Generation of Th1 and Th2 Chemokines by Human Eosinophils: Evidence for a Critical Role of TNF-α This information is current as Lin Ying Liu, Mary Ellen Bates, Nizar N. Jarjour, William of September 28, 2021. W. Busse, Paul J. Bertics and Elizabeth A. B. Kelly J Immunol 2007; 179:4840-4848; ; doi: 10.4049/jimmunol.179.7.4840 http://www.jimmunol.org/content/179/7/4840 Downloaded from References This article cites 43 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/179/7/4840.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 © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Generation of Th1 and Th2 Chemokines by Human Eosinophils: Evidence for a Critical Role of TNF-␣1 Lin Ying Liu,* Mary Ellen Bates,† Nizar N. Jarjour,* William W. Busse,* Paul J. Bertics,† and Elizabeth A. B. Kelly2* Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their micro- environment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-␣ plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation- regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-␥) and CXCL10 (IFN-␥-inducible protein-10), were expressed after stimulation with TNF-␣ plus IFN-␥. Addition of TNF-␣ appeared to be essential for IFN-␥-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Downloaded from Th2-type chemokines. Inhibition of NF-␬B completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-␬B, STAT6, and STAT1 was induced in eosinophils by TNF-␣, IL-4, and IFN-␥, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-␣ plus IL-4 or TNF-␣ plus IFN-␥. Thus, independently activated signaling cascades appear to lead to activation of NF-␬B, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-␣ is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can http://www.jimmunol.org/ promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils. The Journal of Immu- nology, 2007, 179: 4840–4848. here is growing evidence that eosinophils can contribute and CCL22 are typically induced by IL-4 (or IL-13) via a STAT6- to the adaptive immune response by producing cytokines mediated pathway, whereas CXCL9 and CXCL10 are secreted in T such as IL-4 and IFN-␥ (1–5), and, as we have recently response to IFN-␥ via a STAT1-mediated pathway (11). A critical demonstrated (6), by regulating Th1 and Th2 cytokine generation role for STAT1 and STAT6 in differential trafficking of Th1 and by CD4ϩ T cells. Eosinophils have been primarily associated with Th2 cells to the lung has been demonstrated in mice (12, 13). In by guest on September 28, 2021 Th2-type immune responses as follows: IL-5 is important for eo- STAT1-deficient mice, adoptively transferred Ag-specific Th1 sinophil maturation, differentiation, and survival, whereas IL-13 cells failed to home to the airway in response to local allergen contributes to eosinophil recruitment to target organs, via induc- challenge. This failure to home was most likely due to the defect tion of eotaxins (7). in Ag-induced expression of CXCL9 and CXCL10 in the airway, Many factors contribute to the initiation and regulation of in- because intranasal administration of CXCL10 restored trafficking flammatory cells. The characteristics of an inflammatory response of Th1 cells in STAT1 knockout mice (12). Similarly, STAT6- are largely determined by the type of chemokines in the microen- deficient mice failed to express CCL17 and CCL22 and did not vironment. It is generally accepted that Th2 cells are preferentially support trafficking of Th2 cells (12, 13). These studies provide attracted by CCL17 (thymus and activation-regulated chemokine) further in vivo evidence for differential homing of Th1 and Th2 and CCL22 (macrophage-derived chemokine) (8, 9), whereas Th1 cells and suggest a link between STAT1 and induction of Th1- cells are preferentially recruited by CXCL9 (monokine induced by associated chemokines in comparison with STAT6 and Th2-asso- ␥ ␥ IFN- ) and CXCL10 (IFN- -inducible protein-10) (8–10). Regu- ciated chemokines. Thus, existing evidence suggests that an envi- lation of the generation of these chemokine is also distinct. CCL17 ronment rich in IL-4 and IL-13 invokes STAT6-mediated generation of CCL17/CCL22, which in turn recruit Th2-type cells. Conversely, a Th1 environment is supportive of CXCL9/CXCL10 *Section of Allergy, Pulmonary, and Critical Care Medicine, Department of Medi- and further recruitment of Th1-type cells. cine, and †Department of Biomolecular Chemistry, University of Wisconsin School of Medicine, Madison, WI 53792 We and others have demonstrated the presence of increased lev- Received for publication April 6, 2007. Accepted for publication July 18, 2007. els of Th2-associated chemokines, CCL17 and CCL22, in bron- choalveolar lavage fluid of atopic asthmatic subjects after airway 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 Ag challenge (14–16). Of note, bronchoalveolar lavage fluid con- with 18 U.S.C. Section 1734 solely to indicate this fact. centrations of CXCL9 and CXCL10 were also markedly increased. 1 This work was supported in part by an institutional Specialized Center of Research Because allergen challenge induces a striking airway eosinophilia grant (NIH HL56396) and a University of Wisconsin General Clinical Research Cen- ter grant (NIH M01RR03186). that is associated with Th2-type cytokines, including IL-5 and IL- 13, the presence of high concentrations of Th1-type chemokines 2 Address correspondence and reprint requests to Dr. Elizabeth A. B. Kelly, Section of Allergy, Pulmonary, and Critical Care Medicine, 600 Highland Avenue, CSC K4/ was not expected. Based on these observations, we designed stud- 928, University of Wisconsin School of Medicine, Madison, WI 53792-9988. E-mail ies to determine whether human eosinophils are a source of T address: [email protected] cell-associated chemokines, to investigate the conditions under Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 which these chemokines are released from eosinophils, and to www.jimmunol.org The Journal of Immunology 4841 establish several of the intracellular mechanisms responsible for Flow cytometric analysis for detection of cell surface and eosinophil synthesis of CCL17, CCL22, CXCL9, and CXCL10. intracellular cytokine receptors Purified blood eosinophils were cultured in medium (RPMI 1640 with 5% Materials and Methods FCS and 1% penicillin/streptomycin) alone or stimulated with 10 ng/ml ␥ ␣ Research volunteers IFN- or TNF- alone or in combination. After 0, 3, and 24 h incubation, cells were washed and incubated with PE-conjugated mAbs to CD69 Peripheral blood for eosinophil purification was obtained from allergic vol- (Beckman Coulter), IFN-␥R␣ (BD Biosciences), and TNFRI and TNFRII unteers (skin prick test positive with a history of seasonal or perennial (R&D Systems) for cell surface staining. At the 24-h incubation time point, allergic rhinitis) ranging in age from 18 to 58 years. None of the subjects intracellular staining was also performed with the same Abs. Before stain- had taken inhaled corticosteroids, or other medications that would interfere ing, eosinophils were fixed with 2% paraformaldehyde for 10 min at 37°C with the study results, or had evidence of a respiratory infection within the and permeabilized for 30 min on ice by adding 0.5 ml of ice-cold 90% previous 4 wk. Informed consent was obtained from each subject before methanol. For analysis, 10,000 events were collected using a BD Immu- participation. The study was approved by the University of Wisconsin- nocytometry Systems FACScan II, and data analyses were performed using Madison Center for Health Sciences Human Subjects Committee. the CellQuest software package (BD Biosciences). Detection of phosphoproteins by Western blot analysis Eosinophil purification Freshly purified eosinophils (6 ϫ 106 cells/tube) were rested in medium Eosinophils were purified, as