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The Journal of Immunology, 2004, 173: 7565-7574.
Copyright © 2004 by The American Association of Immunologists

Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-{gamma}

Patricia C. Fulkerson*, Nives Zimmermann{ddagger}, Lynn M. Hassman{ddagger}, Fred D. Finkelman{dagger} and Marc E. Rothenberg1,{ddagger}

Departments of * Molecular Genetics, Biochemistry, and Microbiology, and {dagger} Internal Medicine, Division of Immunology, University of Cincinnati College of Medicine, Cincinnati, OH 45257; and {ddagger} Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of distinct chemokines within the asthmatic lung suggests that specific regulatory mechanisms may mediate various stages of asthmatic disease. Global transcript expression profiling was used to define the spectrum and kinetics of chemokine involvement in an experimental murine model of asthma. Seventeen chemokines were induced in the lungs of allergen-inoculated mice, as compared with saline-treated mice. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. Seven (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen-induced chemokines were induced early after allergen challenge and remained induced throughout the experimental period. Three chemokines (CXCL2, CCL3, and CCL17) were induced only during the early phase of the inflammatory response after the initial allergen challenge, while seven chemokines (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13) were increased only after a second allergen exposure. Unexpectedly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent, and many of the identified chemokines were overexpressed in STAT6-deficient mice, providing an explanation for the enhanced neutrophilic inflammation seen in these mice. Notably, IFN-{gamma} and STAT1 were shown to contribute to the induction of two STAT6-independent chemokines, CXCL9 and CXCL10. Taken together, these results show that only a select panel of chemokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6; instead, many of the allergen-induced chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coordinate regulation by STAT1, STAT6, and IFN-{gamma}.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
One hallmark of allergic airway disease is accumulation of eosinophils, neutrophils, lymphocytes, and macrophages in the lung (1). Within the airway mucosa, CD4+ Th2-type lymphocytes (Th2 cells) and other inflammatory leukocytes release a range of inflammatory mediators that contribute both directly and indirectly to remodeling of the airway wall, mucus hypersecretion, airway obstruction, and airway hyperreactivity (2, 3, 4). Chemokines are a large family of chemotactic cytokines that orchestrate the migration and activation of leukocyte populations under baseline (homeostatic) and inflammatory conditions (5, 6, 7, 8). This large family of cytokines has been divided into four groups, designated CXC, CC, C, and CX3C, depending on the spacing of conserved cysteine residues. The CXC chemokines mainly target neutrophils and lymphocytes, whereas the CC chemokines target a variety of cell types, including macrophages, eosinophils, basophils, and dendritic cells. For example, CCL11 is a highly potent eosinophil-selective chemoattractant that induces eosinophil degranulation (9, 10, 11, 12). Although extensive studies have demonstrated a central role for chemokines in controlling multiple aspects of the asthmatic response, the full spectrum of chemokines involved in allergic airway inflammation, the distinct role of specific chemokines at different stages in the evolution of allergic lung inflammation, and their regulation in vivo are only partially understood.

Murine models of allergic airway inflammation have demonstrated that overexpression of cytokine products of Th2 cells, specifically IL-4 and IL-13, is sufficient for the induction of numerous lung chemokines and the development of pulmonary eosinophilia (13, 14). IL-4 and IL-13 share a receptor chain, IL-4R{alpha}, which mediates phosphorylation of JAK1 and JAK3, and, subsequently, phosphorylation of IL-4R{alpha}. STAT6 monomers are then recruited to the phosphorylated docking tyrosine residues in IL-4R{alpha} and phosphorylated by JAKs, resulting in STAT6 dimerization and translocation to the nucleus. STAT6 is required for many IL-4- and IL-13-mediated responses, including CCL11 expression (15, 16, 17). Although STAT6-deficient mice have attenuation of many features of experimental asthma (e.g., pulmonary eosinophilia), they are either only partially protected or not protected at all from other aspects of the disease that are less specific for allergy, such as lung neutrophilia (18, 19). Surprisingly, the mechanism by which STAT6 deficiency promotes neutrophilia has not been established. There are several proposed general mechanisms by which STAT6 regulates inflammatory cell recruitment (e.g., it may be required for induction of specific chemokines and adhesion molecules). However, it remains to be determined exactly how STAT6 regulates inflammatory cell recruitment in experimental asthma. This is not just an academic question, because STAT6 and its related signaling pathway are targets for drug development for asthma. As such, it is critical to characterize allergen-induced lung inflammation in the absence of STAT6, because this could be the state of patients who someday receive IL-4, IL-13, and/or STAT6 antagonists.

Recently, we have taken an empiric approach to define the broad spectrum of genes associated with induction of experimental asthma in mice (20). Of the 291 asthma signature genes identified, we found overexpression of expected Th2-associated cytokines (IL-4, CCL11, CCL2, and CCL8); however, several Th1- and IFN-{gamma}-associated chemokines were also up-regulated. Focusing on the chemokines CXCL9 and CXCL10, we have demonstrated that they negatively regulate eosinophil lung recruitment and function (21). This finding highlights the complex interaction between numerous chemokines in the setting of allergic airway inflammation. Additionally, the presence of IFN-{gamma}- and IL-4/IL-13-associated chemokines within the asthmatic lung suggests the interplay of intricate regulatory mechanisms, which have not yet been fully elucidated. In this study, we took a global approach to identify chemokines associated with the murine model of experimental asthma. Furthermore, we aimed to dissect the coordinated kinetic expression and transcriptional regulation of these chemokines. We identified 17 allergen-induced chemokines and demonstrated that they are regulated by distinct kinetic patterns. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. In addition, we report a profound negative regulatory role for STAT6 on the expression of several chemokines in the allergic lung; 7 of the 17 chemokines were surprisingly enhanced following allergen challenge in the absence of STAT6. Only expression of 3 chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent. Also, expression of 2 chemokines, CXCL9 and CXCL10, was dependent on the Th1-associated cytokine and transcription factor IFN-{gamma} and STAT1, respectively. Taken together, these results demonstrate that allergic airway inflammation is accompanied by the interplay of a large number of chemokines with distinct kinetic and transcriptional regulation. Notably, despite the central role of IL-4, IL-13, and STAT6 in promoting those aspects of airway inflammation that are specific for allergy and asthma, these molecules are not responsible for global induction of the majority of chemokines in the allergen-challenged lung; rather they, directly or indirectly, reduce expression of several chemokines that promote a less specific inflammatory response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental asthma induction

BALB/c mice were obtained from the National Cancer Institute (Frederick, MD), STAT6-deficient mice (BALB/c background) from The Jackson Laboratory (Bar Harbor, ME), and STAT1-deficient mice (SVEV background) from Taconic Farms (Germantown, NY). All mice were housed under specific pathogen-free conditions. Asthma-like lung disease was induced by two i.p. injections with 100 µg of OVA adsorbed to 1 mg of aluminum hydroxide (alum), followed by two 50 µg of OVA or saline intranasal challenges 3 days apart, as previously described (22). Mice were sacrificed 3 or 18 h following the first or second allergen challenge. Wild-type and STAT6-deficient mice were treated with 1 mg of a neutralizing monoclonal anti-murine IFN-{gamma} (XMG-6) (23) or rat IgG1 control mAb 24 h before the first allergen challenge. Subsequently, the bronchoalveolar lavage fluid (BALF)2 and/or lung tissue were harvested 18–24 h after challenge. The left lobe of the lungs was fixed in Formalin, embedded in paraffin, and stained with H&E using standard histological techniques.

Preparation of RNA and microarray hybridization

RNA was extracted using the TRIzol reagent as per the manufacturer’s instructions. Following TRIzol purification, RNA was repurified with phenol-chloroform extraction and ethanol precipitation. Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at Cincinnati Children’s Hospital Medical Center. Briefly, RNA quality was first assessed using the Agilent bioanalyzer (Agilent, Palo Alto, CA), and only those samples with 28S/18S ratios between 1.3 and 2 were subsequently used. RNA was converted to cDNA with Superscript choice for cDNA synthesis (Invitrogen Life Technologies, Carlsbad, CA), and subsequently converted to biotinylated cRNA with Enzo High Yield RNA Transcript labeling kit (Enzo Biochem, Farmingdale NY). After hybridization to the murine U74Av2 GeneChip (Affymetrix, Santa Clara, CA), the gene chips were automatically washed and stained with streptavidin-PE using a fluidics system. The chips were scanned with a Hewlett-Packard GeneArray Scanner (Palo Alto, CA). This analysis was performed with one mouse per chip (n ≥ 3 for each allergen challenge condition, and n ≥ 2 for each saline challenge condition).

Northern blot analysis

RNA was electrophoresed in an agarose-formaldehyde gel, transferred to Gene Screen transfer membranes (NEN, Boston, MA) in 10x SSC, and cross-linked by UV radiation, as previously reported (24, 25). The cDNA probes, generated by PCR or from commercially available vectors (I.M.A.G.E. Consortium obtained from American Tissue Culture Collection (Manassas, VA) or Incyte Genomics (Palo Alto, CA)), were sequence confirmed, radiolabeled with 32P, and hybridized using standard conditions.

Microarray data analysis

From data image files, gene transcript levels were determined using algorithms in the Microarray Analysis Suite software (Affymetrix). Global scaling was used to compare genes from chip to chip; thus, each chip was normalized to an arbitrary value (1500). Differences between saline- and allergen-treated mice were also determined using GeneSpring software (Silicon Genetics, Redwood City, CA). Data were normalized to the average of the saline-treated mice. Gene lists were created, as previously reported (20). Analysis of chemokine induction was performed from previously reported gene lists (20). Fold increase for each chemokine was calculated by dividing the mean of the average difference values of the OVA-challenged mice (n = 3) by the mean of the average difference values of the saline-challenged mice (n = 2) at each of the time points. Relative fold change over time was calculated by dividing the mean of the average difference values at 18 h after the first and second OVA challenge by the mean of the average difference values at 3 h after the first OVA challenge for each of the chemokines.

BALF collection and analysis

Mice were euthanized by CO2 inhalation. A midline neck incision was made, and the trachea was cannulated. The lungs were lavaged twice with 1.0 ml of PBS containing 1% FCS and 0.5 mM EDTA. The recovered BALF was centrifuged at 400 x g for 5 min at 4°C, and resuspended in 200 µl. Lysis of RBC was conducted using RBC lysis buffer (Sigma-Aldrich, St. Louis, MO), according to the manufacturer’s recommendations. Total cell numbers were counted with a hemacytometer. Cytospin preparations of 1 x 105 cells were stained with the Hema 3 Staining System (Fisher Diagnostics, Middletown, VA), and differential cell counts were determined.

Cytokine quantitation

Cytokine protein concentration in the BALF of allergen- and saline-challenged mice was quantified by using ELISA kits specific for CXCL9 (R&D Systems, Minneapolis, MN); the detection limit was 0.9 pg/ml. IFN-{gamma} concentration was measured using rat mAbs to mouse IFN-{gamma} (5 µg/ml clone R4-6A2, 1 µg/ml biotinylated clone XMG1.2; BD Pharmingen, San Diego, CA); detection limit was 8 pg/ml for IFN-{gamma}.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experimental asthma induction

We were first interested in identifying genes that were differentially expressed in a well-established model of experimental asthma. In our model, mice were i.p. sensitized with the allergen OVA in the presence of the adjuvant alum on two occasions separated by 14 days (Fig. 1A). Subsequently, mice were challenged with intranasal OVA or control saline on two occasions separated by 3 days. Three or 18 h after the first allergen challenge and 18 h after the second allergen challenge, the lungs were examined for inflammatory cellular infiltration or harvested for RNA analysis. Total cell numbers recovered from the airway lumen were significantly increased (from 5.2 ± 0.9 x 104 to 29.9 ± 12.7 x 104) following allergen challenge compared with saline controls, and the presence of neutrophils, lymphocytes, and, most dramatically, eosinophils was increased in the airway of the allergic lung (Fig. 1B). Examination of the lung demonstrated profound peribronchial and perivascular inflammation (Fig. 1C).



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  		FIGURE 1. Murine model of experimental asthma. A, A schematic representation of the allergen challenge protocol is depicted. Mice received two i.p. injections of OVA and alum. Subsequently, mice were challenged with OVA or saline intranasally and analyzed 3 or 18 h after the first or second allergen challenge. B, Cellular composition of BALF 18 h after the second OVA challenge. A representative experiment (n = 2) with four mice/group is shown. *, p < 0.01. C, H&E-stained saline- and allergen-challenged lungs (18 h after second OVA challenge); magnification = x100. Higher power magnification (x400) shows infiltration around lung blood vessel.

 
Identification of the chemokine panel expressed in experimental asthma

Leukocyte recruitment to the lung during allergic inflammation is partially dependent on chemokines (8, 26). We were interested in identifying a broad spectrum of chemokines that contributed to the marked inflammatory response in our experimental allergic airway model. Lung RNA was subjected to microarray analysis using the Affymetrix chip U74Av2 that contains oligonucleotide probe sets representing 12,422 genetic elements (20). Of the allergen-induced genes, it was notable that chemokines represented a large subset; 17 of the 28 chemokines represented on the chip were induced compared with saline-challenged control mice. Seven CXC and 10 CC chemokines were induced 2-fold or more at their peak of expression compared with saline-challenged mice (Fig. 2A).



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  		FIGURE 2. Identification of allergen-induced chemokines. A, Peak fold increase for allergen-induced CC and CXC chemokines with a mean average difference fold increase of >2 in allergen-challenged lung in comparison with saline-challenged control lungs (n = 2 for saline-challenged lungs; n = 3 for OVA-challenged lungs). B, Chemokine expression at three different time points in asthma model. Gene lists of chemokines induced >2-fold in the allergen-challenged lung in comparison with saline-challenged lung were generated at 3 h after the first OVA challenge, and 18 h after the first and second OVA challenge. Overlap of the gene lists is represented in the Venn diagram format.

 
We next analyzed the chromosomal location of the chemokines represented on the chip and the chemokines induced following allergen challenge. Transcripts from chromosomes 5, 6, 8, and 11 were increased with allergen challenge. Most of the members of the CXC chemokine loci on chromosome 5 (CXCL1, CXCL2, CXCL5, CXCL9, CXCL10, and CXCL13) that were present on the chip were among the increased chemokine genes. In addition, 8 of the 11 CC chemokines included in the CC chemokine cluster on chromosome 11 were among the increased genes.

Stratification of allergen-induced chemokines by their kinetic induction profile

We were next interested in testing the hypothesis that related chemokines would be induced with similar kinetic expression patterns. To test this, we examined chemokine expression at three different time points in our asthma model. Several (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen-induced chemokines were induced 2-fold or more early after the first allergen challenge when compared with saline controls and remained induced throughout the experimental period (Fig. 2B). Three chemokines (CXCL2, CCL3, and CCL17) were induced only early on in the allergic inflammatory response, while another large subset of the allergen-induced chemokines was increased only after the second allergen challenge (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13; Fig. 2B). This analysis revealed that chemokines are coregulated (kinetically) despite diverse structure and chromosomal locations.

To gain a better understanding of the temporal regulation of each chemokine, we examined the change in chemokine expression from an early point after the first allergen challenge through a later time point following the second allergen challenge. Consistent with changes in BALF cellular infiltration over time, three distinct temporal patterns of chemokine expression were identified: early (Fig. 3A), late (Fig. 3C), and stable (Fig. 3E, and data not shown). Five chemokines (CXCL1, CXCL2, CXCL5, CCL3, and CCL17) were grouped in the early induction pattern with peak expression occurring 3 h after the first allergen challenge (Fig. 3A). Four of these chemokines, CXCL1, CXCL2, CXCL5, and CCL3, are potent neutrophil chemoattractants in the mouse, consistent with the early influx of neutrophils into the airway following the initial allergen challenge (data not shown). Northern blot analysis confirmed the early induction of CXCL1, CXCL2, and CXCL5 (Fig. 3B). The late pattern of chemokine induction in the allergic pulmonary response was characterized by the induction of seven chemokines (CCL6, CCL8, CCL9, CCL11, CCL12, CXCL9, and CXCL13) with their expression increasing over time and their peak expression occurring 18 h after the second allergen challenge (Fig. 3C). Peak expression of eosinophil active chemokines (CCL8 and CCL11) in the late induction pattern is consistent with the eosinophil-rich inflammatory cell accumulation 18 h after the second allergen challenge (Fig. 1B). Northern blot analysis confirmed the increasing expression of CCL6 and CCL11 over time in the allergic lung (Fig. 3D). The expression of five chemokines (CCL2, CCL7, CCL22, CXCL10, and CXCL12) did not change >2-fold in relation to their expression at the early time point in our asthma model (Fig. 3E). Northern blot analysis confirmed the relatively steady induction of CCL2 and CCL22. We have previously reported a very high correlation between our gene chip data and Northern blot analysis of RNA levels (27). Indeed, 10 of the 12 chemokines probed by Northern blot analysis were completely consistent with the gene chip data (Fig. 3, and data not shown). CCL2, by Northern blot analysis, was modestly different, revealing an early pattern of induction rather than steady induction derived from the gene chip analysis. However, it is important to note that CCL2 was indeed still induced after the early time point (Fig. 3F). Additionally, CXCL12 induction was more comparable with a late induction pattern (data not shown). Although both Northern blot and gene chip analysis are based on two different techniques to assess mRNA levels, it is remarkable how well they correlated. Taken together, these data indicate that a large panel of chemokines is induced in the allergic lung, and that expression of the chemokines is temporally controlled.



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  		FIGURE 3. Temporal regulation of allergen-induced chemokines. Analysis of the mean average difference fold change of each chemokine at later time points (18 h after first and second OVA challenge) in asthmatic lung in relation to the early time point (3 h after first OVA challenge) revealed three distinct temporal profiles of chemokine expression: early induction (A) chemokines, the late induction (C) chemokines, and the stable induction (E) chemokines. Northern blot analysis (B, D, and F) confirmed expression of representative chemokines from each profile.

 
STAT6 positively and negatively regulates allergen-induced chemokines

Having identified a profile of chemokines associated with allergic airway responses, we were next interested in identifying factors involved in regulating their expression. We hypothesized that STAT6 would be required for the induction of many chemokines in the asthmatic lung because this is an essential transcription factor for the development of Th2-associated responses (15, 18, 28). However, we surprisingly found that most (14 of 17) allergen-induced chemokines did not require STAT6 for their expression; in fact, 7 of the 14 STAT6-independent chemokines were expressed more in the absence of STAT6. For example, the mRNA level of an early CXC chemokine (CXCL2) was enhanced in the absence of STAT6 following allergen challenge (Fig. 4A). Furthermore, expression of all of the allergen-induced late and stable pattern CXC chemokines was increased, when compared with wild-type control mice, suggesting that STAT6 acts as a negative regulator of this family of chemokines, especially later in the developing allergic inflammatory response (Fig. 4A). Consistent with the enhanced transcription in the absence of STAT6, analysis of protein expression revealed an increase in CXCL9 in the lungs of OVA-challenged STAT6-deficient mice (1882 ± 421 pg/ml; mean ± SD, n = 4 mice/group) in comparison with wild-type (280 ± 154 pg/ml; mean ± SD, n = 4 mice/group). We also examined the expression of allergen-induced CC chemokines in STAT6-deficient mice. Like most of the CXC chemokines, expression of CCL3 and CCL12 was enhanced in the absence of STAT6 (Fig. 4B). Although transcription of allergen-induced CCL2, CCL6, CCL7, CCL8, and CCL9 was similar in wild-type and STAT6-deficient lungs, only eosinophil-specific chemokine, CCL11, expression and Th2 T cell chemoattractants, CCL22 and CCL17, expression were significantly reduced or eliminated in the absence of STAT6 (Fig. 4B). These results offer a possible explanation for the inflammation seen in allergen-inoculated STAT6-deficient mice. Indeed, while STAT6-deficient mice had reduced lung eosinophilia, there was an increase in BALF neutrophils following allergen challenge (Fig. 4C). Examination of the lung tissue revealed that modest levels of allergen-induced peribronchial and perivascular inflammation were present in STAT6-deficient mice (Fig. 4D). Taken together, these results demonstrate that STAT6 is required for induction of Th2-specific chemokines, CCL17, CCL22, and CCL11. In addition, STAT6 acts as a negative regulator (directly or indirectly) of expression for chemokines associated with a general inflammatory response. As such, certain aspects of the inflammatory process might be exaggerated in the absence of STAT6.



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  		FIGURE 4. STAT6 regulation of allergen-induced chemokines. A, STAT6 negatively regulates most CXC chemokines. B, STAT6 is required for only 3 of 10 allergen-induced CC chemokines. Northern blot analysis of CC and CXC chemokine expression in STAT6-deficient lungs is shown. Ethidium bromide (EtBr)-stained gel is also shown. Each lane represents a separate mouse. C, Cellular composition of the BALF in the STAT6-deficient lung is shown following induction of experimental asthma. A representative experiment is shown (n = 2) with three to four mice in each group. *, p < 0.02. D, H&E-stained STAT6-deficient allergic lungs (18 h after second OVA challenge); magnification = x100.

 
IFN-{gamma} is required for the expression of the allergen-induced chemokines CXCL9 and CXCL10

In the classic model of allergen-induced chemokine expression in the lung, the Th2 cytokines IL-4 and IL-13 have a dominant role in orchestrating inflammatory cell recruitment. Our finding that Th1-associated chemokines were also increased during the induction of experimental asthma and that STAT6 had a prominent inhibitory role implicated a role for IFN-{gamma} in our experimental model. Indeed, several studies have proposed that the asthmatic lung is associated with Th1- and Th2-regulated processes (29). Consistent with this, many chemokines from our panel of allergen-induced chemokines have been shown to be induced by IFN-{gamma} (e.g., CXCL1, CXCL2, CXCL9, CXCL10, CCL2, CCL3, CCL8). We therefore measured IFN-{gamma} protein levels in the BALF of OVA-challenged wild-type and STAT6-deficient mice. Although IFN-{gamma} levels in the airways of wild-type mice remained below the level of detection (8 pg/ml), IFN-{gamma} was significantly increased from undetectable to 51.5 ± 17.5 pg/ml (mean ± SD, n = 4 mice/group) in the BALF of OVA-challenged STAT6-deficient mice compared with saline-challenged mice. To test the hypothesis that IFN-{gamma} was contributing to chemokine induction in the asthmatic lung, we treated OVA-sensitized mice with a neutralizing anti-IFN-{gamma} mAb before the first allergen challenge, and then examined the levels of leukocytes and chemokine expression in the late-phase lung (18 h after the second OVA challenge). The ability of the Ab to neutralize IFN-{gamma} was confirmed by the reduction of IFN-{gamma} protein levels in the BALF 18 h after the second OVA challenge (IFN-{gamma} levels in the BALF of OVA-challenged STAT6-deficient mice were <8 pg/ml after anti-IFN-{gamma} treatment). Next, we examined the effect of anti-IFN-{gamma} treatment on the levels of allergen-induced chemokines in the lung. Allergen-induced expression of the chemokines CXCL9 and CXCL10 was profoundly reduced in both wild-type and STAT6-deficient mice following anti-IFN-{gamma} treatment compared with mice treated with an isotype control Ab (Fig. 5A). Consistent with the reduction in mRNA accumulation, protein levels of CXCL9 were dramatically reduced in the BALF of OVA-challenged mice treated with anti-IFN-{gamma} compared with control IgG treatment in wild-type (35.9 ± 19.7 vs 128.4 ± 43.9 pg/ml) and in STAT6-deficient mice (113.8 ± 30.5 vs 1142.5 ± 822.2 pg/ml). Expression of the CXC chemokines CXCL12 and CXCL13 was unaffected by inhibition of IFN-{gamma} in the allergic lung (Fig. 5A), indicating that the overexpression of these two chemokines in STAT6-deficient mice was not IFN-{gamma} dependent. We also examined the role of IFN-{gamma} in allergen-induced expression of several CC chemokines, including CCL2, CCL8, CCL9, CCL11, and CCL12. Expression of the eosinophil-specific chemokine CCL11 and the other CC chemokines was unchanged by anti-IFN-{gamma} treatment (Fig. 5B).



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  		FIGURE 5. IFN-{gamma} regulates expression of CXCL9 and CXCL10. A and B, Northern blot analysis of CXC (A) and CC (B) chemokine expression in allergic lung after treatment with anti-IFN-{gamma} or control IgG1 (cIgG) is shown. Ethidium bromide-stained gel is also shown. Each lane represents a separate mouse. C, Eosinophils and neutrophils in the BALF from wild-type (WT; {square}) and STAT6-deficient ({blacksquare}) mice after induction of experimental asthma. A representative experiment is shown (n = 2) with three to four mice in each group. *, p < 0.04.

 
Finally, we were interested in determining whether the ability of anti-IFN-{gamma} to inhibit CXCL9 and CXCL10 was associated with a change in the infiltration of leukocytes into the allergic airway. Based on our prior findings that these chemokines negatively regulate eosinophil recruitment into the lung, we hypothesized that anti-IFN-{gamma} would increase the level of BALF eosinophils. Indeed, anti-IFN-{gamma} treatment increased eosinophil levels in both wild-type and STAT6-deficient mice in comparison with control IgG-treated mice (Fig. 5C). An increase in allergen-induced airway neutrophilia was also observed in the STAT6-deficient mice following anti-IFN-{gamma} treatment, while mononuclear cells, monocytes, and lymphocytes remained unchanged (Fig. 5C, and data not shown). These findings demonstrate that IFN-{gamma} functions as a regulator of infiltrating leukocytes into the allergic lung. In particular, IFN-{gamma} negatively regulates eosinophil recruitment into the lung by inducing expression of the inhibitory chemokines CXCL9 and CXCL10.

STAT1 regulation of allergen-induced chemokines

To further characterize the role of IFN-{gamma} in allergen-induced chemokine expression in the lung, we also examined STAT1-deficient mice because IFN-{gamma} signaling is partly dependent on this transcription factor (30). Notably, IFN-{gamma}-induced expression of some chemokines has been shown to be STAT1 independent in vitro, highlighting the importance of analyzing the role of STAT1 in vivo (30). To determine whether STAT1 has a prominent role in the regulation of IFN-{gamma}-dependent chemokine expression in the allergic lung, we induced allergic airway disease in STAT1-deficient mice. BALF eosinophils were significantly increased not only with allergen challenge in the STAT1-deficient mice, but also in comparison with OVA-challenged wild-type control mice (Fig. 6A). We next examined IFN-{gamma}-dependent chemokine expression in the lungs of STAT1-deficient mice following allergen challenge. Allergen-induced expression of CXCL9 and CXCL10 was completely absent in the lungs of STAT1-deficient mice, demonstrating the importance of this transcription factor in the expression of the eosinophil inhibitory chemokines CXCL9 and CXCL10 in the asthmatic lung (Fig. 6B). Also, protein levels of CXCL9 in the BALF of OVA-challenged STAT1-deficient mice were below the level of detection, confirming the importance of this transcription factor in the induction of CXCL9 in response to allergen challenge. Together, these data suggest a role for IFN-{gamma} and STAT1 in the regulation of eosinophil accumulation in allergic inflammation because they are important for expression of the eosinophil inhibitory chemokines CXCL9 and CXCL10.



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  		FIGURE 6. STAT1 is required for allergen-induced expression of CXCL9 and CXCL10. A, BALF cellular composition in wild-type (WT) and STAT1-deficient lungs after induction of experimental asthma. A representative experiment is shown (n = 3) with three to four mice in each group. *, p < 0.03. B, Northern blot analysis of CXCL9 and CXCL10 expression in wild-type (WT) and STAT1-deficient lungs following saline and allergen (OVA) challenge is shown. Ethidium bromide-stained gel is also shown. Each lane represents a separate mouse.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chemokines, produced by both airway epithelial and infiltrating cells, are believed to orchestrate the accumulation and activation of different leukocyte populations in the lung (12, 31). In this study, we aimed to identify the spectrum of chemokines expressed in the murine lung in response to allergen challenge. To further understand the role of each chemokine in the evolution of the allergic response, we focused on the kinetic expression profile for each allergen-induced chemokine. In addition, we examined the contribution of the cytokine IFN-{gamma} and the transcription factors STAT1 and STAT6 in regulating the expression of these chemokines (Table I). Although considerable information is known about chemokines in allergic lung disease, in this study, we have uncovered several new findings with biological significance. First, in addition to identifying a panel of 17 chemokines temporally induced during the development of experimental asthma, we have identified two chemokines that have not been previously associated with allergic airway disease. Although our global analysis of chemokine induction in the allergic lung is extensive, it is not exhaustive because several murine chemokines (CXCL3, CXCL6, CXCL11, CXCL15, CCL16, CCL20, CCL24, and CCL28) were not represented on the chip used in our analysis. Second, we demonstrate that STAT6 up-regulates only a select group of chemokines associated with eosinophilia and Th2 cell recruitment, while most of the chemokines expressed in the allergic lung are STAT6 independent. Third, we demonstrate that the infiltration of leukocytes, in particular eosinophils, is regulated by the coordinated interplay of STAT6, IFN-{gamma}, and STAT1. And fourth, our results offer an explanation for the allergen-induced inflammation that develops in STAT6-deficient mice because we have determined that a large number of allergen-induced chemokines are negatively regulated by STAT6. This later finding identifies a pathway that explains the allergen-induced inflammation seen in the absence of classic Th2 cell signaling.


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  		Table I. Allergen-induced chemokine regulation by STAT6, IFN-{gamma}, and STAT1a

 
To our knowledge, this is the first report of an overwhelmingly negative regulatory role for STAT6 in chemokine expression in the allergic lung. The downstream effects of STAT6 deficiency in the developing allergic response are complex. It is important to note that the absence or presence of STAT6 had both dramatic effects on chemokine expression (e.g., CCL11 and CXCL13) as well as subtle consequences (e.g., CCL17 and CCL22). As such, we cannot rule out the possibility that the inhibitory effects of STAT6 are indirect because chemokine mRNA levels can be modified by a variety of mechanisms, including mRNA stability (32). In addition, Th2 cytokines, IL-4 and IL-13, have been shown to induce the expression of suppressors of cytokine signaling (SOCS)1 in a lung cell line, and SOCS1 has been demonstrated to regulate cytokine-induced chemokine expression by fibroblasts (33, 34, 35, 36). In our experimental asthma model, SOCS1 and SOCS3 expression was induced 18 h after the first and second allergen challenge in wild-type mice, but there was no significant induction in the STAT6-deficient asthmatic lungs (27). Thus, we can speculate that the STAT6-dependent inhibition of chemokine expression in allergic airway disease may be mediated, at least in part, by SOCS proteins. Our data are surprising because prior work has shown that IL-4 and IL-13 can induce a variety of chemokines (in addition to CCL11, CCL17, and CCL22) (37). Thus, while IL-4 and IL-13 overexpression is sufficient for induction of CCL2, CCL6, CCL7, CCL8, and CCL12, it is not required for the induction of these chemokines in experimental allergic airway inflammation (at least in the OVA model) (37).

Although the negative effects of STAT6 on IFN-{gamma} production and signaling are well-established, as are the positive effects of the IFNs and STAT1 on CXCL9 and CXCL10 in other models, our data demonstrating overexpression of several chemokines in STAT6-deficient mice suggest that these pathways actually contribute to a specific type of inflammation in allergen-treated mice that only occurs in the absence of STAT6 (30, 38, 39, 40). For example, enhanced neutrophil-active chemoattractant expression (e.g., CXCL2 and CCL3) may lead to increased cell activation and mediator release, perpetuating the inflammatory response and contributing to tissue damage in lung diseases, such has been shown in chronic obstructive pulmonary disease (41, 42). CXCL12, another chemokine with enhanced expression in the absence of STAT6, has been demonstrated to be constitutively expressed in a wide range of tissues, including the lung (43). Although cytokine-mediated regulation of its receptor, CXCR4, has been examined in eosinophils and lung epithelial cells, regulation of CXCL12 in inflammatory processes remains unclear (44, 45). The CXCL12/CXCR4 axis has been shown to contribute to the inflammatory response in a murine model of asthma (46). The level of CXCL12 protein, as measured by immunohistochemistry, was reported as unchanged, but mRNA levels were unexamined (46). Another consequence of STAT6 deficiency in the allergic lung may include altering the effector activity of infiltrating leukocytes due to the increased chemokine levels. Indeed, we have found the induction of numerous other inflammation-associated genes in the allergen-challenged lungs of STAT6-deficient mice (27) (data not shown). Notably, these genes include macrophage-associated transcripts, such as platelet-activating factor acetylhydrolase, complement component C1q, and CCR2. In addition, 19 allergen-induced genes were unique to the STAT6-deficient asthmatic lungs when compared with wild-type controls, including several lymphocyte-associated transcripts (27). Additional studies are needed to determine the relationship between chemokine expression and the new genetic program induced in STAT6-deficient mice.

In our experimental model, IFN-{gamma} induces the expression of CXCL9 and CXCL10, by a STAT1-dependent mechanism. These results implicate an important role for the Th1-associated cytokine IFN-{gamma} and signaling molecule STAT1 in the regulation of chemokine expression and ultimately the recruitment of inflammatory cells, especially eosinophils, in the developing allergic response in the lung. These collective results suggest the codevelopment of both Th1 and Th2 responses during allergic airway inflammation. Indeed, although asthma is a Th2-associated disease, numerous studies have shown coinvolvement of Th1 and Th2 cells in the pathogenesis and/or effector phase of human asthma and experimental asthma in rodents (13, 47, 48, 49, 50). The interplay between Th2 (STAT6) and Th1 (IFN-{gamma} and STAT1) signaling is complex. STAT6 signaling can suppress IFN-{gamma} (and STAT1 responses) through effects on T cells, while IFN-{gamma} (and STAT1) can inhibit Th2 differentiation and the activity of IL-4 (through SOCS) (51, 52, 53, 54). Defining the role of Th1 responses in the development of experimental asthma may have important clinical implications because numerous therapeutic strategies (including conventional allergen immunotherapy) are being designed to promote Th1 responses in an attempt to inhibit Th2 responses.

In summary, we have identified a panel of allergen-induced chemokines with three distinct kinetic expression profiles (early, late, and stable). Surprisingly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent. Notably, expression of many of the allergen-induced chemokines was enhanced in the absence of STAT6. Taken together, these results show that only a select panel of chemokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6, indicating the importance of these chemokines for the inflammatory allergic response. We also demonstrate a role for IFN-{gamma} and STAT1 in the induction of two chemokines (CXCL9 and CXCL10). Thus, allergic airway inflammation is orchestrated by the interplay of a large number of chemokines with distinct kinetic and transcriptional regulation. These results raise concern that pharmacological inhibition of a single STAT molecule such as STAT6 or its upstream signaling molecules (e.g., IL-13) may convert one type of airway inflammation (e.g., allergic eosinophilic) into another (Ag-driven neutrophilic). It remains to be determined whether this shift would occur in humans treated with IL-4, IL-13, and/or STAT6 antagonists and whether the net effects of such a shift would be beneficial or harmful.


   Footnotes
 
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. Marc E. Rothenberg, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: Marc.Rothenberg{at}cchmc.org Back

2 Abbreviations used in this paper: BALF, bronchoalveolar lavage fluid; SOCS, suppressor of cytokine signaling. Back

Received for publication April 5, 2004. Accepted for publication August 30, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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