Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-γ This information is current as Patricia C. Fulkerson, Nives Zimmermann, Lynn M. of September 28, 2021. Hassman, Fred D. Finkelman and Marc E. Rothenberg J Immunol 2004; 173:7565-7574; ; doi: 10.4049/jimmunol.173.12.7565 http://www.jimmunol.org/content/173/12/7565 Downloaded from References This article cites 54 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/173/12/7565.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Pulmonary Chemokine Expression Is Coordinately Regulated by STAT1, STAT6, and IFN-␥ Patricia C. Fulkerson,* Nives Zimmermann,‡ Lynn M. Hassman,‡ Fred D. Finkelman,† and Marc E. Rothenberg1‡ 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 Downloaded from 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-␥ 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 che- mokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6; instead, many of the allergen-induced http://www.jimmunol.org/ chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coor- dinate regulation by STAT1, STAT6, and IFN-␥. The Journal of Immunology, 2004, 173: 7565–7574. ne hallmark of allergic airway disease is accumulation of kines at different stages in the evolution of allergic lung inflam- eosinophils, neutrophils, lymphocytes, and macrophages mation, and their regulation in vivo are only partially understood. O in the lung (1). Within the airway mucosa, CD4ϩ Th2- Murine models of allergic airway inflammation have demon- type lymphocytes (Th2 cells) and other inflammatory leukocytes strated that overexpression of cytokine products of Th2 cells, spe- release a range of inflammatory mediators that contribute both di- cifically IL-4 and IL-13, is sufficient for the induction of numerous by guest on September 28, 2021 rectly and indirectly to remodeling of the airway wall, mucus hy- lung chemokines and the development of pulmonary eosinophilia (13, persecretion, airway obstruction, and airway hyperreactivity (2– 14). IL-4 and IL-13 share a receptor chain, IL-4R␣, which mediates 4). Chemokines are a large family of chemotactic cytokines that phosphorylation of JAK1 and JAK3, and, subsequently, phosphory- orchestrate the migration and activation of leukocyte populations lation of IL-4R␣. STAT6 monomers are then recruited to the phos- under baseline (homeostatic) and inflammatory conditions (5–8). phorylated docking tyrosine residues in IL-4R␣ and phosphorylated This large family of cytokines has been divided into four groups, by JAKs, resulting in STAT6 dimerization and translocation to the designated CXC, CC, C, and CX3C, depending on the spacing of nucleus. STAT6 is required for many IL-4- and IL-13-mediated re- conserved cysteine residues. The CXC chemokines mainly target sponses, including CCL11 expression (15–17). Although STAT6-de- neutrophils and lymphocytes, whereas the CC chemokines target a ficient mice have attenuation of many features of experimental asthma variety of cell types, including macrophages, eosinophils, ba- (e.g., pulmonary eosinophilia), they are either only partially protected sophils, and dendritic cells. For example, CCL11 is a highly potent or not protected at all from other aspects of the disease that are less eosinophil-selective chemoattractant that induces eosinophil de- specific for allergy, such as lung neutrophilia (18, 19). Surprisingly, granulation (9–12). Although extensive studies have demonstrated the mechanism by which STAT6 deficiency promotes neutrophilia a central role for chemokines in controlling multiple aspects of the has not been established. There are several proposed general mecha- asthmatic response, the full spectrum of chemokines involved in nisms by which STAT6 regulates inflammatory cell recruitment (e.g., allergic airway inflammation, the distinct role of specific chemo- 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 Departments of *Molecular Genetics, Biochemistry, and Microbiology, and †Internal Medicine, Division of Immunology, University of Cincinnati College of Medicine, asthma. This is not just an academic question, because STAT6 and its Cincinnati, OH 45257; and ‡Division of Allergy and Immunology, Cincinnati Chil- related signaling pathway are targets for drug development for dren’s Hospital Medical Center, University of Cincinnati College of Medicine, Cin- cinnati, OH 45229 asthma. As such, it is critical to characterize allergen-induced lung Received for publication April 5, 2004. Accepted for publication August 30, 2004. inflammation in the absence of STAT6, because this could be the state of patients who someday receive IL-4, IL-13, and/or STAT6 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 antagonists. with 18 U.S.C. Section 1734 solely to indicate this fact. Recently, we have taken an empiric approach to define the broad 1 Address correspondence and reprint requests to Dr. Marc E. Rothenberg, Division spectrum of genes associated with induction of experimental of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Univer- sity of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229- asthma in mice (20). Of the 291 asthma signature genes identified, 3039. E-mail address: [email protected] we found overexpression of expected Th2-associated cytokines Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7566 ASTHMA CHEMOKINE PROFILE (IL-4, CCL11, CCL2, and CCL8); however, several Th1- and IFN- murine U74Av2 GeneChip (Affymetrix, Santa Clara, CA), the gene chips ␥-associated chemokines were also up-regulated. Focusing on the were automatically washed and stained with streptavidin-PE using a flu- chemokines CXCL9 and CXCL10, we have demonstrated that idics system. The chips were scanned with a Hewlett-Packard GeneArray Scanner (Palo Alto, CA). This analysis was performed with one mouse per they negatively regulate eosinophil lung recruitment and function chip (n Ն 3 for each allergen challenge condition, and n Ն 2 for each saline (21). This finding highlights the complex interaction between nu- challenge condition). merous chemokines in the setting of allergic airway inflammation. Additionally, the presence of IFN-␥- and IL-4/IL-13-associated Northern blot analysis chemokines within the asthmatic lung suggests the interplay of RNA was electrophoresed in an agarose-formaldehyde gel, transferred to Gene intricate regulatory mechanisms, which have not yet been fully Screen transfer membranes (NEN, Boston, MA) in 10ϫ SSC, and cross-linked elucidated. In this study, we took a global approach to identify by UV radiation, as previously reported (24, 25). The cDNA probes, generated chemokines associated with the murine model of experimental by PCR or from commercially available vectors (I.M.A.G.E. Consortium ob- asthma. Furthermore, we aimed to dissect the coordinated kinetic tained from American Tissue Culture Collection (Manassas, VA) or Incyte Genomics (Palo Alto, CA)), were sequence