressed the generation of Th effectors that secrete IL-4. During the first 24 h of CD3/CD28 stimulation in vitro, GIF secreted from naïve CD4 cells acted on the same cells, maintained nuclear factor of activated T cells (NFAT)c2 in the nucleus, and repressed IL-4 mRNA levels. Thus, GIF represents a self-regulatory mechanism of Th2 cell generation from naïve CD4 cells, in which the posttranslational modification plays a crucial role.

Fig. 1.

GIF inhibits Th2-dependent immune responses. (A) GIF+/+ and −/− mice were immunized with 1 μg Ova in alum i.p. on days 0 and 21. Serum levels of anti-Ova Ab titers on day 24 are shown. *, P < 0.03; **, P < 0.002; ***, P < 0.001. Columns and error bars represent the mean ± SD (n = 8 per group). (B) GIF+/+ and −/− mice were immunized with 25 μg TNP-Ficoll i.p. on day 0. Serum levels of anti-TNP Ab titers on day 8 are shown. n = 8 per group. (C–F) Allergic airway inflammation model. (C) Lungs from PBS or Ova-challenged mice were isolated and tissue sections were stained with haematoxylin-eosin (H&E) or periodic acid-schiff (PAS) reagents. (D) 24 h after the last challenge with inhaled Ova, mice were exposed to 25 mg/ml methacholine and AHR was assessed by whole-body plethysmography. Average baseline “enhanced pause” (PenH) was 0.39 for both GIF+/+ and GIF−/− mice. (E) 48 h after the last challenge with Ova, eosinophils in bronchoalveolar lavage fluids were counted. (F) Peribronchial lymph node cells from Ova-challenged mice were cultured in the presence of 100 μg/ml Ova for 4 days and secretion of IL-5 and IL-13 was determined by ELISA. *, P < 0.05; **, P < 0.02; n = 6 per group. Representative results of three independent experiments are shown.

Fig. 2.

Modified GIF inhibits IL-4 secretion. (A) CD4⁺CD25⁻ cells purified from GIF+/+ and −/− mice were stimulated with anti-CD3 and anti-CD28. Cytokine secretion was determined by ELISA. Errors for individual points were <15% of the means. (B and C) CD4 cells purified from GIF−/− mice were stimulated with anti-CD3 and anti-CD28 in the presence of 0–500 ng/ml modified (B) or unmodified (C) GIF for 6 days. Cells were restimulated with anti-CD3 and anti-CD28 for 24 h and secretion of IL-4 and IFN-γ was determined by ELISA. Columns and error bars represent the mean ± SD. The results are representative of three independent experiments.

Fig. 3.

Modified GIF is mainly secreted by CD4⁺CD25⁻ cells. The secretion of GIF in vitro was determined by ELISA that specifically detects C60-modified GIF (see Materials and Methods). (A) CD4⁺CD25⁻ (closed circles) and CD4⁺CD25⁺ (open circles) cells were purified and stimulated with plate-bound anti-CD3, soluble anti-CD28 and 3 ng/ml IL-2. (B) B cells were purified and stimulated with either LPS and IL-4 (closed circles) or anti-CD40 and IL-4 (open circles). Error bars indicate SD. Each experiment was repeated three times and representative data are shown.

Fig. 4.

T cell-derived GIF inhibits IL-4 production in vivo. Lethally irradiated GIF−/− mice were reconstituted with bone marrow cells from GIF+/+ mice, GIF−/− mice, or a 1:1 mixture of bone marrow cells from GIF−/− and TCRβ−/− GIF+/+ mice. Eight weeks later, all mice were immunized with Ova in CFA. Five days after immunization, spleens were taken. Cytokine secreting cells were enumerated by ELISPOT. *, P < 0.05; **, P < 0.02. Columns and error bars represent the mean ± SD (n = 6 per group). The results are representative of three independent experiments.

Fig. 5.

GIF suppresses initiation of Th2 differentiation. (A and B) CD4⁺CD25⁻ cells purified from GIF+/+ and −/− mice were stimulated with anti-CD3 and anti-CD28. IL-4 (A) and GATA-3 (B) mRNA were measured by quantitative real time PCR. The level of L32 mRNA in each sample was arbitrarily set to 1 × 10⁶ units, and values are relative to this. Columns and error bars represent the mean ± SD. (C) The reporter CD4 cells from 4get knockin GIF−/− mice were labeled with Alexa Fluor 633, mixed with CD4⁺CD25⁻ cells from either GIF+/+ or −/− mice at 1:4 and stimulated for indicated times with anti-CD3 and anti-CD28. Levels of IL-4 mRNA were assessed by the expression of enhanced GFP (eGFP). Dot plots are gated on Alexa Fluor 633+ cells. eGFP⁺ cells are enclosed and numbers beside rectangles indicate percentage in the reporter populations. (D) GIF+/+ and −/− CD4 cells were stimulated with anti-CD3 and anti-CD28 for 3 or 24 h, and nuclear (Nuc) and cytosolic (Cyt) fractions were prepared. Levels of NFATc2, NFATc1, NF κB p50, and JunB were examined by immunoblotting. Lamin B, control nucleus-specific protein, is shown. (E) GIF−/− CD4 cells were stimulated with anti-CD3 and anti-CD28 for 24 h in the presence or absence of 500 ng/ml modified or unmodified mouse GIF. Nuclear extracts were examined for NFATc2, NFATc1, NF κB p50, and JunB by immunoblotting. Data are representative of at least 2 independent experiments.