Production of Both IL-27 and IFN-γ after the Treatment with a Ligand for Invariant NK T Cells Is Responsible for the Suppression of Th2 Response and Allergic Inflammation in a Mouse Experimental Asthma Model

Mice

Female BALB/c mice were purchased from Charles River Laboratories Japan and used at 7–14 wk of age. Vα14 NKT-deficient (Jα-281^−/−) mice of the BALB/c background were provided by M. Taniguchi (RIKEN Research Center for Allergy and Immunology (RCAI), Yokohama, Japan). All mice were bred and maintained in the animal facilities at the RIKEN RCAI under specific pathogen-free conditions. Animal care was followed in accordance with the guidelines of RIKEN.

Reagents

α-GalCer, KRN7000, was purchased from Kirin Brewery. Recombinant murine (rm) IFN-γ was from Chemicon International and rmIL-27 was from R&D Systems. rmCD1d:Ig dimer was purchased from BD Biosciences Pharmingen. Anti-CD11c microbeads, anti-CD11b microbeads, and rat anti-mouse IgG1 microbeads were purchased from Miltenyi Biotec. Anti-mouse IFN-γ-neutralizing mAb (11) was provided by Dr. R. Nakagawa (RIKEN RCAI).

Immunization, treatment, and airway challenge

BALB/c mice were immunized by an i.p. injection of 100 μg of OVA (Sigma-Aldrich) adsorbed to 2 mg of alum (Pierce). α-GalCer (2 μg) was injected i.p. 7 days after the OVA priming. After another 7 days, mice were challenged by intranasal administration of 50 μg of OVA on 3 consecutive days. Recovery of bronchoalveolar lavage (BAL) fluids and lung histopathology were done 24 h after the last Ag challenge.

BAL fluid

Mice were fully anesthetized by i.p. injection of Nembutal. BAL was performed by cannulation of the trachea. Lungs were lavaged three times with 0.5 ml of PBS supplemented with 10% FBS. The numbers of cells in the BAL fluids were enumerated with a hemocytometer. Differential counts for 200 cells were done by Eosinostain-Hansel (Torii and Co.) staining of cells on cytospin slides.

ELISA

Microtiter plates (Corning) were coated overnight with 50 μg/ml OVA protein in 50 mM sodium carbonate buffer (pH 9.0) at 4°C. After blocking and washing, appropriate dilutions of serum samples or serial dilutions of a standard serum pool from OVA-immunized mice were added to the plate. After incubation for 3 h at 37°C, anti-OVA IgG1 Abs bound to the plate were measured by HRP-conjugated anti-mouse IgG1 (Zymed) and tetramethylbenzidine-positive substrate system (DakoCytomation). ELISA kits for anti-OVA-IgE Abs were purchased from Dainippon Sumitomo Pharma. Commercial ELISA kits were used for IFN-γ (Endogen) and IL-4, IL-5, IL-13, and IL-27p28 (R&D Systems).

Histopathology

Lungs 24 h after OVA challenge were infused with formalin and embedded in paraffin. Lung sections were cut and stained with periodic acid-Schiff (PAS) for light microscopy examination.

Cell preparations and cultures

Spleen cells were swollen by infusion of 1 mg/ml collagenase D (Roche Molecular Biochemicals) in HBSS containing 10 mM HEPES buffer (pH 7) and incubated for 45 min at 37°C. For the isolation of iNKT cells, dimeric rmCD1d:Ig was loaded with a molar excess of α-GalCer for 16 h at 37°C according to the manufacturer’s instructions (BD Biosciences). Spleen or lymph node cells were incubated with α-GalCer-loaded rmCD1d:Ig dimer for 30 min at 4°C. After washing, the cells were incubated with rat anti-mouse IgG1 microbeads for 30 min at 4°C. iNKT cells were recovered by binding to a magnet. From the remaining cells, dendritic cells (DCs) were first recovered using anti-CD11c microbeads, followed by macrophage recovery using anti-CD11b microbeads.

Peripheral lymph nodes of the neck, axilla, inguen, and hilum pulmonis were recovered 7 days after α-GalCer treatment. CD4⁺ T cells in the peripheral lymph nodes were purified using CD4⁺ T cell isolation kits (Miltenyi Biotec). CD4⁺ peripheral lymph node T cells (3 × 10⁵) from the OVA-primed mice were cultured with irradiated (20 Gy) syngeneic splenocytes (3 × 10⁶) and OVA (0, 1, 10, or 100 μg/ml) in 96-well microtiter plates for 72 h. The concentrations of cytokines in the culture supernatants were determined by ELISA. For sorting of iNKT cells, the spleen cells after the blockage by anti-CD16/CD32 (2.4G2; BD Biosciences) were incubated with anti-TCRβ-FITC (H57-597; BD Biosciences), anti-CD19-PE (1D3; BD Biosciences), and α-GalCer-rmCD1d:Ig dimmer plus anti-mouse IgG1-allophycocyanin (BD Biosciences) for 30 min at 4°C. Cell sorting was done using FACSAria (BD Biosciences).

RT-PCR

Total RNA of isolated cells was extracted by RNAeasy (Qiagen). The following RT-PCR primer sets were used for mouse genes: IL-27EBI3, 5′-cagagtgcaatgccatgcttctc-3′ and 5′-ctgtgaggtcctgagctgac-3′; IL-27p28, 5′-cttcaagagctgcgcagggaattc-3′ and 5′-ctgaaagcggaggtgcctgtgcag-3′; IFN-γ, 5′-tgaacgctacacactgcatcttgg-3′ and 5′-cgactccttttccgcttcctgag-3′; IL-4, 5′-atgggtctcaacccccagctagt-3′ and 5′-gctctttaggctttccaggaagtc-3′; and GAPDH, 5′-accacagtccatgccatcac-3′ and 5′-tccaccaccctgttgctgta-3′. The PCR amplification used 35 cycles for IL-27EBI3 (532 bp) and IL-27p28 (344 bp), 30 cycles for IFN-γ and IL-4, and 25 cycles for GAPDH. The amounts of cDNA were standardized by quantification of the housekeeping gene GAPDH using primers for mouse samples.

Statistical analysis

The statistical significance of differences between the experimental groups was determined by Student’s t test.

Systemic administration of α-GalCer into OVA-primed mice suppresses Th2 cell function and airway inflammation

Preliminary experiments with OVA-primed mice confirmed that splenic CD4⁺ T cells obtained 7 days after the priming formed high levels of IL-4, IL-5, and IL-13, but not IFN-γ, upon stimulation with OVA in the presence of irradiated spleen cells. α-GalCer was injected i.p. into BALB/c mice 7 days after priming with alum-adsorbed OVA. Seven days later, CD4⁺ T cells were recovered from the lymph nodes of the OVA-primed mice and cultured with irradiated syngeneic splenocytes and OVA. As shown in Fig. 1⇓A, the IL-5 and IL-13 levels produced by the cells of the α-GalCer-treated mice were substantially lower than those from T cells of saline-treated control mice. IL-4 was undetectable in all culture supernatants (data not shown). OVA-specific IgE and IgG1 serum Ab levels of the α-GalCer-treated mice, before or after repeated intranasal challenges of OVA, were significantly lower than those of saline-treated mice (Fig. 1⇓B). After intranasal challenge with OVA of the two groups, PAS⁺ goblet cell hyperplasia was observed in the lungs of the saline-treated mice, but it was not observed in the α-GalCer-treated mice (Fig. 1⇓C). The numbers of total cells and eosinophils in BAL fluids from the α-GalCer-treated mice were significantly lower than those from the saline-treated mice (Fig. 1⇓D). The results indicate that systemic administration of α-GalCer after the Ag priming suppressed the production of cytokines from already polarized Th2 cells upon antigenic stimulation.

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FIGURE 1.

Systemic α-GalCer administration regulates established Th2 cells. BALB/c mice (n = 8) were primed with alum-adsorbed OVA, then received an i.p. injection of α-GalCer or saline 7 days after the primary immunization. A, Seven days after the treatment, CD4⁺ T cells from the lymph nodes of treated mice were cultured with OVA-pulsed APCs for 72 h. IL-5 and IL-13 in culture supernatants were determined by ELISA. NS, p ≧ 0.05 and ∗, p < 0.05 compared with saline control. B, The OVA-primed mice were challenged with repeated intranasal OVA challenges. Serum concentrations of OVA-specific IgE and IgG1 Abs before and after the challenges were determined by ELISA. Each dot represents the concentration from five to six mice per group. Experiments were repeated three times with similar results. ∗∗, p < 0.01 compared with saline control. C, Lung tissue sections were stained with PAS (original magnification: ×100 and ×200, respectively). D, After challenges, total cells, macrophages (Mac), eosinophils (Eos), neutrophils (Neu), and lymphocytes (Lym) in BAL fluids were counted. Results are mean ± SD (n = 3–5 mice). Results shown are representative of six independent experiments. ∗∗, p < 0.01 compared with saline control.

IFN-γ alone is insufficient to attenuate polarized Ag-specific Th2 cell functions in vivo

In view of previous reports, IFN-γ from α-GalCer-activated iNKT cells was responsible for the suppression of Th2 cell development and Th2 cytokines production from polarized Th2 cells (2, 3, 4, 5). To clarify the role of IFN-γ in our experimental model, anti-IFN-γ-neutralizing Ab was administered at the time of α-GalCer injection into OVA-primed mice. Sera were recovered 3 h after the treatment and then the IFN-γ concentration was determined by ELISA. As shown in Fig. 2⇓A, elevation of the IFN-γ concentration in sera after the α-GalCer administration was completely inhibited by the coadministration of anti-IFN-γ Ab. In the same experiment, however, the numbers of total cells and eosinophils in the BAL fluids of α-GalCer-treated mice were significantly lower than those in saline-treated mice (Fig. 2⇓B). These results suggested that the suppressive effects by the systemic α-GalCer administration into OVA-primed mice might not be explained by the only IFN-γ production induced by α-GalCer-stimulated iNKT cells. Next, we tested whether the administration of rmIFN-γ into Ag-primed mice may reproduce the suppression of Th2 cell function and airway inflammation as shown in Fig. 1⇑. Thus, rmIFN-γ was injected i.p. into the OVA-primed BALB/c mice. In preliminary experiments, the serum concentration of IFN-γ after the administration of 5 μg of rmIFN-γ reached a level comparable to that obtained by the α-GalCer treatment and fell below detectable levels within 12 h (Fig. 2⇓C). To maintain the serum concentration of IFN-γ comparable to that obtained by the administration of α-GalCer, rmIFN-γ was injected i.p. three times every 6 h into OVA-primed mice. After OVA-intranasal challenges of the rmIFN-γ-treated mice, the numbers of total cells and eosinophils in the BAL fluids of rmIFN-γ-treated animals were comparable to those from saline-treated mice (Fig. 2⇓D). CD4⁺ T cells were recovered from their lymph nodes 7 days after the rmIFN-γ treatment and stimulated with OVA using the procedures described in Fig. 1⇑A. As shown in Fig. 2⇓E, IL-5 and IL-13 production was not suppressed by the rmIFN-γ treatment. Furthermore, histochemical analysis also showed that IFN-γ were not involved in the suppression of PAS⁺ goblet cell hyperplasia (Fig. 2⇓F). However, in vitro treatment with rmIFN-γ could suppress IL-13 production from OVA-stimulated CD4⁺ T cells derived from the lymph nodes of OVA-primed mice in a dose-dependent manner (data not shown). These results indicate that IFN-γ alone does not account for the suppression of polarized Th2 cell responses in the α-GalCer-treated mice.

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FIGURE 2.

IFN-γ is insufficient for suppression of polarized Th2 cells in vivo. A, Saline, α-GalCer (2 μg), rmIFN-γ (5 μg), and α-GalCer (2 μg) plus anti-IFN-γ mAb(250 μg) were injected i.p. into BALB/c mice (n = 10), respectively. The concentration of IFN-γ in the serum 3 h after the injection was determined by ELISA. ∗∗, p < 0.01 compared with α-GalCer treatment. B, Saline, α-GalCer (2 μg), and α-GalCer (2 μg) plus anti-IFN-γ mAb (250 μg) were injected i.p. into BALB/c mice 7 days after the priming with OVA (10 μg) in alum. After repeated OVA challenges, cells in BAL fluids were counted. Results are representative of two independent experiments: mean ± SD (n = 2–4/group). ∗∗, p < 0.01 compared with saline control. C, Serial diluted rmIFN-γ (0.2, 1, or 5 μg) was injected i.p. into BALB/c mice. The concentration of IFN-γ in the serum was determined by ELISA. D, Saline or rmIFN-γ (5 μg) was injected i.p. into BALB/c mice 7 days after OVA immunization. After repeated OVA challenges, cells in BAL fluids were counted. Results are representative of two independent experiments: mean ± SD (n = 2–4/group). ∗, p < 0.05 compared with saline control. E, Production of IL-5 and IL-13 by CD4⁺ peripheral lymph node T cells was determined as shown in Fig. 1. F, Lung tissue sections of the mice injected with α-GalCer plus anti-IFN-γ or rmIFN-γ 24 h after the OVA challenge were stained with PAS (original magnification: ×100 and ×200, respectively). Mac, Macrophages; Eos, eosinophils; Neu, neutrophils; Lym, lymphocytes.

IL-27 expression by a-GalCer-activated iNKT cells

We wondered whether some of the cytokines produced by the stimulation of iNKT cells with α-GalCer were responsible for the attenuation of Th2 responses. Normal spleen cells were cultured in the absence or presence of α-GalCer and cytokines in the culture supernatant were measured by ELISA. As expected, IFN-γ, IL-4, and IL-10 were produced after stimulation of normal spleen cells with α-GalCer. It was also noted that substantial quantities of IL-27p28 were detected in the culture supernatants 24 h after the addition of α-GalCer (Fig. 3⇓).

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FIGURE 3.

Cytokine productions of α-GalCer-activated splenocytes. Whole spleen cells (4 × 10⁵ cells) of normal BALB/c mice were cultured with serially diluted α-GalCer for 24, 48, or 72 h. The concentrations of IFN-γ, IL-4, IL-10, IL-13, and IL-27 in the culture supernatants were determined by ELISA.

In vivo production of IL-27p28 was observed as well after an i.p. injection of α-GalCer into OVA-primed mice. The serum concentrations of IFN-γ, IL-4, and IL-27 peaked 6 h after an i.p. injection of α-GalCer and then declined to undetectable levels after 48 h (Fig. 4⇓A). Since the IL-27 heterodimer is composed of p28 and EBI3 subunits, we next examined EBI3 expression of cells derived from α-GalCer-treated mice. To identify the cell sources of IL-27EBI3, spleens were recovered either before or 1, 3, 6, or 12 h after the α-GalCer treatment; iNKT cells, CD11c⁺ DCs, and CD11b⁺ macrophages were obtained from the splenocytes of α-GalCer-treated BALB/c mice. After isolation of total RNA from each cell fraction, RT-PCR was conducted. As shown in Fig. 4⇓B, the transcript of the IL-27EBI3 subunit was detected in the iNKT cells 3 h after α-GalCer stimulation, but not in the macrophages or DCs. To confirm the purity of each cell fraction, the cells of each fraction were stimulated with PMA plus ionomycin. The only fraction of DCs produced IL-12 in the culture supernatant and macrophages and DCs could also produce IL-27p28, although the expression levels of IL-27p28 in these cells were lower than those of iNKT cells, suggesting that splenic DCs could produce intact IL-27 composed of p28 and EBI3 after the stimulation with PMA plus ionomycin (Fig. 4⇓C). To exclude the possibility of CD11c⁺ DCs in the fraction of iNKT cells, the isolation of the cells was next conducted by flow cytometer, followed by RT-PCR analysis of p28 and EBI3 transcripts. Spleen cells of normal or α-GalCer-treated mice were stained with anti-CD19 mAb, anti-TCRβ mAb, and α-GalCer-loaded CD1d:Ig. iNKT cells were as CD19⁻TCRβ⁺ α-GalCer-CD1d:Ig⁺ cells. As shown, a representative sorting result of the cells derived from α-GalCer-treated mice in Fig. 4⇓D, the highly purified iNKT cell fraction was isolated. The sorted iNKT cells from normal mice were divided into two aliquots, which were subsequently cultured in the absence or presence of PMA and ionomycin. RT-PCR analysis displayed that the expression of the EBI3 transcript depended on the stimulation with in vivo α-GalCer treatment and in vitro PMA plus ionomycin, whereas that of the p28 transcript was almost comparable among all fractions (Fig. 4⇓E). Furthermore, IL-27p28 was also detected in the culture supernatant of the sorted iNKT cells derived from normal mice upon stimulation with PMA plus ionomycin (data not shown). These results collectively suggest that splenic iNKT cells of α-GalCer-treated mice could potentially produce IL-27 heterodimer composed of p28 and EBI3.

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FIGURE 4.

Serum concentrations of IFN-γ, IL-4, and IL-27 from α-GalCer-treated mice. α-GalCer (2 μg) was injected i.p. into BALB/c (WILD) or iNKT^−/− BALB/c background mice (NKT^−/−) 7 days after OVA immunization. A, Serum IFN-γ, IL-4, and IL-27p28 concentrations were assessed by ELISA at various times after α-GalCer injection (3, 6, 12, 24, and 48 h). B, Before or after α-GalCer injection (1, 3, 6, and 12 h), α-GalCer-loaded CD1d:Ig-bound cells (NKT), anti-CD11c microbead-bound cells (DC), and anti-CD11b-bound cells (macrophage (Mac)) were sequentially isolated from whole spleen cells by magnetic microbeads. Transcripts of IL-27 (EBI-3), IFN-γ, IL-4, and GAPDH as a standard were analyzed by RT-PCR. Experiments were repeated twice with similar results. C, Each cell preparation (2 × 10⁵ cells) prepared in Fig. 4B was cultured in the presence of PMA (20 ng/ml) and ionomycin (500 nM). After 48 h, the concentration of IL-12 and IL-27p28 in the culture supernatants was determined by ELISA. D, Spleen cells were recovered 6 h after α-GalCer-injection from the BALB/c mice and stained with anti-CD19-PE, anti-TCRβ-FITC mAbs, and α-GalCer-CD1d:Ig plus anti-mouse IgG1-allophycocyanin. CD19⁻TCRβ⁺α-GalCer-CD1d:Ig⁺ cells were sorted by flow cytometer. E, CD19⁻TCRβ⁺α-GalCer-CD1d:Ig⁺ cells derived from α-GalCer-treated BALB/c mice (lane a) and the cells derived from nontreated BALB/c mice, which were cultured for 6 h in the absence (lane b) or presence (lane c) of PMA plus ionomycin were prepared. Transcripts of IL-27 (EBI-3), IL-27 (p28), and GAPDH as a standard were analyzed by RT-PCR.

Administration of IL-27 along with IFN-γ suppresses polarized Th2 cell responses in vivo

In view of a recent report indicating that IL-27 suppresses Th2 cell development and Th2 cytokines production by polarized Th2 cells (11), we suspected that IL-27 production of α-GalCer-treated mice might be responsible for the suppression of Th2 response and allergic inflammation in α-GalCer-treated mice. Thus, the effect of IL-27 alone on the responses of polarized Th2 cells was tested. Preliminary experiments showed that two i.p. injections of 10 μg of rmIL-27 every 6 h gave an IL-27 concentration comparable to that obtained by the injection of 2 μg of α-GalCer (Fig. 5⇓A). After repeated rmIL-27 injections into OVA-primed mice, CD4⁺ T cells were recovered from their lymph nodes 7 days after the rmIL-27 treatment and stimulated with OVA by the method described in Fig. 1⇑. Unexpectedly, the production of IL-5 and IL-13 by the OVA-primed CD4⁺ T cells was not suppressed by the rmIL-27 treatment. The treatment failed to inhibit and reversely enhanced the infiltration of total cells and eosinophils into BAL fluids (Fig. 5⇓B). In contrast, in vitro treatment with rmIL-27 could suppress IL-13 production by the OVA-stimulated CD4⁺ T cells derived from the lymph nodes of OVA-primed mice in a dose-dependent manner (Fig. 5⇓C).

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FIGURE 5.

Combination of IL-27 plus IFN-γ suppresses polarized Th2 cell functions in vivo. A, rIL-27 (1 or 10 μg) was injected i.p. into BALB/c mice. The concentration of IL-27 in the serum was determined by ELISA. B, IL-27 (10 μg) alone were injected i.p. into BALB/c mice 7 days after OVA immunization. IL-5 and IL-13 production by CD4⁺ peripheral lymph node T cells from the treated and untreated mice was determined as shown in Fig. 1. After repeated OVA challenges, cells in BAL fluids were counted. C, BALB/c mice were immunized with alum-adsorbed OVA. After 7 days, CD4⁺ peripheral lymph node T cells (3 × 10⁵) from the mice were cultured with OVA (100 μg/ml)-pulsed irradiated (20 Gy) syngeneic splenocytes (3 × 10⁶) in the presence of rIL-27 in 96-well microtiter plates for 72 h. IL-5 and IL-13 in the culture supernatants were determined by ELISA. D, IL-27 (10 μg) with IFN-γ (5 μg) were injected i.p. into BALB/c mice 7 days after OVA immunization. IL-5 and IL-13 production by CD4⁺ peripheral lymph node T cells from the treated and untreated mice was determined as shown in Fig. 1. After repeated OVA challenges, cells in BAL fluids were counted. Data shown are representative data from one of two independent experiments: mean ± SD (n = 2–4/group). ∗, p < 0.05 and ∗∗, p < 0.01 compared with saline control. E, Lung tissue sections of the mice injected saline, rmIL-27, or rmIL-27 plus rmIFN-γ 24 h after the OVA challenge were stained with PAS (original magnification: ×100 and ×200, respectively). Mac, Macrophages; Eos, eosinophils; Neu, neutrophils; Lym, lymphocytes.

We wondered whether both IL-27 and IFN-γ might be indispensable for the suppression of polarized Th2 cell responses. Thus, simultaneous administration of both rmIL-27 and rmIFN-γ into OVA-primed mice was conducted. As shown in Fig. 5⇑D, both the cytokine productions by OVA-stimulated CD4⁺ T cells and the eosinophilic airway infiltration of the OVA-primed mice were substantially suppressed by the combined treatment with rmIL-27 and rmIFN-γ. OVA-specific IgE and IgG1 serum Ab levels of the mice treated with rmIL-27 and rmIFN-γ, before or after repeated intranasal challenges of OVA, were significantly suppressed compared with those treated with rmIL-27 alone or saline control (data not shown). Histochemical analysis clearly showed that airway contraction of the lungs derived from the mice treated with rmIL-27 alone was augmented more than that of the saline-control, while that with the combination of rmIL-27 plus IFN-γ was recovered (Fig. 5⇑E). These results suggest that the production of both IL-27 and IFN-γ in α-GalCer-treated mice is responsible for the suppression of polarized Th2 cell responses and airway inflammation.