Enhanced Th2 Cell-Mediated Allergic Inflammation in Tyk2-Deficient ...

Enhanced Th2 Cell-Mediated Allergic Inflammation in Tyk2-Deficient Mice This information is current as of June 20, 2015.

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J Immunol 2003; 170:1077-1083; ; doi: 10.4049/jimmunol.170.2.1077 http://www.jimmunol.org/content/170/2/1077

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References

Yohei Seto, Hiroshi Nakajima, Akira Suto, Kazuya Shimoda, Yasushi Saito, Keiichi I. Nakayama and Itsuo Iwamoto

The Journal of Immunology

Enhanced Th2 Cell-Mediated Allergic Inflammation in Tyk2-Deficient Mice1 Yohei Seto,* Hiroshi Nakajima,2* Akira Suto,* Kazuya Shimoda,† Yasushi Saito,* Keiichi I. Nakayama,‡ and Itsuo Iwamoto*

A

llergic airway inflammation is associated with intense eosinophil and T cell infiltration into the submucosal tissue of airways, and the inflammatory cells are believed to cause epithelial damage and then airway hyperreactivity (1, 2). In a murine model of allergic airway inflammation, we and others have provided evidence that CD4⫹ T cells and IL-5 mediate Ag-induced eosinophil recruitment into the airways of sensitized mice (3, 4). In addition, it has been shown that IL-13 is a key cytokine that induces goblet cell hyperplasia and airway hyperreactivity (5, 6). Conversely, IFN-␥ (7, 8) and IL-12 (9, 10) downregulate Ag-induced allergic inflammation in the airways. Taken together, these observations suggest that Ag-induced allergic inflammation is regulated by the balance between Th1 and Th2 cells, in which Th2 cells up-regulate but Th1 cells down-regulate the allergic inflammation by producing cytokines. The receptor binding of cytokines results in the activation of the Janus family of protein tyrosine kinases (Janus kinases (Jaks))3 and subsequently the activated Jaks phosphorylate signal transducers and activators of transcription (Stats) (11, 12). There are four *Department of Internal Medicine II, Chiba University School of Medicine, Chiba, Japan; and †Medicine and Biosystemic Science, Graduate School of Medical Sciences, and ‡Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Received for publication June 24, 2002. Accepted for publication November 6, 2002. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1

This work was supported in part by grants from the Ministry of Education, Science, and Culture (Japan), and Health Science Research Grants (Japan). Y.S. was supported in part by grants from Asahi General Hospital. 2

Address correspondence and reprint requests to Dr. Hiroshi Nakajima, Department of Internal Medicine II, Chiba University School of Medicine, 1-8-1 Inohana, Chiba City, Chiba 260-8670, Japan. E-mail address: [email protected] 3 Abbreviations used in this paper: Jak, Janus kinase; Stat, signal transducer and activator of transcription; WT, wild type; alum, aluminum hydroxide; PAS, periodic acid-Schiff; BALF, bronchoalveolar lavage fluid.

Copyright © 2003 by The American Association of Immunologists, Inc.

mammalian Jaks, Jak1, Jak2, Jak3, and Tyk2, and they are differentially activated in response to various cytokines (11, 12). Tyk2 has been initially identified as an essential molecule for mediating IFN-␣␤ signaling (13) and subsequently shown to be activated in response to IL-6 (14), IL-10 (15), IL-12 (16), and IL-13 (17). Recently, to address the specific and nonredundant role of Tyk2, mutant mice lacking Tyk2 (Tyk2⫺/⫺ mice) were generated by homologous recombination, and using Tyk2⫺/⫺ mice, it has been demonstrated that Tyk2 is not essential for many of the biological responses upon IFN-␣␤, IL-6, and IL-10 stimulation (18, 19). In contrast, Tyk2 has been shown to be required for IL-12-induced IFN-␥ production in activated T cells (18, 19). However, the role of Tyk2 in in vivo immune responses to exogenous Ags is unknown. To determine the role of Tyk2 in immune responses to exogenous Ags, we examined Ag-specific Ab production and Ag-induced allergic inflammation in the airways in Tyk2⫺/⫺ mice. We also examined the role of Tyk2 in Ag-induced Th cell differentiation using TCR transgenic Tyk2⫺/⫺ mice. We found that immune responses to Ag were biased toward Th2-type in Tyk2⫺/⫺ mice in vitro as well as in vivo. Consequently, Ag-specific IgE production and Ag-induced eosinophil recruitment into the airways were enhanced in Tyk2⫺/⫺ mice. These results indicate that Tyk2 plays an important role in regulating Th1/Th2 balance toward Th1-type and thus down-regulating Th2 cell-mediated allergic inflammation.

Materials and Methods Mice As the degree of Ag-induced eosinophil recruitment into the airways differs depending on the genetic background of the mice (20), Tyk2-deficient mice (Tyk2⫺/⫺ mice; Ref. 18) were backcrossed for ⬎4 generations onto BALB/c mice (Charles River Laboratories, Atsugi, Japan). All mice were H-2d/d and littermate wild-type (WT) mice were used as controls. Backcrossing onto BALB/c mice for four generations results in responses that are indistinguishable to those seen in BALB/c mice in our assays (Ref. 21 0022-1767/03/$02.00


Allergic inflammation is mediated by Th2 cell-derived cytokines, including IL-4, IL-5, and IL-13, and down-regulated by IFN-␥ and IL-12. Tyk2 is a member of the Janus family of protein tyrosine kinases and is activated by a variety of cytokines: IFN-␣␤, IL-6, IL-10, IL-12, and IL-13. In this study, we investigated the role of Tyk2 in the regulation of Ag-induced Th cell differentiation and Ag-induced allergic inflammation in the airways using Tyk2-deficient (Tyk2ⴚ/ⴚ) mice. When splenocytes were stimulated with antigenic peptide, IL-12-mediated Th1 cell differentiation was decreased, but IL-4-mediated Th2 cell differentiation was increased in Tyk2ⴚ/ⴚ mice. In vivo, Ag-specific IgE and IgG1 production was increased, but Ag-specific IgG2a production was decreased in Tyk2ⴚ/ⴚ mice as compared with those in control mice. In addition, Ag-induced eosinophil and CD4ⴙ T cell recruitment, as well as the production of Th2 cytokines in the airways, was increased in Tyk2ⴚ/ⴚ mice. Adoptive transfer experiments revealed that CD4ⴙ T cells were responsible for the enhanced Ag-induced eosinophil recruitment in Tyk2ⴚ/ⴚ mice. In contrast, although the level of IL-13 was increased in the airways of Tyk2ⴚ/ⴚ mice after Ag inhalation, the number of goblet cells, as well as Muc5ac mRNA expression, was decreased in Tyk2ⴚ/ⴚ mice. Together, these results indicate that Tyk2 plays a bilateral role in the regulation of allergic inflammation in the airways: Tyk2 plays a role in the down-regulation of Th2 cell-mediated Ab production and eosinophil recruitment in the airways by regulating Th1/Th2 balance toward Th1-type, while Tyk2 is necessary for the induction of IL-13-mediated goblet cell hyperplasia in the airways. The Journal of Immunology, 2003, 170: 1077–1083.

1078 and our unpublished data). OVA-specific DO11.10 (DO10⫹) TCR transgenic mice (22) were backcrossed over ten generations onto BALB/c mice and then crossed with Tyk2⫺/⫺ mice. DO10⫹Tyk2⫺/⫺ mice (H-2d/d) and the littermate DO10⫹ mice (H-2d/d) were analyzed in the following experiments. Mice were housed in microisolator cages under pathogen-free conditions. All experiments were performed according to the guidelines of Chiba University.

Immunization Mice (aged 7– 8 wk) were immunized i.p. twice with 4 ␮g of OVA (SigmaAldrich, St. Louis, MO) in 4 mg of aluminum hydroxide (alum) at a 2-wk interval. As a control, mice were injected i.p. with 4 mg of alum alone.

Ag-induced allergic inflammation in the airways

Cytokine levels in BALF The amounts of IL-4, IL-5, IFN-␥, and TNF-␣ in the BALF were determined by the enzyme immunoassay using murine IL-4, IL-5, IFN-␥, and TNF-␣ ELISA kits from BD PharMingen (San Diego, CA). The amounts of IL-13 in the BALF were determined by ELISA kit from R&D Systems (Minneapolis, MN). The assays were performed in duplicate according to the manufacturers’ instruction. The detection limits of these assays were 15 pg/ml IL-4 and IL-5, 50 pg/ml IFN-␥ and TNF-␣, and 30 pg/ml IL-13.

Determination of Ag-specific IgE, IgG1, and IgG2a Abs in serum Two weeks after the second immunization, the titer of OVA-specific IgE Ab in mouse serum was assessed by a 24-h passive cutaneous anaphylaxis reaction as described previously (21). The amount of OVA-specific IgG1 and IgG2a Abs in serum was measured by ELISA as described previously (21).

Flow cytometric analysis Cells from the BALF and spleen were stained and analyzed on a FACSCalibur (BD Biosciences, San Jose, CA) using CellQuest software. For direct staining, the following conjugated Abs were purchased from BD PharMingen: anti-CD4 FITC, PE, APC (H129.19), anti-CD8 PE (53.6.7), and anti-B220 FITC (RA3-6B2). Before staining, FcRs were blocked with anti-CD16/32 Ab (2.4G2; BD PharMingen).

Measurement of airway reactivity Two weeks after the second immunization, sensitized mice were challenged with the inhaled OVA three times at a 24-h interval. Twenty-four hours after the final OVA challenge, airway reactivity to aerosolized methacholine (3–50 mg/ml) was measured using the Buxco whole body plethysmograph (Buxco Electronics, Sharon, CT) as described by Hamelmann et al. (24).

RT-PCR Total RNA was isolated from lung tissues using Isogen solution (Nippon Gene, Tokyo, Japan), and RT-PCR for Muc5ac mRNA was performed using primer pairs as described elsewhere (25). RT-PCR for ␤-actin mRNA was also performed to control the sample-to-sample variation in RNA isolation and integrity, RNA input, and reverse transcription. All PCR amplifications were performed at least three times with multiple sets of experimental RNAs.

Adoptive transfer experiments for Ag-induced eosinophil and T cell infiltration in the airways Splenocytes from OVA-sensitized Tyk2⫺/⫺ mice and littermate WT mice were stimulated with OVA (200 ␮g/ml) at 37°C for 72 h. After dead cells were removed by centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ), cells (1 ⫻ 107 cells/mouse) were transferred to nonimmunized BALB/c SCID mice. The frequency of cell populations of transferred cells was 60 –70% of CD4⫹ T cells, 10 –15% of CD8⫹ T cells, and 20 –25% of B220⫹ cells, and no significant difference was observed between WT mice and Tyk2⫺/⫺ mice. In some experiments, CD4⫹ T cells were removed by magnetic cell sorting using FITC-labeled anti-CD4 Ab (RM4-5; BD PharMingen) and anti-FITC magnetic microbeads (Miltenyi Biotec, Sunnyvale, CA), and the CD4⫹ T cell-depleted populations were transferred to BALB/c SCID mice. These mice were then challenged with inhaled OVA (50 mg/ml) at 0, 24, and 48 h after the cell transfer, and at 24 h after the last OVA inhalation, the number of eosinophils and CD4⫹ T cells in the BALF was evaluated as described above. In preliminary experiments, we found that the in vitro Ag stimulation of splenocytes was required for the induction of Ag-induced eosinophil recruitment in the airways.

Ag-induced T cell differentiation Splenocytes (1 ⫻ 106/ml) from DO10⫹Tyk2⫺/⫺ mice and the littermate DO10⫹ mice were stimulated with OVA323–339 peptide (50 ␮M) in a 24well microtiter plate at 37°C for 48 h. Where indicated, IL-12 (7.5 ng/ml; R&D Systems) was added to polarize toward Th1 cells (Th1 condition), and IL-4 (7.5 ng/ml; R&D Systems) was added to polarize toward Th2 cells (Th2 condition). Cells were washed with PBS and cultured for another 3 days in Th0 (nonpolarizing), Th1, or Th2 condition in the presence of IL-2 (5 ng/ml). Intracellular cytokine analyses for IL-4 vs IFN-␥ were performed as described previously (26).

Data analysis Data are summarized as mean ⫾ SD. The statistical analysis of the results was performed by the unpaired t test. Values of p ⬍ 0.05 were considered significant.

Results Ag-induced T cell differentiation is biased toward Th2-type in TCR transgenic Tyk2⫺/⫺ mice It has been demonstrated that IL-4 and IL-12 mediate Th2 and Th1 cell differentiation, respectively (27, 28). IL-4 stimulation results in the activation of Jak1 and Jak3 and subsequently in the phosphorylation of Stat6, whereas IL-12 activates Jak2 and Tyk2 and then phosphorylates Stat4 (11, 12). Although the importance of Stat4 and Stat6 in the differentiation of Th1 cells and Th2 cells is well documented (27), the roles of Jaks in the Th cell differentiation are still largely unknown. To address the role of Tyk2 in Ag-induced Th cell differentiation, Tyk2⫺/⫺ mice were crossed with OVA-specific DO10⫹ TCR transgenic mice, and Ag-induced Th cell differentiation in DO10⫹Tyk2⫺/⫺ mice was compared with that in the littermate DO10⫹ mice. As shown in Fig. 1, when splenocytes from DO10⫹Tyk2⫺/⫺ mice were stimulated with antigenic peptide (OVA323–339) in nonpolarizing Th0 condition, Th2 cells (IL-4⫹IFN-␥⫺ cells) were increased as compared with those in DO10⫹ mice (DO10⫹Tyk2⫺/⫺ mice, 11.2 ⫾ 3.0%, vs DO10⫹ mice, 5.8 ⫾ 2.2%, mean ⫾ SD; n ⫽ 5 mice in each group; p ⬍ 0.01) (Fig. 1, A and B). Moreover, IL-4 and IL-5 levels were increased in the culture supernatant in DO10⫹Tyk2⫺/⫺ mice (data not shown). In contrast, the number of Th1 cells (IL-4⫺IFN-␥⫹ cells) was not affected by the absence of Tyk2 in Th0 condition.


Allergic airway inflammation was induced by the inhalation of OVA in sensitized mice as described previously (3). Briefly, 12–14 days after the second immunization, the sensitized mice were given aerosolized OVA (50 mg/ml) dissolved in 0.9% saline by a DeVilbiss 646 nebulizer (DeVilbiss, Somerset, PA) for 20 min. OVA solution contained ⬍1 ng/ml endotoxin. As a control, 0.9% saline alone was administered by the nebulizer. In experiments shown in Figs. 3B and 6, the sensitized mice were given aerosolized OVA (50 mg/ml) three times at a 24-h interval. At indicated times after the inhalation, trachea and a sagittal block of right lung were excised, fixed in 10% buffered-formalin, and embedded in paraffin. The specimens (3-␮m thick) of the trachea were stained with Luna and H&E solutions. The number of eosinophils in the submucosal tissue of trachea was counted in Luna-stained sections and expressed as the number of eosinophils per the length of the basement membrane of trachea, which was measured with a digital curvimeter. Lung sections were stained with H&E and periodic acid-Schiff (PAS) according to standard protocols. A semiquantitative scoring system was used to grade the magnitude of inflammatory cell infiltration in the perivascular and peribronchial spaces on H&E-stained lung sections as described previously (23). Goblet cells were counted on PASstained lung sections using a scoring system as described elsewhere (6). The number of eosinophils, lymphocytes, neutrophils, and macrophages recovered in the bronchoalveolar lavage fluid (BALF) was evaluated as described previously (21). In short, after bronchoalveolar lavage was performed with 3 ml of PBS, BALF was centrifuged at 400 ⫻ g for 5 min at 4°C, and differential cell counts were performed on cytospin cell preparations stained with Wright-Giemsa solution. A fraction of the cells were subjected to a flow cytometric analysis for the lymphocyte surface phenotyping of CD4 and CD8 as described below.

ROLE OF Tyk2 IN ALLERGIC INFLAMMATION


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However, interestingly, when IL-12 was added to the culture (Th1 condition), IL-12-mediated Th1 cell differentiation was significantly reduced in DO10⫹Tyk2⫺/⫺ mice (DO10⫹Tyk2⫺/⫺ mice, 18.3 ⫾ 4.5%, vs DO10⫹ mice, 55.3 ⫾ 8.3%; n ⫽ 5; p ⬍ 0.001) (Fig. 1, C vs D). The finding of reduced IL-12-mediated Th1 cell differentiation is consistent with a previous finding using anti-TCR Ab-stimulated CD4⫹ T cells in Tyk2⫺/⫺ mice (18). Furthermore, in the presence of IL-4 (Th2 condition), the number of Th2 cells in DO10⫹Tyk2⫺/⫺ mice was more increased than that in DO10⫹ mice (DO10⫹Tyk2⫺/⫺ mice, 34.6 ⫾ 5.9%, vs DO10⫹ mice, 19.8 ⫾ 4.2%; n ⫽ 5; p ⬍ 0.01) (Fig. 1, E and F). Taken together, these results indicate that Tyk2 regulates Th1/Th2 balance toward Th1-type during Ag-induced Th cell differentiation. Ag-specific IgE production is increased in Tyk2⫺/⫺ mice Because Th cell differentiation is biased toward Th2-type in Tyk2⫺/⫺ mice in vitro (Fig. 1), we next attempted to determine

FIGURE 2. Ag-specific IgE production is increased in Tyk2⫺/⫺ mice. Tyk2⫺/⫺ mice and WT mice were immunized i.p. twice with OVA in alum at a 2-wk interval, and 14 days after the second immunization, the levels of Ag-specific IgE, IgG1, and IgG2a in sera were evaluated. As controls, Tyk2⫺/⫺ mice and WT mice were injected with alum alone. Anti-OVA IgE Ab was assessed by a 24-h passive cutaneous anaphylaxis reaction, and anti-OVA IgG1 and IgG2a Abs were assessed by ELISA. Data are means ⫾ SD for 10 mice in each group. The mean values in Tyk2⫺/⫺ mice are significantly different from the mean value of WT mice. ⴱ, p ⬍ 0.02; ⴱⴱ, p ⬍ 0.01.

FIGURE 3. Ag-induced eosinophil recruitment into the airways is increased in Tyk2⫺/⫺ mice. A, OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged with the inhalation of OVA or saline (as a control), and the number of eosinophils infiltrating into the submucosal tissue of trachea was evaluated at 24 h after the inhalation. Unsensitized Tyk2⫺/⫺ mice and WT mice were used as controls. Data are means ⫾ SD for eight mice in each group. ⴱ, p ⬍ 0.01. B, OVA-sensitized mice were challenged with the inhaled OVA three times at a 24-h interval. At 24 h after the last OVA inhalation, lung was removed and stained with H&E. Inflammatory cell infiltration into the perivascular and peribronchial spaces was scored as described previously (23). Data are means ⫾ SD for 10 mice in each group. ⴱ, p ⬍ 0.01. C, OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged with the inhaled OVA or saline, and the number of eosinophils, lymphocytes, CD4⫹ T cells, neutrophils, and macrophages in BALF was evaluated at 36 h after the inhalation. Data are means ⫾ SD for eight mice in each group. ⴱ, p ⬍ 0.01; ⴱⴱ, p ⬍ 0.005.

whether Tyk2 regulates in vivo Th1/Th2 response to exogenous Ag. Given that Ig class-switching is regulated by cytokines from Th cells (29, 30), we analyzed Ag-specific Ab production in Tyk2⫺/⫺ mice. Tyk2⫺/⫺ mice and the littermate WT mice were immunized i.p. twice with OVA in alum at a 2-wk interval, and 14 days after the second immunization, the levels of Ag-specific IgE, IgG1, and IgG2a in sera were evaluated. In agreement with the enhanced Th2 cell differentiation by the absence of Tyk2 (Fig. 1), Tyk2⫺/⫺ mice exhibited increased levels of OVA-specific IgE and IgG1 production as compared with those in WT mice (n ⫽ 10; p ⬍ 0.02 and p ⬍ 0.01, respectively) (Fig. 2). In contrast, OVA-specific IgG2a production was decreased in Tyk2⫺/⫺ mice (n ⫽ 10, p ⬍ 0.01)


FIGURE 1. Development of Th2 cells is increased in TCR transgenic Tyk2⫺/⫺ mice. Splenocytes from DO10⫹ mice (A, C, and E) or DO10⫹Tyk2⫺/⫺ mice (B, D, and F) were stimulated with OVA323–339 peptide (50 ␮M) for 48 h in Th0- (A and B), Th1- (C and D), or Th2- (E and F) polarizing condition. Cells were cultured in the presence of IL-2 for another 3 days and then re-stimulated with plate-bound anti-CD3 mAb for 6 h. Intracellular cytokine profiles for IL-4 vs IFN-␥ were determined on CD4⫹ T cells. Shown are representative FACS profiles from five mice in each group.

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Table I. Cytokine levels in BALF a Cytokines (pg/ml)

IL-4 WT Tyk2⫺/⫺ IL-5 WT Tyk2⫺/⫺ IL-13 WT Tyk2⫺/⫺ IFN-␥ WT Tyk2⫺/⫺ TNF-␣ WT Tyk2⫺/⫺

Saline

OVA

ND ND

80.0 ⫾ 17.1 120.5 ⫾ 20.4*

ND ND

250.6 ⫾ 47.2 630.5 ⫾ 99.4**

ND ND

78.3 ⫾ 27.9 155.9 ⫾ 31.3**

ND ND

2105.5 ⫾ 370.4 2080.5 ⫾ 290.2

ND ND

330.2 ⫾ 37.9 290.7 ⫾ 51.8

(Fig. 2). These results suggest that systemic immune response to exogenous Ag is biased toward Th2-type in Tyk2⫺/⫺ mice. Ag-induced allergic inflammation in the airways is enhanced in Tyk2⫺/⫺ mice Next, we evaluated Ag-induced eosinophil recruitment into the airways, another characteristic of Th2 cell-mediated immune response (2– 4), in Tyk2⫺/⫺ mice. OVA-sensitized Tyk2⫺/⫺ mice and littermate WT mice were challenged with inhaled OVA, and eosinophil recruitment into the submucosal tissue of trachea was evaluated at 24 h after Ag inhalation. As shown in Fig. 3A, Aginduced eosinophil recruitment into the trachea was significantly increased by 97% in Tyk2⫺/⫺ mice (Tyk2⫺/⫺ mice, 34.8 ⫾ 6.3, vs WT mice, 17.7 ⫾ 3.5 eosinophils/mm; n ⫽ 8 mice in each group; p ⬍ 0.01). The inhaled OVA challenge did not significantly induce eosinophil recruitment into the trachea in either unsensitized WT mice or Tyk2⫺/⫺ mice (Fig. 3A). Ag-induced eosinophil recruitment into the trachea at 48 h after Ag inhalation was also significantly increased in Tyk2⫺/⫺ mice (data not shown). Histological analysis showed that inflammatory cell infiltration in the lung was

FIGURE 4. IFN-␥ inhibits Ag-induced eosinophil recruitment into the airways in Tyk2⫺/⫺ mice. OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged with the inhaled OVA or saline, and the number of eosinophils in BALF was evaluated at 36 h after the inhalation. Recombinant murine IFN-␥ (60,000 U/mouse) or PBS was administered i.p. 2 h before and 10 and 22 h after the inhaled OVA challenge. Data are means ⫾ SD for four mice in each group. ⴱ, p ⬍ 0.001.

also significantly enhanced in Tyk2⫺/⫺ mice after Ag inhalation as compared with that of WT mice (Fig. 3B). As anticipated, without the inhaled Ag challenge, no inflammatory cell infiltration was observed in either WT mice or Tyk2⫺/⫺ mice (Fig. 3B). To determine which cell type infiltrates in the airways of Tyk2⫺/⫺ mice, cell populations in BALF of sensitized mice were determined after Ag inhalation (Fig. 3C). Consistent with the results shown in Fig. 3A, the number of eosinophils recovered in BALF at 36 h after Ag inhalation was significantly increased in Tyk2⫺/⫺ mice (Tyk2⫺/⫺ mice, 38.4 ⫾ 7.7, vs WT mice, 25.1 ⫾ 5.4 ⫻ 104 cells/mice; n ⫽ 8 mice in each group; p ⬍ 0.01) (Fig. 3C). The number of lymphocytes recovered in BALF was also increased by 117% in Tyk2⫺/⫺ mice (n ⫽ 8, p ⬍ 0.005) (Fig. 3C). FACS analysis revealed that the majority of lymphocytes in BALF were CD4⫹ T cells, and consequently the number of CD4⫹ T cells was increased by 110% in Tyk2⫺/⫺ mice (Fig. 3C; p ⬍ 0.005). In contrast, the number of neutrophils or macrophage in the BALF was not affected by the absence of Tyk2 (Fig. 3C). These results indicate that Tyk2 plays a role in the down-regulation of Ag-induced eosinophil and CD4⫹ T cell recruitment into the airways. We then examined cytokine levels in the BALF of sensitized Tyk2⫺/⫺ mice and WT mice after Ag inhalation. IL-4 and IL-5 levels in the BALF at 36 h after Ag challenge were increased in Tyk2⫺/⫺ mice by 50 and 152%, respectively (n ⫽ 5; p ⬍ 0.05 and p ⬍ 0.01, respectively) (Table I). IL-13 levels were also increased in the BALF of Tyk2⫺/⫺ mice, by 99% (n ⫽ 5, p ⬍ 0.01) (Table I). In contrast, IFN-␥ and TNF-␣ levels were indistinguishable between WT mice and Tyk2⫺/⫺ mice (Table I). As expected, the inhaled Ag challenge did not significantly induce cytokine production in the airways in unsensitized WT mice or Tyk2⫺/⫺ mice (data not shown). Taken together, these results suggest that Tyk2 plays a role in the down-regulation of Ag-induced Th2 cell differentiation in vivo and that the enhanced Ag-induced eosinophil recruitment in Tyk2⫺/⫺ mice results in part from the enhanced IL-5 production in the airways. It has been shown that endogenously produced IFN-␥ negatively regulates Ag-induced eosinophil recruitment into the airways (7, 8). To exclude the possibility that the enhanced eosinophil recruitment into the airways in Tyk2⫺/⫺ mice results from the defect in IFN-␥ production, we examined the effect of rIFN-␥ on Ag-induced eosinophil recruitment into the airways in Tyk2⫺/⫺ mice.


a OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged with the inhalation of OVA or saline, and the cytokine levels in BALF were evaluated at 36 h after the inhalation. The amount of IL-4, IL-5, IL-13, IFN-␥, and TNF-␣ in the BALF was determined by ELISA. Data are means ⫾ SD for five mice in each group. The detection limits of these assays were 15 pg/ml IL-4 and IL-5, 50 pg/ml IFN-␥ and TNF-␣, and 30 pg/ml IL-13. ND, Not detectable. ⴱ and ⴱⴱ, Significantly different from the mean value of WT mice; ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.

FIGURE 5. CD4⫹ T cells are responsible for the enhanced eosinophil recruitment in the airways in Tyk2⫺/⫺ mice. Splenocytes from OVA-sensitized Tyk2⫺/⫺ mice and WT mice were stimulated with OVA (200 ␮g/ ml) for 72 h and then injected i.v. to nonimmunized BALB/c SCID mice (1 ⫻ 107 cells/mice). Where indicated, CD4⫹ T cells were depleted by magnetic cell sorting before injection. These mice were then challenged with the inhaled OVA three times at a 24-h interval. Twenty-four hours after the last OVA inhalation, the number of eosinophils and CD4⫹ T cells in the BALF was evaluated. Data are means ⫾ SD for five mice in each group. ⴱ, p ⬍ 0.005.


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Consistent with a previous report (7), the injection of rIFN-␥ to sensitized WT mice significantly decreased Ag-induced eosinophil recruitment into the airways (n ⫽ 4, p ⬍ 0.001) (Fig. 4). Similarly, the injection of rIFN-␥ decreased Ag-induced eosinophil recruitment into the airways in sensitized Tyk2⫺/⫺ mice (n ⫽ 4, p ⬍ 0.001) (Fig. 4). These results suggest that there is no significant defect in IFN-␥-mediated inhibition of Ag-induced allergic inflammation in Tyk2⫺/⫺ mice. CD4⫹ T cells are responsible for the enhanced eosinophil recruitment into the airways in Tyk2⫺/⫺ mice It has been shown that Tyk2 is expressed not only in lymphoid cells but also in nonlymphoid cells (11, 12). To determine which cell type is responsible for the enhanced allergic inflammation in the airways of Tyk2⫺/⫺ mice, we performed the adoptive transfer experiments. To eliminate the interference of endogenous immune response in recipient mice during the inhaled Ag challenge, we performed these experiments using BALB/c SCID mice as recip-

ient mice. Splenocytes from OVA-sensitized Tyk2⫺/⫺ mice or littermate WT mice were stimulated with OVA for 3 days and then transferred i.v. to nonimmunized BALB/c SCID mice. These mice were challenged with the inhaled OVA three times at a 24-h interval, and the number of eosinophils and CD4⫹ T cells in the BALF was evaluated at 24 h after the last OVA inhalation. As shown in Fig. 5, when splenocytes from WT mice were transferred to BALB/c SCID mice, eosinophil recruitment into the airways was induced by the inhaled Ag challenge (Fig. 5). In the absence of the cell transfer (PBS alone was injected), few eosinophils were detected in the BALF (Fig. 5), suggesting that the splenocytes from donor mice are required for the airway eosinophilia in this system. Interestingly, Ag-induced eosinophil recruitment into the airways was significantly increased in mice transferred with splenocytes from Tyk2⫺/⫺ mice (n ⫽ 5, p ⬍ 0.005) (Fig. 5). In addition to eosinophils, Ag-induced CD4⫹ T cell recruitment was also increased in mice transferred with splenocytes from Tyk2⫺/⫺ mice (Fig. 5). Moreover, IL-4 production in the BALF was increased, but IFN-␥


FIGURE 6. Ag-induced goblet cell hyperplasia is decreased in Tyk2⫺/⫺ mice. A, OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged with inhaled OVA or saline three times at a 24-h interval. As controls, unsensitized Tyk2⫺/⫺ mice and WT mice were challenged with the inhaled OVA or saline. At 24 h after the last OVA inhalation, lung was removed and the degree of goblet cell hyperplasia was scored on PAS-stained sections as described previously (6). Data are means ⫾ SD for 8 –10 mice in each group. ⴱ, p ⬍ 0.01. B, Representative photomicrographs of PAS-stained lung sections from WT mice (a, b, c, and d) and Tyk2⫺/⫺ mice (e, f, g, and h) are shown (magnification ⫻200). C, OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged with the inhaled OVA or saline as described above. Total RNA was prepared from lung tissues, and RT-PCR for Muc5ac mRNA as well as ␤-actin mRNA (as a control) was performed. Shown are representative data from five independent experiments. D, OVA-sensitized Tyk2⫺/⫺ mice and WT mice were challenged three times with the inhaled OVA or saline at a 24-h interval. As controls, unsensitized Tyk2⫺/⫺ mice and WT mice were challenged three times with inhaled OVA at a 24-h interval. Airway hyperreactivity was measured at 24 h after the final OVA challenge using a Buxco system where mice were exposed to increasing concentrations of methacholine (3–50 mg/ml). Airway reactivity is expressed as enhanced pause (Penh) values for each concentration of methacholine over baseline response. Data are means ⫾ SD for six mice in each group.

1082 production was decreased in mice transferred with splenocytes from Tyk2⫺/⫺ mice (data not shown). Furthermore, when CD4⫹ T cell-depleted splenocytes from either WT mice or Tyk2⫺/⫺ mice were transferred to BALB/c SCID mice, not only Ag-induced CD4⫹ T cell recruitment but also Ag-induced eosinophil recruitment into the airways was significantly decreased (Fig. 5). These results indicate that the Ag-induced eosinophil recruitment into the airways is mediated by CD4⫹ T cells and thus suggest that CD4⫹ T cells are responsible for the enhanced eosinophil recruitment into the airways in Tyk2⫺/⫺ mice. Ag-induced goblet cell hyperplasia and airway hyperresponsiveness in Tyk2⫺/⫺ mice

Discussion In the present study, we show that the absence of Tyk2 results in enhanced Th2 cell-mediated Ab production and allergic inflammation. We found that Ag-induced eosinophil and CD4⫹ T cell recruitment into the airways as well as Th2 cytokine production in the airways was increased in Tyk2⫺/⫺ mice (Fig. 3 and Table I). We also found that Ag-induced Th cell differentiation was biased toward Th2-type in TCR transgenic Tyk2⫺/⫺ mice (Fig. 1). Furthermore, CD4⫹ T cells were responsible for the enhanced eosinophil recruitment into the airways in Tyk2⫺/⫺ mice (Fig. 5). Therefore, these results indicate that Tyk2 plays an important role in the down-regulation of Th2 cell-mediated allergic inflammation. Tyk2 was first identified as an essential component in IFN-␣ signaling (13). However, recent studies with Tyk2⫺/⫺ mice have shown that the defective IFN-␣ responses in Tyk2⫺/⫺ embryonic fibroblasts are overcome when high doses of IFN-␣ are provided (18, 19). Moreover, Tyk2⫺/⫺ mice respond normally to IL-10 and IL-6 (18, 19), both of which activate Tyk2 (14, 15). Thus, unlike other Jaks, the nonredundant functions of Tyk2 in IFN-␣, IL-10, and IL-6 signaling are modest. In contrast, IL-12-induced IFN-␥ production was severely impaired in Tyk2⫺/⫺CD4⫹ T cells (Fig. 1 and Refs. 18 and 19), and the defect was not overcome even by a high concentration of IL-12 (20 ng/ml) (data not shown), suggesting that IL-12 signaling depends more profoundly on Tyk2 than IFN-␣ signaling. Interestingly, we found that the levels of IL-4 and IL-5 in the BALF of Ag-inhaled Tyk2⫺/⫺ mice were increased as compared with those in WT mice but IFN-␥ levels were comparable to those in WT mice (Table I). Because it has been shown that IL-12 and subsequent Stat4 activation is required for IFN-␥ production in CD4⫹ T cells but not in CD8⫹ T cells (32), Tyk2 may not be essential for IFN-␥ production from CD8⫹ T cells. Therefore, it is possible that IFN-␥ production from non-CD4⫹ T cell populations,

including CD8⫹ T cells, masks the possible defect in IFN-␥ production of CD4⫹ T cells in the airways of Tyk2⫺/⫺ mice. However, we also found that, when Ag-specific CD4⫹ T cells were stimulated with antigenic peptide in the absence of exogenous cytokines, Th2 cells were increased but Th1 cells were not decreased by the absence of Tyk2 (Fig. 1). These results suggest that Tyk2 may be involved in the regulation of the balance between Th1 cells and Th2 cells beyond its role in IL-12 signaling for IFN-␥ production. In this regard, it has been demonstrated that some of the IFN-␥-dependent signaling is impaired in Tyk2⫺/⫺ embryonic fibroblasts (19), although no association of Tyk2 with the IFN-␥R has been reported. Therefore, it is plausible that Tyk2 may be involved in IFN-␥-mediated inhibition of Th2 cell differentiation independent of IFN-␥ production, thus accounting for the enhanced Th2 cell differentiation without the decreased IFN-␥ production in Tyk2⫺/⫺CD4⫹ T cells. This possibility is under investigation in our laboratory. In contrast to the enhanced eosinophil and CD4⫹ T cell recruitment in Tyk2⫺/⫺ mice, we also found that Ag-induced goblet cell hyperplasia and Muc5ac mucin gene expression were decreased in Tyk2⫺/⫺ mice (Fig. 6). Because IL-13, a key cytokine for epithelial goblet cell hyperplasia and the induction of mucin gene expression (2, 5, 6, 31), was increased in the BALF of Tyk2⫺/⫺ mice after Ag inhalation, these results suggest that Tyk2 may be involved in IL-13-mediated goblet cell hyperplasia in the airways. This observation is in agreement with the previous finding that Tyk2 is associated with IL-13R␣1 chain and phosphorylated upon IL-13 stimulation (17, 33). In contrast, we found that Tyk2⫺/⫺ mice exhibited a response to methacholine-induced airway hyperreactivity similar to that of WT mice (Fig. 6D), whereas Ag-induced goblet cell hyperplasia was decreased in Tyk2⫺/⫺ mice. Because airway hyperreactivity is believed to be mediated not only by epithelial damage but also by the inflammatory change in the airways (34), the increased inflammatory cell infiltration in the airways may compensate for the decreased epithelial damage in Tyk2⫺/⫺ mice (Fig. 6A) and then Tyk2⫺/⫺ mice exhibit a response to methacholine-induced airway hyperreactivity similar to that of WT mice (Fig. 6D). Although there has been no information available on the expression and/or activation levels of Tyk2 protein in allergic diseases, our results raise the possibility that the impaired expression and/or activation of Tyk2 may be involved in the pathogenesis of Th2 cell-mediated allergic diseases. Further studies are required to address the role of Tyk2 in the pathogenesis of allergic diseases in humans.

Acknowledgments We thank Drs. Y. Maezawa, K. Hirose, K. Suzuki, S.-i. Kagami, K. Kumano, K. Kurasawa, and S. Yoshida for valuable discussions.

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Because the levels of IL-13, a key cytokine that induces goblet cell hyperplasia and airway hyperreactivity (5, 6), were increased by inhaled Ag challenge in Tyk2⫺/⫺ mice (Table I), we next examined the number of epithelial goblet cells as well as airway hyperreactivity to methacholine after Ag inhalation in Tyk2⫺/⫺ mice. Surprisingly, Ag-induced epithelial goblet cell hyperplasia was significantly decreased in Tyk2⫺/⫺ mice as compared with that in WT mice (n ⫽ 8 –10, p ⬍ 0.02) (Fig. 6, A and B). In addition, Ag-induced mRNA expression of Muc5ac, one of the mucin genes that are mainly produced by goblet cells in response to IL-13 (31), was decreased in Tyk2⫺/⫺ mice as compared with that in WT mice (Fig. 6C). Given the increased IL-13 production in the airways of Tyk2⫺/⫺ mice (Table I), these results suggest that Tyk2 may be involved in IL-13-mediated hyperplasia of epithelial goblet cells. In contrast, no significant difference was observed in the responses to methacholine-induced airway hyperreactivity between Tyk2⫺/⫺ mice and WT mice (n ⫽ 6 for each group; Fig. 6D).



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