Locally Via Alteration of Chemokines Changes in Allergic Airway ...

The Journal of Immunology

STAT4 Signal Pathways Regulate Inflammation and Airway Physiology Changes in Allergic Airway Inflammation Locally Via Alteration of Chemokines Kavita Raman,* Mark H. Kaplan,† Cory M. Hogaboam,* Aaron Berlin,* and Nicholas W. Lukacs1* Mice homozygous for the STAT4-null mutation were sensitized to cockroach Ag, challenged intratracheally 21 days later, and compared with STAT4-competent allergic mice. The STAT4ⴚ/ⴚ mice showed significant decreases in airway hyperreactivity (AHR) and peribronchial eosinophils compared with wild-type controls. In addition, pulmonary levels of chemokines were decreased in the STAT4ⴚ/ⴚ mice, including CC chemokine ligand (CCL)5, CCL6, CCL11, and CCL17. However, levels of Th2-type cytokines, such as IL-4 and IL-13, as well as serum IgE levels were similar in the two groups. Transfer of splenic lymphocytes from sensitized wild-type mice into sensitized STAT4ⴚ/ⴚ mice did not restore AHR in the mutant mice. Furthermore, chemokine production and peribronchial eosinophilia were not restored during the cellular transfer experiments. Thus, it appears that STAT4 expression contributes to a type 2 process such as allergen-induced chemokine production and AHR. In additional studies, competent allergic mice were treated with anti-IL-12 locally in the airways at the time of allergen rechallenge. These latter studies also demonstrated a decrease in AHR. Altogether, these data suggest that STAT4-mediated pathways play a role locally within the airway for the exacerbation of the allergen-induced responses. The Journal of Immunology, 2003, 170: 3859 –3865.

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he induction of atopic airway responses requires a complex series of mechanisms that depend upon the cytokine environment that is created by the specific immune response to allergens. Specifically, the Th1/Th2 phenotype that is established in the airways is thought to determine the severity of the subsequent atopic and asthmatic reactions (1). A significant number of publications have clearly identified that Th2 cytokines, IL-4, IL-5, and IL-13, play an important role in initiating and maintaining the asthmatic airway and causing many of the detrimental pathophysiologic responses observed during disease (2–10). In addition, these same cytokines are known to induce a number of specific chemokines that are closely related to the asthmatic response and appear to specifically recruit leukocytes that initiate a more severe response (11–18). In particular, the recruitment of eosinophils and Th2-type lymphocytes may be especially pertinent to the development of chronic debilitating asthmatic responses (19 –22). A significant effort to establish a therapy to skew the local pulmonary response away from a Th2-type response and toward a physiologically less detrimental Th1-type response to allergens is a major focus of several academic and pharmaceutic programs. Studies have clearly established that introduction of exogenous or adenoviral overexpression of Th1-type cytokines, IL-12 or IFN-␥, can significantly reduce the Th2 cytokine-mediated pulmonary responses in murine models of asthma (23–26). However, the levels

*Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109; and †Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202 Received for publication July 17, 2002. Accepted for publication January 10, 2003. 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 Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, 5214 Medical Science I, Ann Arbor, MI 48109-0602. E-mail address: nlukacs@ umich.edu

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

that have been introduced have been substantial and not physiologically comparable to normal biological levels. The systemic neutralization of IL-12 before and during allergen challenges significantly increased the Th2-mediated effects and altered the pathophysiology (27). Other studies that have attempted to transfer Th1- and/or Th2-type lymphocytes to determine the impact of these responses on the local allergen response have met with mixed responses. Although the Th2-type, but not Th1-type, lymphocytes appear to induce a much more severe disease, Th1-type lymphocyte transfers were unable to down-regulate the Th2 lymphocyte-induced pathophysiology (28, 29). In fact, it appeared the Th1-type lymphocyte transferred along with the Th2-type cell transfer only creates more inflammation. In additional studies using IL-12-deficient mice, a significant decrease in the pulmonary allergic response in systemically sensitized mice, especially eosinophil accumulation, was dependent upon alteration of VCAM-1 expression (30). In contrast, a study using a model of airway sensitization with OVA demonstrated an opposite effect in IL-12⫺/⫺ mice and observed an increase in eosinophilia (31). Thus, physiologic type I cytokines within the lung may have a significant impact on the development of disease. In the present studies, we have investigated the role of the specific STAT molecule STAT4 and one of the activators, IL-12. This specific signal is closely linked to the development of Th1-type responses, and strategies to augment this pathway during Th2-type asthmatic responses may be advantageous to drive the local reaction away from the allergic/asthmatic phenotype. In contrast to the original hypothesis of STAT4 having a regulatory role on the Th2associated allergic airway response, it appears that STAT4 signaling may be required for the implementation of a fully active allergic immune response. The data provided in these studies suggest that STAT4-mediated signaling is required locally to provide for appropriate chemokine production, leading to the detrimental inflammatory response and altered airway physiology. 0022-1767/03/$02.00

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Materials and Methods Mice BALB/c (wild-type (wt)2) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). STAT4⫺/⫺ mice were obtained and backcrossed on a BALB/c background as previously described (32).

Cockroach Ag challenge Allergic mice were immunized with sterile clinical grade cockroach allergen (Bayer, Elkhart, IN) with no endotoxin contamination as previously described (33–35). Briefly, mice were immunized with 10 ␮g of cockroach allergen in IFA on day 0. On day 14, the mice were given an intranasal challenge of 10 ␮g of cockroach allergen in 10 ␮l of diluent to localize the response to the airway. This initial intranasal challenge with Ag induced little cellular infiltrate into the lungs of the mice upon histological examination. Mice were then rechallenged 6 days later by intratracheal administration of 10 ␮g of cockroach allergen in 50 ␮l of sterile PBS or with PBS alone (vehicle).

Measurement of airway hyperreactivity (AHR) AHR in anesthetized mice was measured as previously described (33–35) with a mouse plethysmograph (Buxco, Troy, NY) using a direct ventilation method specifically designed for low tidal volumes. A single optimal methacholine dose was used, which elicited a minimal response in control BALB/c mice but gave a significant hyperreactive response in allergic mice. There was no difference in background methacholine responses in STAT4⫺/⫺ vs wt BALB/c mice (data not shown).

ELISAs Whole lungs were homogenized in 1 ml of lysis buffer (PBS with 0.01% Triton X-100 nonionic detergent) containing protease inhibitors. Debrisfree supernatants were isolated and the cytokines were measured by ELISA as described (35, 36). Ab pairs from R&D Systems (Minneapolis, MN) were used for ELISAs. The sensitivity of the analyses was ⬃10 pg/ml. No cross-reactivity to any other chemokine or cytokine was detected in individual assays.

Cell transfer Spleens were harvested from sensitized wt or STAT4⫺/⫺ mice and teased to a single-cell suspension. After lysing the RBCs, the adherent cell populations were depleted by incubating in tissue culture dishes for 1 h at 37°C. The isolated lymphocytes (⬃70% CD3⫹ and ⬃26% CD19⫹) were then transferred into fully allergen-sensitized STAT4⫺/⫺ mice (2 ⫻ 107 cells/mouse). The mice were then challenged intratracheally with allergen at 24 h post-cell transfer and then analyzed for AHR at 24 h post-allergen rechallenge.

Anti-IL-12 treatment of mice Sensitized allergic mice were given anti-IL-12 polyclonal Ab intranasally (150 ␮g of purified IgG in 15 ␮l) within 15 min of intratracheal allergen challenge to anesthetized mice. Control mice were given appropriate control purified IgG Ab. Both the anti-IL-12 and control IgG were harvested from specific pathogen-free rabbits in the University of Michigan facility. The serum was harvested, the IgG fraction was collected over protein G columns, and the Ab was tested for endotoxin contamination. The IL-12 IgG Ab was tested for specificity by direct ELISA titers and reacted significantly only to IL-12. Treated mice were checked for AHR at ⬃18 h post-allergen challenge.

STAT4 REGULATES ALLERGIC AIRWAY INFLAMMATION erates quantitative data based on the PCR at early cycles when PCR fidelity is the highest. Just as important, the real-time PCR system has a linear dynamic range of at least five orders of magnitude, reducing the need for serial dilutions. These computer-linked operations, using the specialized software, make the quantitation of PCR products achievable, and because each well has its own internal standard (GAPDH) with a different fluorescent dye marker, the product can be instantly quantitated and compared with other samples.

Statistics Statistical significance was determined by ANOVA. Values of p ⬍ 0.05 were considered significant.

Results Attenuation of AHR in STAT4-deficient mice A number of investigations have indicated that STAT4 is necessary for the development of type I immune responses via IL-12mediated activation pathways (32, 37, 38). Thus, the absence of STAT4 signaling pathways should be detrimental in a Th2-associated response such as allergic pulmonary (asthmatic) inflammation. To examine this aspect, we have used a cockroach allergeninduced model of Th2-type airway inflammation that recapitulates many aspects of human asthma. After sensitization and airway rechallenge, the airway physiology responses of the STAT4⫺/⫺ mice were compared with wt STAT4⫹/⫹ mice on a common BALB/c background. The data in Fig. 1 illustrates that, after 24 and 48 h of allergen response, the STAT4⫺/⫺ mice demonstrated a significant decrease in AHR compared with that of the STAT4⫹/⫹ control allergic mice. This was counterintuitive to what would be expected in this Th2 cytokine-mediated model of airway inflammation. Examination of the histology demonstrated that the most striking aspect of these responses was the diminished peribronchial inflammation that was generated in the STAT4⫺/⫺ vs the wt control response (Fig. 2). Thus, absence of STAT4 during an allergic response had a physiologically beneficial effect in allergic mice after allergen rechallenge. Absence of STAT4 signaling pathway has little effect on Th2-associated responses As mentioned previously, the primary role that has been described for STAT4 signal pathways has been the regulation of Th1 vs Th2 cytokine pathways. Therefore, we examined the levels of cytokines within the lungs that are related to these pathways. The data in Fig. 3 indicates little alteration in the type 1/type 2 cytokine profile in the STAT4⫺/⫺ vs wt control challenge mice. Interestingly, IL-12 levels as well as IL-5 levels were significantly reduced, whereas IL-4 and IFN were not. The alteration of IL-5,

Real-time PCR of lungs from allergic mice for IL-12 Five micrograms of total RNA from specific samples was reversed transcribed into cDNA using a prescribed reverse transcriptase kit from PE Biosystems (Foster City, CA). Primers and probe sets for IL-12 and GADPH were developed by PE Biosystems using a patented technique for optimal and specific amplification. Briefly, during PCR, a fluorogenic probe, consisting of an oligonucleotide with both a reporter and a quencher dye attached, anneals specifically between the forward and reverse primers. When the probe is cleaved by the 5⬘ nuclease activity of the DNA polymerase, the reporter dye is separated from the quencher dye, and a sequence-specific signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored during the PCR. This real-time detection gen2 Abbreviations used in this paper: wt, wild type; AHR, airway hyperreactivity; CCL, CC chemokine ligand.

FIGURE 1. Alteration of AHR in STAT4⫺/⫺ mice. BALB/c mice with and without STAT4 expression were immunized and rechallenged with cockroach allergen (see Materials and Methods). Twenty-four and 48 h post-allergen challenge, the change in airway resistance was measured after an i.v. methacholine challenge (100 ␮g/kg). The data represent the mean ⫾ SE from eight mice per group. ⴱ, p ⬍ 0.05.


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FIGURE 2. Histologic examination of STAT4⫺/⫺ demonstrates a reduction in peribronchial inflammation. Lungs from STAT4⫹/⫹ and STAT4⫺/⫺ allergic mice were examined 48 h after allergen challenge, processed for histology, and stained with H&E. Representative airways depict decreased inflammation in the STAT4⫺/⫺ mice.

although modest, may have contributed to the decrease in peribronchial eosinophil accumulation. In this Th2 model, the IFN-␥ levels are relatively low, and therefore it may not be surprising that they were not altered. In addition to the Th1/Th2 cytokine profiles, we also examined an additional aspect of allergic disease, serum IgE levels. Isotype switching to IgE in B cells is dependent upon IL-4-mediated switching, clearly a Th2-type response. The levels of IgE were not altered in the STAT4⫺/⫺ mice (Fig. 4), which followed the findings in Fig. 3 of equivalent levels of IL-4 in the STAT4⫺/⫺ and wt mice. Thus, the overall cytokine environment and sensitization of the animals appeared to be quite similar, both having a sufficient Th2-related response.

nificantly altered. Therefore, we next examined the chemotactic mediators to determine whether altered chemokine levels could explain the reduced leukocyte recruitment and activation within the lungs of the mice. The data in Fig. 5 clearly indicates that a number of critical CC chemokines were significantly lower in the STAT4⫺/⫺ animals. In particular, CC chemokine ligand (CCL)5, CCL6, CCL11, and CCL17 were all significantly reduced in the STAT4⫺/⫺ animals. Thus, although the Th2 cytokine environment appeared to be largely maintained, the local production of chemokines was significantly altered, suggesting that STAT4 plays a role in the production of these chemokines.

Alteration of chemokines in the STAT4⫺/⫺ mice Although the cytokine profiles and serum IgE levels appeared to be generally intact, the pathophysiology and inflammation were sig-

FIGURE 3. Absence of STAT4 during allergen challenge has only a marginal effect on cytokine levels in the lungs of allergic mice. Whole lung homogenates were examined for levels of cytokines, IL-4, IL-5, IL-12, IL-13, and IFN-␥, using specific ELISAs. The data represents mean ⫾ SE in lungs from six mice per group. ⴱ, p ⬍ 0.05.

FIGURE 4. STAT4⫺/⫺ mice have no alteration in serum IgE levels. Total serum IgE levels were compared in STAT4⫺/⫺ mice and in allergic wt mice. There were negligible levels of IgE found in nonallergic wt and STAT4⫺/⫺ mouse serum. Data represent the mean ⫾ SE from six mice per group.

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STAT4 REGULATES ALLERGIC AIRWAY INFLAMMATION

FIGURE 5. Alteration of chemokines in STAT4⫺/⫺ mice. Chemokine levels in whole lung homogenates were examined in wt and STAT4⫺/⫺ allergic mice using specific ELISAs. The data represent the mean ⫾ SE of chemokine levels from six mice per group. ⴱ, p ⬍ 0.05.

Transfer of STAT4-sufficient lymphocytes does not restore allergic airway responses To further examine the possible mechanism of the response, we addressed the question of whether the STAT4⫺/⫺ animals had an altered lymphocyte response that reduced the overall activation of the lung environment. Although the cytokine phenotype, Th1/Th2, appeared to be intact, it was possible that, in the absence of STAT4, there would be a lymphocyte defect. To initially examine this aspect, we used splenic lymphocyte populations from sensitized wt or STAT4⫺/⫺ mice that contained both B and T lymphocytes (70% CD3⫹ and 26% CD19⫹). In this way, we would be able to examine whether reconstitution of sensitized lymphoid populations would be sufficient to regain the expected allergenmediated response. The transfer of splenic lymphocytes (2 ⫻ 107/ mouse) 24 h before final rechallenge demonstrated no reconstitution of the response. Neither the AHR (Fig. 6) nor the peribronchial eosinophil accumulation (Fig. 7A) and chemokine levels (Fig. 7B) were restored with the transfer of sensitized lymphocytes from STAT4-sufficient animals. Transfer of lymphocytes from sensitized STAT4⫺/⫺ mice into STAT4⫺/⫺ mice also did not reconstitute any of the responses. In additional studies, transfer of splenic lymphocytes from sensitized wt mice into naive wt mice induced a significant AHR response upon allergen challenge com-

FIGURE 6. Transfer of STAT4-sufficient lymphocytes does not reconstitute allergen-induced AHR responses. Splenic lymphocytes, T and B cells, were isolated from STAT4-sufficient and -deficient animals and transferred via i.v. tail vein injection (2 ⫻ 107/mouse) into STAT4-deficient mice. After 24 h, the mice were challenged with cockroach allergen as previously described. The AHR responses were measured at 24 h postchallenge. The data were combined from two repeat experiments. ⴱ, p ⬍ 0.05.

FIGURE 7. Transfer of STAT4-sufficient lymphocytes does not reconstitute eosinophil accumulation or chemokine levels. The association of the inflammatory response with changes in airway physiology was examined by enumeration of the peribronchial eosinophils (A) as well as by determining the level of chemokines within the lungs of the allergic animals (B), as previously described. The data were combined from two repeat experiments. ⴱ, p ⬍ 0.05.

pared with that of control mice (15.3 ⫾ 2.3 cm H2O/ml/s vs 3.2 ⫾ 0.8 cm H2O/ml/s, respectively). Thus, although the transfer of lymphocytes from sensitized mice can effectively induce an allergic response, it was not sufficient to reconstitute the STAT4⫺/⫺ mice. Overall, it appeared that there was a dysregulation of the local activation of chemokines.

FIGURE 8. IL-12 mRNA expression in allergic mice during the allergen response assessed by real-time PCR. At various time points, whole lung mRNA was isolated from allergic mice challenged with cockroach allergen or saline. Reversed-transcribed cDNA samples were subjected to real-time PCR analysis and the experiment, and the fold-increase of cockroach-challenged animals was determined compared with controls. The data represent the mean ⫾ SE from three to five mice per time point.


FIGURE 9. Local neutralization of IL-12 significantly attenuates cockroach allergen (CRA)-induced AHR during this Th2-type response. Purified rabbit anti-mouse IL-12 or control IgG (150 ␮g/mouse) was administered intranasally 15 min post-allergen challenge. The animals were examined for changes in airway physiology at 24 h post-allergen challenge. The data represent the mean ⫾ SE from five mice per group.

Neutralization of IL-12 in allergic mice alters AHR similar to STAT4⫺/⫺ mice A number of previous studies have clearly established IL-12 as a potent inducer of Th1 responses and administration of high doses of rIL-12 (100 –1000⫻ physiologic levels) can alter allergen-induced responses. Furthermore, systemic inhibition of IL-12 appears to significantly increase the Th2-mediated responses (27). However, the role of IL-12 produced locally in an established allergic response has not been thoroughly investigated. Because the STAT4⫺/⫺ mice display an altered allergic response, we hypothesized that the IL-12 produced during the allergic response may contribute to the local pulmonary pathogenesis. Initially, we assessed the expression pattern of IL-12 p40 in allergen-challenged mice using quantitative real-time PCR (Fig. 8). These data demonstrated that expression was increased by ⬎15 fold at 4 h postchallenge compared with that of control-treated animals. To address whether IL-12 produced during the response had any effect on the pulmonary function, we administered purified IgG polyclonal anti-IL-12 or control IgG by intranasal delivery (150 ␮g in 15 ␮l saline/mouse). The data in Fig. 9 demonstrate that local administration of anti-IL-12 during the allergic response significantly attenuated the AHR. The magnitude of the reduced response was not equal to that observed in the STAT4⫺/⫺ mice. When local cytokine and chemokine production was assessed, there were no significant differences (data not shown). Altogether, these results indicate that IL-12 and STAT4 have a role locally in the lung during allergic airway responses.

Discussion The activation and coordination of allergic inflammatory/immune responses depend upon a complex interaction of multiple cell populations and diverse mechanisms. According to dogma, allergic airway responses are dependent upon Th2-type cytokines, including local production of IL-4, IL-5, and IL-13 (39 – 41). In contrast, a number of investigations have demonstrated that addition of superphysiologic levels of exogenous Th1-associated cytokines, such as IL-12 and IFN, down-regulate the allergic response and AHR in murine models of asthmatic disease (23–26). Several studies have suggested that type 1 cytokines actually contribute to the overall allergic environment and play a significant role in the development of the detrimental responses (30, 42). In fact, a recent study demonstrated that IL-12⫺/⫺ animals had reduced airway inflammation, especially eosinophils, suggesting that IL-12 may have a role during the development of allergic airway responses (30). This latter view has been supported in the present studies

3863 where the absence of STAT4, a major type 1 immune signal, significantly alters the development of an allergic airway response via dysregulation of chemokine production, airway inflammation, and attenuation of increased airway physiology. Interestingly, the IL-12 and IL-5 levels were reduced but not those of IFN-␥, IL-4, or IL-13. The reduction observed in IL-12 is consistent with previous observations in the STAT4⫺/⫺ mice (32). The alteration appeared to be centered primarily upon chemokine production. The chemokines that were reduced have been shown to be regulated by STAT6 signal transduction pathways (18, 43); however, in the absence of STAT4 signals, the chemokines appeared to be reduced. Given the fact that lymphocyte transfer did not reconstitute any of the responses, one must assume that the source of the STAT4-related chemokines could be from structural cells or nonlymphoid leukocytes, such as airway epithelial cells and macrophages. Both airway epithelial cells and resident macrophages may be the most significant source of chemokines in the lungs during local responses (44). Additional studies are under way to further investigate the role of these two cell populations in the lung for their role in this STAT4-related response. A previous study indicated that systemic neutralization of IL-12 48 h before and during allergen challenge resulted in a significant increase in the Th2-mediated OVA-induced response (27). Whereas our neutralization protocol targeted local airway IL-12 only at the time of allergen challenge, the previous study altered systemic IL-12 that includes lymph nodes and may have altered lymphocyte activation/differentiation (45– 47). Furthermore, other Th2-mediated models have found that local IL-12 significantly contributes to the pathogenesis of the response possibly by altering chemokines (30, 42, 48). Interestingly, in the present studies, although anti-IL-12 delivered to the airway reduced AHR, the treatment had no significant effect on cytokine or chemokine production. The STAT4⫺/⫺ mice would have a complete signal blockade, whereas anti-IL-12 would have only a partial inhibition because other cytokines, such as type I IFNs, IL-18, and IL-23, can signal via STAT4 (49 –54). We have previously shown that IL-18 can locally induce CCL11, one of the chemokines reduced in the STAT4⫺/⫺ mice (35). The fact that other STAT4-associated cytokines are also present during the response may help explain the anti-IL-12 results. STAT4 has been extensively studied in T lymphocyte activation for the development of Th1-type cells related to IFN-␥ production. Original studies have identified that the STAT4 protein is a major signaling molecule required for the development of Th1 responses both in vitro and in vivo through IL-12R signaling (32, 37, 47, 55). However, other cell populations have not been examined as closely for STAT4 signal transduction responses. Recent studies have indicated that macrophages can also have STAT4-mediated responses associated with activation of anti-pathogen and inflammatory responses (56, 57). Although chemokine production in lymphocytes does not appear to be dependent upon STAT4 activation (18), chemokine production may be one aspect of macrophage activation regulated by STAT4-mediated pathways. Other studies have indicated that IL-12, a specific STAT4 activator, may be an effective component in sepsis and during antifungal responses in vivo via its ability to up-regulate chemokine production (58, 59). Although STAT4 has not been reported to induce epithelial cell activation, airway epithelial cells can produce several of the chemokines that were attenuated in this model. Thus, one must consider that STAT4-mediated mechanisms may be relevant for epithelial cell-derived chemokine responses. Interestingly, IL-12 production has recently been described from airway epithelial cells (60) possibly contributing to the local activation within the airway. In addition, type I IFNs, IL-18, and IL-23 can activate STAT4 and

3864 may have a role within the allergic airway responses for activation of local cell populations (49 –54, 61). Previous studies on STAT protein responses in models of asthmatic inflammation have concentrated primarily on STAT6, which provides a major stimulus for not only development of a complete Th2-type response with significant eosinophilia and AHR but also mucus production within the airway (38, 62– 66). As STAT6 is the primary signal induced by Th2 cytokines, these studies verified the requirement for IL-4/IL-13 receptor signaling during asthmatictype inflammation. Other investigators have also found that constitutive STAT1 activation, normally associated with IFN (-␣, -␤, and -␥), also appears to be related to the severity of the asthmatic response (67). This corresponds well with cell transfer experiments that failed to regulate, but rather enhanced Th2-type allergic responses when Th1-type cells were transferred into challenged animals (29). Thus, the Th2-type cytokines during asthmatic/allergic responses may not be the only cytokines needed for inflammatory responses in the development of severe airway asthma. Support of these concepts can be found during viral infections, which are the most common cause of asthma exacerbations and induce significant levels of type I cytokines (20, 68 –72). In these latter responses, a significant level of chemokines and inflammatory cell accumulation is observed. One could imagine that the increase in chemokines after a viral infection in an asthmatic individual with peripheral eosinophilia and a pre-existing Th2-type lung environment would have additional problems with recruited and activated eosinophils within the airway. Thus, the airway milieu would be greatly impacted by the Th1-type response that induces chemokines with eosinophil chemotactic function. Overall, the data in the present studies outline a role for STAT4 in the development of the local allergic airway response. The mechanism is centered on the production of chemokines and enhanced accumulation of the appropriate effector cells. Future studies will examine the relative role of specific STAT4 protein-mediated signaling in specific cell populations for chemokine production.

References 1. Busse, W. W., S. Banks-Schlegel, and S. E. Wenzel. 2000. Pathophysiology of severe asthma. J. Allergy Clin. Immunol. 106:1033. 2. Lukacs, N. W., R. M. Strieter, S. W. Chensue, and S. L. Kunkel. 1994. Interleukin-4-dependent pulmonary eosinophil infiltration in a murine model of asthma. Am. J. Respir. Cell Mol. Biol. 10:526. 3. Kips, J. C., G. G. Brusselle, G. F. Joos, R. A. Peleman, R. R. Devos, J. H. Tavernier, and R. A. Pauwels. 1995. Importance of interleukin-4 and interleukin-12 in allergen-induced airway changes in mice. Int. Arch. Allergy Immunol. 107:115. 4. Cohn, L., R. J. Homer, A. Marinov, J. Rankin, and K. Bottomly. 1997. Induction of airway mucus production by T helper 2 (Th2) cells: a critical role for interleukin 4 in cell recruitment but not mucus production. J. Exp. Med. 186:1737. 5. Hogan, S. P., A. Koskinen, and P. S. Foster. 1997. Interleukin-5 and eosinophils induce airway damage and bronchial hyperreactivity during allergic airway inflammation in BALB/c mice. Immunol. Cell Biol. 75:284. 6. Grunig, G., M. Warnock, A. E. Wakil, R. Venkayya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, and D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282: 2261. 7. Wang, J., K. Palmer, J. Lotvall, S. Milan, X. F. Lei, K. I. Matthaei, J. Gauldie, M. D. Inman, M. Jordana, and Z. Xing. 1998. Circulating, but not local lung, IL-5 is required for the development of antigen-induced airways eosinophilia. J. Clin. Invest. 102:1132. 8. Schwarze, J., G. Cieslewicz, E. Hamelmann, A. Joetham, L. D. Shultz, M. C. Lamers, and E. W. Gelfand. 1999. IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection. J. Immunol. 162:2997. 9. Henderson, W. R., Jr., E. Y. Chi, and C. R. Maliszewski. 2000. Soluble IL-4 receptor inhibits airway inflammation following allergen challenge in a mouse model of asthma. J. Immunol. 164:1086. 10. Gavett, S. H., D. J. O’Hearn, X. Li, S. K. Huang, F. D. Finkelman, and M. Wills-Karp. 1995. Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice. J. Exp. Med. 182:1527.

STAT4 REGULATES ALLERGIC AIRWAY INFLAMMATION 11. Andrew, D. P., M. S. Chang, J. McNinch, S. T. Wathen, M. Rihanek, J. Tseng, J. P. Spellberg, and C. G. Elias III. 1998. STCP-1 (MDC) CC chemokine acts specifically on chronically activated Th2 lymphocytes and is produced by monocytes on stimulation with Th2 cytokines IL-4 and IL-13. J. Immunol. 161:5027. 12. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D’Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, and F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129. 13. Mochizuki, M., J. Bartels, A. I. Mallet, E. Christophers, and J. M. Schroder. 1998. IL-4 induces eotaxin: a possible mechanism of selective eosinophil recruitment in helminth infection and atopy. J. Immunol. 160:60. 14. Li, L., Y. Xia, A. Nguyen, L. Feng, and D. Lo. 1998. Th2-induced eotaxin expression and eosinophilia coexist with Th1 responses at the effector stage of lung inflammation. J. Immunol. 161:3128. 15. Li, L., Y. Xia, A. Nguyen, Y. H. Lai, L. Feng, T. R. Mosmann, and D. Lo. 1999. Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells. J. Immunol. 162:2477. 16. MacLean, J. A., A. Sauty, A. D. Luster, J. M. Drazen, and G. T. De Sanctis. 1999. Antigen-induced airway hyperresponsiveness, pulmonary eosinophilia, and chemokine expression in B cell-deficient mice. Am. J. Respir. Cell Mol. Biol. 20:379. 17. Teran, L. M., M. Mochizuki, J. Bartels, E. L. Valencia, T. Nakajima, K. Hirai, and J. M. Schroder. 1999. Th1- and Th2-type cytokines regulate the expression and production of eotaxin and RANTES by human lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 20:777. 18. Zhang, S., N. W. Lukacs, V. A. Lawless, S. L. Kunkel, and M. H. Kaplan. 2000. Cutting edge: differential expression of chemokines in Th1 and Th2 cells is dependent on Stat6 but not Stat4. J. Immunol. 165:10. 19. O’Byrne, P. M. 1996. Airway inflammation and asthma. Aliment. Pharmacol. Ther. 10 (Suppl. 2):18. 20. Kay, A. B. 1997. T cells as orchestrators of the asthmatic response. Ciba Found. Symp. 206:56. 21. Kon, O. M., and A. B. Kay. 1999. T cells and chronic asthma. Int. Arch. Allergy Immunol. 118:133. 22. Busse, W. W., and R. F. Lemanske, Jr. 2001. Asthma. N. Engl. J. Med. 344:350. 23. Bruselle, G. G., J. C. Kips, R. A. Peleman, G. F. Joos, R. R. Devos, J. H. Tavernier, and R. A. Pauwels. Role of IFN-␥ in the inhibition of the allergic airway inflammation caused by IL-12. Am. J. Respir. Cell Mol. Biol. 17:767. 24. Hogan, S. P., P. S. Foster, X. Tan, and A. J. Ramsay. Mucosal IL-12 gene delivery inhibits allergic airways disease and restores local antiviral immunity. Eur. J. Immunol. 28:413. 25. Lee, Y. L., C. L. Fu, Y. L. Ye, and B. L. Chiang. Administration of interleukin-12 prevents mite Der p 1 allergen-IgE antibody production and airway eosinophil infiltration in an animal model of airway inflammation. Scand. J. Immunol. 49: 229. 26. Li, X. M., R. K. Chopra, T. Y. Chou, B. H. Schofield, M. Wills-Karp, and S. K. Huang. Mucosal IFN-␥ gene transfer inhibits pulmonary allergic responses in mice. J. Immunol. 157:3216. 27. Keane-Myers, A., M. Wysocka, G. Trinchieri, and M. Wills-Karp. 1998. Resistance to antigen-induced airway hyperresponsiveness requires endogenous production of IL-12. J. Immunol. 161:919. 28. Hansen, G., G. Berry, R. H. DeKruyff, and D. T. Umetsu. 1999. Allergen-specific Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation. J. Clin. Invest. 103:175. 29. Randolph, D. A., R. Stephens, C. J. Carruthers, and D. D. Chaplin. 1999. Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation. J. Clin. Invest. 104:1021. 30. Wang, S., Y. Fan, X. Han, J. Yang, L. Bilenki, and X. Yang. 2001. IL-12dependent vascular cell adhesion molecule-1 expression contributes to airway eosinophilic inflammation in a mouse model of asthma-like reaction. J. Immunol. 166:2741. 31. Zhao, L. L., A. Linden, M. Sjostrand, Z. H. Cui, J. Lotvall, and M. Jordana. 2000. IL-12 regulates bone marrow eosinophilia and airway eotaxin levels induced by airway allergen exposure. Allergy 55:749. 32. Kaplan, M. H., Y. L. Sun, T. Hoey, and M. J. Grusby. 1996. Impaired IL-12 responses and enhanced development of Th2 cells in Stat4-deficient mice. Nature 382:174. 33. Campbell, E. M., S. L. Kunkel, R. M. Strieter, and N. W. Lukacs. 1998. Temporal role of chemokines in a murine model of cockroach allergen-induced airway hyperreactivity and eosinophilia. J. Immunol. 161:7047. 34. Campbell, E. M., I. F. Charo, S. L. Kunkel, R. M. Strieter, L. Boring, J. Gosling, and N. W. Lukacs. 1999. Monocyte chemoattractant protein-1 mediates cockroach allergen-induced bronchial hyperreactivity in normal but not CCR2⫺/⫺ mice: the role of mast cells. J. Immunol. 163:2160. 35. Campbell, E., S. L. Kunkel, R. M. Strieter, and N. W. Lukacs. 2000. Differential roles of IL-18 in allergic airway disease: induction of eotaxin by resident cell populations exacerbates eosinophil accumulation. J. Immunol. 164:1096. 36. Lukacs, N. W., K. K. Tekkanat, A. Berlin, C. M. Hogaboam, A. Miller, H. Evanoff, P. Lincoln, and H. Maassab. 2001. Respiratory syncytial virus predisposes mice to augmented allergic airway responses via IL-13-mediated mechanisms. J. Immunol. 167:1060. 37. Szabo, S. J., N. G. Jacobson, A. S. Dighe, U. Gubler, and K. M. Murphy. 1995. Developmental commitment to the Th2 lineage by extinction of IL-12 signaling. Immunity 2:665. 38. Kaplan, M. H., and M. J. Grusby. 1998. Regulation of T helper cell differentiation by STAT molecules. J. Leukocyte Biol. 64:2. 39. Umetsu, D. T., and R. H. DeKruyff. 1997. Th1 and Th2 CD4⫹ cells in the pathogenesis of allergic diseases. Proc. Soc. Exp. Biol. Med. 215:11.

The Journal of Immunology 40. Wills-Karp, M. 1999. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17:255. 41. Erb, K. J., and G. Le Gros. 1996. The role of Th2 type CD4⫹ T cells and Th2 type CD8⫹ T cells in asthma. Immunol. Cell Biol. 74:206. 42. Pearlman, E., J. H. Lass, D. S. Bardenstein, E. Diaconu, F. E. Hazlett, Jr., J. Albright, A. W. Higgins, and J. W. Kazura. 1997. IL-12 exacerbates helminthmediated corneal pathology by augmenting inflammatory cell recruitment and chemokine expression. J. Immunol. 158:827. 43. Goebeler, M., B. Schnarr, A. Toksoy, M. Kunz, E. B. Brocker, A. Duschl, and R. Gillitzer. 1997. Interleukin-13 selectively induces monocyte chemoattractant protein-1 synthesis and secretion by human endothelial cells: involvement of IL4R␣ and Stat6 phosphorylation. Immunology 91:450. 44. Strieter, R. M., and S. L. Kunkel. 1997. Chemokines in the lung. In Lung: Scientific Foundations, 2nd Ed. R. Crystal, J. West, E. Weibel, and P. Barnes, eds. Raven Press, New York, p. 155. 45. Grusby, M. J. 1997. Stat4- and Stat6-deficient mice as models for manipulating T helper cell responses. Biochem. Soc. Trans. 25:359. 46. Kaplan, M. H., A. L. Wurster, and M. J. Grusby. 1998. A signal transducer and activator of transcription (Stat)4-independent pathway for the development of T helper type 1 cells. J. Exp. Med. 188:1191. 47. Jacobson, N. G., S. J. Szabo, R. M. Weber-Nordt, Z. Zhong, R. D. Schreiber, J. E. Darnell, Jr., and K. M. Murphy. 1995. Interleukin 12 signaling in T helper type 1 (Th1) cells involves tyrosine phosphorylation of signal transducer and activator of transcription (Stat)3 and Stat4. J. Exp. Med. 181:1755. 48. Magone, M. T., S. M. Whitcup, A. Fukushima, C. C. Chan, P. B. Silver, and L. V. Rizzo. 2000. The role of IL-12 in the induction of late-phase cellular infiltration in a murine model of allergic conjunctivitis. J. Allergy Clin. Immunol. 105:299. 49. Freudenberg, M. A., T. Merlin, C. Kalis, Y. Chvatchko, H. Stubig, and C. Galanos. 2002. Cutting edge: a murine, IL-12-independent pathway of IFN-␥ induction by Gram-negative bacteria based on STAT4 activation by type I IFN and IL-18 signaling. J. Immunol. 169:1665. 50. Nguyen, K. B., W. T. Watford, R. Salomon, S. R. Hofmann, G. C. Pien, A. Morinobu, M. Gadina, J. J. O’Shea, and C. A. Biron. 2002. Critical role for STAT4 activation by type 1 interferons in the interferon-␥ response to viral infection. Science 297:2063. 51. Biber, J., S. Jabbour, R. Parihar, J. Dierksheide, Y. Hu, H. Baumann, P. Bouchard, M. Caligiuri, and W. Carson. 2002. Administration of two macrophage-derived interferon-␥-inducing factors (IL-12 and IL-15) induces a lethal systemic inflammatory response in mice that is dependent on natural killer cells but does not require interferon-␥. Cell. Immunol. 216:31. 52. Farrar, J. D., J. D. Smith, T. L. Murphy, S. Leung, G. R. Stark, and K. M. Murphy. 2000. Selective loss of type I interferon-induced STAT4 activation caused by a minisatellite insertion in mouse Stat2. Nat. Immunol. 1:65. 53. Rogge, L., D. D’Ambrosio, M. Biffi, G. Penna, L. J. Minetti, D. H. Presky, L. Adorini, and F. Sinigaglia. 1998. The role of Stat4 in species-specific regulation of Th cell development by type I IFNs. J. Immunol. 161:6567. 54. Cho, S. S., C. M. Bacon, C. Sudarshan, R. C. Rees, D. Finbloom, R. Pine, and J. J. O’Shea. 1996. Activation of STAT4 by IL-12 and IFN-␣: evidence for the involvement of ligand-induced tyrosine and serine phosphorylation. J. Immunol. 157:4781. 55. Thierfelder, W. E., J. M. van Deursen, K. Yamamoto, R. A. Tripp, S. R. Sarawar, R. T. Carson, M. Y. Sangster, D. A. Vignali, P. C. Doherty, G. C. Grosveld, and J. N. Ihle. 1996. Requirement for Stat4 in interleukin-12-mediated responses of natural killer and T cells. Nature 382:171.

3865 56. Fukao, T., D. M. Frucht, G. Yap, M. Gadina, J. J. O’Shea, and S. Koyasu. 2001. Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses. J. Immunol. 166:4446. 57. Frucht, D. M., M. Aringer, J. Galon, C. Danning, M. Brown, S. Fan, M. Centola, C. Y. Wu, N. Yamada, H. El Gabalawy, and J. J. O’Shea. 2000. Stat4 is expressed in activated peripheral blood monocytes, dendritic cells, and macrophages at sites of Th1-mediated inflammation. J. Immunol. 164:4659. 58. Matsukawa, A., M. H. Kaplan, C. M. Hogaboam, N. W. Lukacs, and S. L. Kunkel. 2001. Pivotal role of signal transducer and activator of transcription (Stat)4 and Stat6 in the innate immune response during sepsis. J. Exp. Med. 193:679. 59. Kawakami, K., M. H. Qureshi, T. Zhang, Y. Koguchi, K. Shibuya, S. Naoe, and A. Saito. 1999. Interferon-␥ (IFN-␥)-dependent protection and synthesis of chemoattractants for mononuclear leucocytes caused by IL-12 in the lungs of mice infected with Cryptococcus neoformans. Clin. Exp. Immunol. 117:113. 60. Walter, M. J., N. Kajiwara, P. Karanja, M. Castro, and M. J. Holtzman. 2001. Interleukin 12 p40 production by barrier epithelial cells during airway inflammation. J. Exp. Med. 193:339. 61. Oppmann, B., R. Lesley, B. Blom, J. C. Timans, Y. Xu, B. Hunte, F. Vega, N. Yu, J. Wang, K. Singh, et al. 2000. Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12. Immunity 13:715. 62. Kaplan, M. H., U. Schindler, S. T. Smiley, and M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313. 63. Akimoto, T., F. Numata, M. Tamura, Y. Takata, N. Higashida, T. Takashi, K. Takeda, and S. Akira. 1998. Abrogation of bronchial eosinophilic inflammation and airway hyperreactivity in signal transducers and activators of transcription (STAT)6-deficient mice. J. Exp. Med. 187:1537. 64. Kuperman, D., B. Schofield, M. Wills-Karp, and M. J. Grusby. 1998. Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production. J. Exp. Med. 187:939. 65. Mattes, J., M. Yang, A. Siqueira, K. Clark, J. MacKenzie, A. N. McKenzie, D. C. Webb, K. I. Matthaei, and P. S. Foster. 2001. IL-13 induces airways hyperreactivity independently of the IL-4R ␣-chain in the allergic lung. J. Immunol. 167:1683. 66. Tekkanat, K. K., H. F. Maassab, D. S. Cho, J. J. Lai, A. John, A. Berlin, M. H. Kaplan, and N. W. Lukacs. 2001. IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent. J. Immunol. 166:3542. 67. Sampath, D., M. Castro, D. C. Look, and M. J. Holtzman. 1999. Constitutive activation of an epithelial signal transducer and activator of transcription (STAT) pathway in asthma. J. Clin. Invest. 103:1353. 68. Martinez, F. D. 1997. Definition of pediatric asthma and associated risk factors. Pediatr. Pulmonol. Suppl. 15:9. 69. Nicholson, K. G., J. Kent, and D. C. Ireland. 1993. Respiratory viruses and exacerbations of asthma in adults. BMJ 307:982. 70. Grunberg, K., M. C. Timmers, H. H. Smits, E. P. de Klerk, E. C. Dick, W. J. Spaan, P. S. Hiemstra, and P. J. Sterk. 1997. Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo. Clin. Exp. Allergy 27:36. 71. Pizzichini, M. M., E. Pizzichini, A. Efthimiadis, A. J. Chauhan, S. L. Johnston, P. Hussack, J. Mahony, J. Dolovich, and F. E. Hargreave. 1998. Asthma and natural colds: inflammatory indices in induced sputum: a feasibility study. Am. J. Respir. Crit. Care Med. 158:1178. 72. Hogg, J. C. 1999. Childhood viral infection and the pathogenesis of asthma and chronic obstructive lung disease. Am. J. Respir. Crit. Care Med. 160:S26.