Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 This information is current as Promoter of September 27, 2021. Rhian M. Phillips, Victoria E. L. Stubbs, Mandy R. Henson, Timothy J. Williams, James E. Pease and Ian Sabroe J Immunol 2003; 170:6190-6201; ; doi: 10.4049/jimmunol.170.12.6190 Downloaded from http://www.jimmunol.org/content/170/12/6190 References This article cites 43 articles, 24 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/170/12/6190.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *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 © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Variations in Eosinophil Chemokine Responses: An Investigation of CCR1 and CCR3 Function, Expression in Atopy, and Identification of a Functional CCR1 Promoter1 Rhian M. Phillips,2* Victoria E. L. Stubbs,2* Mandy R. Henson,† Timothy J. Williams,* James E. Pease,3,4* and Ian Sabroe4*† We previously showed in a small group of donors that eosinophils from a subgroup of individuals responded equipotently to CC chemokine ligand (CCL)11/eotaxin and CCL3/macrophage-inflammatory protein-1␣ in assays of eosinophil shape change (CCL3/ macrophage-inflammatory protein-1␣-highly responsive (MHR) donors). In this study, we investigated the functional role of CCL3 in eosinophil responses in 73 donors. MHR donors, identified by their eosinophil shape change responses, represented ϳ19% of the donor pool. Eosinophils from these donors showed increased eosinophil CCR1 expression and also underwent CCL3-mediated chemotaxis and up-regulation of CD11b. All MHR donors gave a history of atopy-associated diseases. In a further Downloaded from study, we prospectively recruited 110 subjects, subdivided into nonatopics or atopics, and investigated expression of CCR1 and CCR3 on eosinophils, basophils, monocytes, and neutrophils. Eosinophil CCR1 expression was non-normally distributed in atop- ics, although higher CCR1 expression levels were not predictive of a diagnosis of atopy or atopic disease. We identified the CCR1 promoter and investigated its function. We found a minimal promoter within 177 bp of the transcription start site, and an upstream enhancer region that facilitated expression in leukocyte cell lines. Collectively, these data demonstrate that MHR http://www.jimmunol.org/ individuals form an important subgroup that, when associated with a diagnosis of allergic disease, may require tailored therapy to modulate eosinophil recruitment. Identification of a functional CCR1 promoter will facilitate the study of possible genetic determinants underlying this potentially important clinical phenotype. The Journal of Immunology, 2003, 170: 6190–6201. he allergic airway inflammation of asthma is character- CCL11-induced eosinophil recruitment to the skin (8), and recruit- ized by the recruitment of eosinophils from the blood into ment of eosinophils to the lung is impaired in CCR3-deficient mice, T the airways. Eosinophils are able to contribute to the in- which fail to develop airway hyperresponsiveness following OVA flammatory response by release of mediators that induce broncho- sensitization (9), although this depends on the route of sensitiza- constriction, increased microvascular permeability, and mucus for- tion (10). by guest on September 27, 2021 mation, and through the release of toxic granule contents that cause The closely related receptor, CCR1, which is thought to share a tissue damage in the lungs (1, 2). Eosinophils may further contribute common ancestry with CCR3, is expressed by basophils, mono- to the inflammatory response through their abilities to function as cytes, and memory T cells (11, 12). We have previously shown APC (3). Eosinophil accumulation is principally regulated by the CC that high levels of CCR1 are expressed by eosinophils from a chemokines that signal through the major eosinophil chemokine re- proportion of individuals (ϳ15–20% of donors) (13). Eosinophils ceptor CCR3, including CC chemokine ligand (CCL)511/eotaxin, from these donors are highly responsive to the CCR1 ligand CCL24/eotaxin-2, CCL26/eotaxin-3, and CCL13/monocyte chemo- CCL3/macrophage-inflammatory protein (MIP)-1␣ in functional attractant protein-4 (4–7). Ab blockade of CCR3 in vivo inhibits assays of shape change and calcium mobilization, and the donors were subsequently designated CCL3/MIP-1␣ highly responsive (MHR) (13). CCL3 expression is increased in the human asthmatic *Leukocyte Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London, United Kingdom; and †Division of Genomic Med- lung (14) and may contribute significantly to eosinophil recruit- icine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom ment in MHR individuals, and thus, this variation in donor respon- Received for publication October 21, 2002. Accepted for publication April 3, 2003. siveness to CCL3 is an important consideration when developing The costs of publication of this article were defrayed in part by the payment of page small-molecule antagonists of chemokine receptors for the treat- charges. This article must therefore be hereby marked advertisement in accordance ment of eosinophil-mediated pathologies. with 18 U.S.C. Section 1734 solely to indicate this fact. In this study, we investigated the ability of CCL3 to induce 1 This work was supported by National Asthma Campaign Project Grants 98/065 (to responses in eosinophils from a large panel of 73 donors. In a R.M.P.) and 99/012 (to V.E.L.S.), a Glaxo-SmithKline personal research award (to I.S.), the Medical Research Council (to I.S.), The Wellcome Trust (Program Grant further separate cohort of 110 individuals, we investigated expres- 038775/Z/96/A to J.E.P.), and the National Asthma Campaign (to T.J.W.). sion levels of CCR1 and CCR3 on eosinophils, as well as neutro- 2 R.M.P. and V.E.L.S. contributed equally to this study. phils, basophils, and monocytes. We also examined possible cor- 3 Address correspondence and reprint requests to Dr. James Pease, Leukocyte Biology relations between chemokine receptor expression levels and the Section, Division of Biomedical Sciences, Imperial College London, Sir Alexander diagnosis of atopy, and identified a functional CCR1 promoter. Fleming Building, South Kensington Campus, London, U.K., SW7 2AZ. E-mail ad- dress: [email protected] 4 J.E.P. and I.S. contributed equally to this study as principal investigators. Materials and Methods 5 Abbreviations used in this paper: CCL, CC chemokine ligand; MIP, macrophage- Reagents inflammatory protein; MHR, CCL3/MIP-1␣ highly responsive; MPR, CCL3/MIP-1␣ poorly responsive; PMNL, polymorphonuclear leukocyte; FSC, forward scatter; SSC, General laboratory reagents were from Sigma-Aldrich (Poole, U.K.) unless side scatter; RAST, radioallergosorbent test; RACE, rapid amplification of cDNA otherwise specified. Cell culture reagents were from Invitrogen (Paisley, ends; HEK, human embryonic kidney; ORF, open reading frame. U.K.). CellFix was from BD Immunocytometry Systems (San Jose, CA). Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 6191 Chemokines and cytokines were from PeproTech (London, U.K.). The serum IgE, data were available on 109 subjects. For investigation of the mAbs mouse anti-human CCR1 (2D4; IgG1) and mouse anti-human CCR3 correlation between atopy and chemokine receptor expression, data were (7B11; IgG2a) were generous gifts from Dr. S. Qin (Millennium Pharma- available on 96 subjects. We aimed to recruit ϳ50% symptomatic atopics ceuticals, Cambridge, MA). Abs to HLA-DR (FITC conjugate) were from and 50% normal controls. Subjects were subsequently classified as nona- Sigma-Aldrich. Biotinylated anti-CD123, FITC-conjugated anti-CD14, topic if RASTs were negative and there was no clinical history of atopic and streptavidin-conjugated APC were purchased from eBioscience (San disease (34 subjects). Subjects were classified as atopic if one or more Diego, CA). PE-conjugated anti-CCR1 (clone 53504.111) and anti-CCR3 RASTs were positive (62 subjects). Of the 62 subjects, 13 did not have a (clone 61828.111) Abs were obtained from R&D Systems (Abingdon, history of atopic disease (asymptomatic atopic). We were unable to reliably U.K.). PE-conjugated anti-CD123 was obtained from BD Biosciences classify 14 subjects as atopic or nonatopic, e.g., those with a clinical history (Mountain View, CA), and Abs to CD16 (FITC) and CD11b (PE) were of hay fever in the spring season alone (for which specific RAST analysis from DAKO (Ely, U.K.). Relevant isotype controls were purchased from was not performed). the manufacturer