Promoter Identification of a Functional CCR1 CCR3 Function, Expression in Atopy, and Responses: an Investigation of CCR1 And

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

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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 atopics, 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: 6190Ð6201.

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 (ϳ15Ð20% 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 (4Ð7). 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 address: [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).

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 or Sigma-Aldrich. Leukocyte gated autofluorescence/forward scatter (FSC) assays Analysis of chemokine receptor expression In studies correlating leukocyte chemokine receptor expression with func- Venous blood was sampled according to a St. Mary’s Hospital Local Re- tion, expression was measured in mixed PMNL populations. Cells (5 ϫ search Ethics Committee-approved protocol, and was anti-coagulated with 106/ml) were incubated with unconjugated primary Abs (anti-CCR1 at 10 3.8% trisodium citrate. Polymorphonuclear leukocytes (PMNLs) were sep- ϩ ␮g/ml; anti-CCR3 at 3 ␮g/ml) in staining buffer (PBS without Ca2 and arated from mononuclear cells over a discontinuous plasma-Percoll gradi- ϩ Mg2 containing 10 mM HEPES and 0.25% BSA (pH 7.3Ð7.4)) on ice. ent as previously described (13). Cells were rested at 37¡C for 30 min in ϩ ϩ Cells were washed and resuspended in secondary Ab (PE-conjugated goat- assay buffer (PBS containing Ca2 /Mg2 (pH 7.4) with 10 mM HEPES, 10 anti-mouse F(abЈ) ) for 30 min on ice. Nonspecific binding sites were mM glucose, and 0.1% BSA). The cells were centrifuged and resuspended 2 blocked by incubation with mouse IgG (50 ␮g/ml), followed by incubation in assay buffer as described previously (13), and aliquots of 5 ϫ 105 cells with FITC-conjugated anti-CD16 to distinguish eosinophils from neutro- Downloaded from were incubated with buffer or chemokine for 4 min at 37¡C, transferred to phils. Samples were analyzed vs the appropriate matched isotype controls ice, and fixed with an optimized fixative. Samples were analyzed on a by flow cytometry. To determine chemokine receptor expression in whole FACSCalibur flow cytometer (BD Biosciences), as previously described blood in the prospective study of leukocyte receptor expression patterns, (13), and eosinophils were separated from neutrophils by their autofluo- whole blood was anticoagulated with EDTA (10 mM), and aliquots were rescence. A total of 500 eosinophils per sample were acquired. In some stained with 1) anti-CCR1-PE plus anti-CD16- FITC, 2) anti-CCR3-PE experiments, eosinophils were further purified from granulocyte popula- plus anti-CD16-FITC, 3) anti-CCR1-PE plus anti-HLA-DR-FITC plus anti- tions by negative magnetic selection using a human eosinophil enrichment CD123-biotin, and 4) anti-CCR3-PE plus anti-HLA-DR-FITC plus anti- mixture containing mAbs to CD2, CD14, CD16, CD19, CD56, and gly- http://www.jimmunol.org/ CD123-biotin. Cells were then washed and labeled with streptavidin-APC cophorin A (StemCell Technologies, Vancouver, Canada), according to the (3 and 4). RBCs were lysed using Optilyse B (Beckman Coulter, Fullerton, manufacturer’s instructions. Resulting populations of eosinophils were typ- CA) according to the manufacturer’s instructions. Chemokine receptor ex- ically Ͼ97% pure, with the majority of contaminating cells identified as pression was determined by flow cytometry on eosinophils as CD16-neg- lymphocytes by flow cytometry FSC/side-scatter (SSC) plots. Whole- ative cells in the granulocyte FSC/SSC region, neutrophils as CD16-pos- blood gated autofluorescence/FSC assays were performed using a modifi- itive cells in the granulocyte region, and basophils as CD123-positive, cation of methods described by Bryan et al. (15). Citrate-anticoagulated HLA-DR-negative cells in the PBMC FSC/SSC gates, and monocytes were whole blood was added to aliquots of buffer or chemokine and incubated identified by FSC/SSC gating. Data are expressed as the geometric mean at 37¡C for 4 min in a shaking water bath. Tubes were placed on ice, and fluorescence after subtraction of isotype control values for all cells except 250 ␮l of optimized ice-cold fixative was added. After 1 min, samples were monocytes, for which data are expressed as mean fluorescence. To assess added to 2 ml of cold lysis solution (0.15 M NH Cl and 0.01 M KHCO ), 4 3 chemokine receptor expression levels following cytokine stimulation, pu- by guest on September 27, 2021 vortexed and left on ice until lysis of RBCs had been achieved. Samples rified eosinophils were incubated with IL-3 (10Ð1000 pM), IL-5 (10Ð1000 were centrifuged, and the leukocytes were resuspended in optimized ice- pM), TNF-␣ (100 ng/ml), or IFN-␥ (100 ng/ml) for 24 h at 37¡Cina cold fixative and analyzed by flow cytometry as described previously (15). humidified CO2 incubator, before staining with anti-CCR1-PE. Chemotaxis assays 5Ј Rapid amplification of cDNA ends (RACE) Eosinophils were purified by negative magnetic selection. Cells (1 ϫ 105; in HBSS containing Ca2ϩ and Mg2ϩ, 0.25% BSA, and 30 mM HEPES (pH Total RNA was extracted from human PBMCs, butyric acid-treated (0.5 7.4)) were placed onto the top of a porous filter (5-␮m pore size) of a mM; 5 days) HL-60 clone 15 cells, and THP-1 cells using the RNeasy micro-Boyden chamber (NeuroProbe, Gaithersburg, MD), with chemo- RNA extraction kit (Qiagen, Crawley, U.K.). One microgram of each of the kines in the bottom well of the plate. Plates were incubated at 37¡Cina RNAs were used as templates for 5Ј RACE, using the FirstChoice RLM- humidified CO2 incubator for 1 h. Cells in the lower chamber were counted RACE kit (Ambion, Austin, TX). The sequences of the CCR1 gene-specific by flow cytometry as previously described (5). Migration in response to primers were as follows: primary 5Ј-caggatgtttccaaccaggc-3Ј and nested chemokine was expressed as a ratio of the migration observed in response 5Ј-gttcaccttctggcacggagttgc-3Ј. The 5Ј RACE products were subcloned by to buffer alone (chemotactic index). TA cloning into pCR2.1 (Invitrogen) and sequenced in their entirety (MWG-Biotech, Milton Keynes, U.K.). Analysis of CD11b expression Purified eosinophils were incubated at 5 ϫ 106 cells/ml in assay buffer Cell culture and transfections (PBS containing Ca2ϩ and Mg2ϩ, 0.1% BSA, 10 mM glucose, and 10 mM Human embryonic kidney (HEK) 293 cells were a generous gift from Prof. HEPES (pH 7.4)) containing chemokine/buffer alone for 30 min. Following ϩ ϩ A. Magee (Cell and Molecular Biology Section, Faculty of Medicine, Im- stimulation, cells were washed in PBS without Ca2 /Mg2 , containing perial College London). HL-60 clone 15 cells were a generous gift from 0.25% BSA and 10 mM HEPES (pH 7.4), and stained with PE-conjugated Drs. L. Tiffany and P. Murphy (Laboratory of Host Defenses, National anti-CD11b. CD11b expression by eosinophils was determined by flow Institute of Allergy and Infectious Diseases, National Institutes of Health, cytometry. Data are expressed as the percentage increase in fluorescence Bethesda, MD). RAW 264.7 cells, K562 cells, and THP-1 cells were ob- compared with samples incubated in buffer alone. tained from the American Type Culture Collection (Manassas, VA). HEK Chemokine receptor expression in a defined population 293 cells were transfected in 96-well tissue culture plates using the Poly- fect reagent (Qiagen). HL-60 clone 15 cells were treated with 0.5 mM A total of 110 volunteers were recruited in a study approved by the South butyric acid 5 days before transfection for maximal up-regulation of CCR1 Sheffield Local Research Ethics Committee. Subjects were aged 18Ð60 gene expression (16), and transfected by electroporation as described (17). and either had a history of atopic disease or were healthy, nonatopic con- RAW 264.7 cell transfection was conducted in 96-well tissue culture plates trols. Atopic donors were not taking systemic immunosuppressive therapy, using FuGENE6 transfection reagent (Roche Molecular Biochemicals, but requirement of inhaled and topical corticosteroids was not a contrain- Lewes, U.K.). K562 cells were transfected in 96-well tissue culture plates dication to taking part in this study. A clinical history was taken for atopic using DMRIE-C transfection reagent (Invitrogen). Buffer, IL-3 (10Ð1000 disease (asthma, hay fever, eczema), and blood was taken for measurement pM), TNF-␣ (10Ð100 ng/ml), or IFN-␥ (10Ð100 ng/ml) was added to the of serum IgE and specific IgE Abs to Timothy grass pollen, cat dander, and cells 24 h after the initial 5-h incubation with DMRIE-C/DNA complexes, house dust mite by radioallergosorbent test (RAST). For investigation of and luciferase activity was determined a further 24 h after cytokine addi- the correlation of receptor expression on different leukocyte types and vs tion. Each assay was performed in triplicate. 6192 CCR1-MEDIATED EOSINOPHIL RESPONSES

Reporter gene constructs luciferase activity relative to activity of the Renilla luciferase, which was under the control of the SV40 promoter. A 2-kb region upstream of, and including, a 122-bp region of the untranslated CCR1 exon 1 sequence was obtained from human genomic DNA Dual-Luciferase reporter gene assay (Sigma-Aldrich) by PCR using XhoI-flanked primers: 5Ј-ATATCTCTC ϫ GAGaggtcatccctcttgctgggt and 3Ј-ATATCTCTCGAGggttccaagggacttt HEK 293 and RAW 264.7 cells were lysed in 1 passive lysis buffer gtccg (MWG-Biotech; XhoI sites and linker sequence for optimal enzy- (Promega) 24 h posttransfection; HL-60 clone 15 cells and K562 cells were matic cleavage are indicated in capitals). Internal NheI and SacI sites in the lysed 48 h posttransfection. The Dual-Luciferase reporter gene assay (DLR chromosomal sequence at positions Ϫ1582 and Ϫ177 bp, respectively, assay; Promega) was performed on cell lysates according to the manufac- were used to generate NheI-XhoI-digested (Ϫ1582/ϩ122 bp) and 5Ј-trun- turer’s instructions. Firefly and Renilla luciferase activities were detected cated SacI-XhoI-digested (Ϫ177/ϩ122 bp) fragments of the 2-kb PCR using an Anthos Lucy 1.0 luminescence plate reader with dual injectors product, which were cloned into the appropriate restriction sites of the (Labtech International, East Sussex, U.K.). promoterless pGL3.enhancer vector (Promega, Southampton, U.K.), up- Statistical analysis stream of the firefly luciferase gene (pGL3 Ϫ1582/ϩ122 and pGL3 Ϫ177/ ϩ122, respectively). A 3Ј truncated Ϫ1582/Ϫ177 bp fragment was ob- Distributions of leukocyte CCR1 expression in atopic and nonatopic sub- tained by PCR using the original 5Ј primer and a XhoI-flanked 3Ј primer: ject groups were tested for deviation from normality using a Shapiro- ATATCTCTCGAGctcttgagtcttggccctggg. A 5Ј truncated fragment of Ϫ51/ Wilkes test. Correlations between chemokine receptor expression on dif- ϩ122 bp was obtained by PCR using a NheI-flanked 5Ј primer, ATATCT ferent leukocyte types and between chemokine receptor expression and GCTAGCgaactttgtccctttcttgtc, and the original 3Ј primer. These fragments markers of atopy such as serum IgE were determined using nonparametric were also cloned into pGL3.enhancer (pGL3 Ϫ1582/Ϫ177 and pGL3 Ϫ51/ analysis by calculating the Spearman correlation coefficient and calculating ϩ122). HEK 293, K562, and RAW 264.7 cells were cotransfected with r2 values. Other data originating from two or more groups were analyzed 400 ng of pGL3 vector plus 50 ng of internal control vector (pRL.SV40 by Mann-Whitney U test, one-way ANOVA with Tukey’s posttest, or two-

containing the Renilla luciferase gene; Promega); differentiated HL-60 way ANOVA, as appropriate. Analyses were performed using the Graph- Downloaded from clone 15 cells were cotransfected with 10 ␮g of pGL3 vector plus 5 ␮gof Pad Prism program (GraphPad Software, San Diego, CA). Correlations, pRL.SV40. Transfection efficiency was normalized by calculating firefly analysis of distribution normality, and discriminate analysis of data from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Eosinophil shape change responses to CCL3/MIP-1␣. Responses of MHR donor eosinophils (A and C) and MPR donor eosinophils (B and D) to increasing concentrations of CCL11/eotaxin (f) or CCL3/MIP-1␣ (E). Eosinophil shape change responses in PMNL preparations (A and B) and whole-blood preparations (C and D) are shown. Results are expressed as the mean percent change in FSC relative to the FSC of buffer-stimulated cells (n ϭ 6(A), 30 (B), 12 (C), and 44 (D)) Ϯ SEM. The Journal of Immunology 6193 the prospective study of chemokine receptor expression and atopy were performed by TurnStat (Reading, U.K.).

Results CCL3/MIP-1␣ induces eosinophil shape change, chemotaxis, and CD11b up-regulation in ϳ19% of individuals We investigated chemokine receptor expression levels and functional responses of eosinophils to CCR1 and CCR3 ligands in mixed granulocyte preparations, and compared these results with those from studies examining the effects of CCR1 and CCR3 ligands on eosinophil responses in whole blood and in purified eosinophil preparations. Donors fell into two groups: those in whom eosinophil responses to CCL3 were approximately equipotent to responses to CCL11 and those in whom responses to CCL3 were considerably less than responses to CCL11. Eosinophil responses from a total of 73 donors were studied either in mixed PMNL, whole blood, or both. In total, 14 of the 73 donors (19%) showed the MHR phenotype. Mean data comparing CCL3 and CCL11 Downloaded from responses in MHR donors are shown (Fig. 1, A, in mixed granulocytes, and C, in whole blood). The total number of donors studied is shown in Table I. In the remaining group of donors, eosinophils responded poorly to CCL3 stimulation (Fig. 1, B and D). In order for donors to be classified as either MHR or CCL3/MIP-1␣ poorly responsive (MPR), the phenotype was confirmed on at least http://www.jimmunol.org/ two (often more) separate occasions. Some MHRs were tested up to four times, each result showing absolute concordance, and one MPR donor was tested nine times. Donors were typically tested FIGURE 2. CCL3/MIP-1␣-induced eosinophil migration and CD11b over a period of many months, with some donors tested for up to up-regulation. A and B, Chemotactic response of MHR donor (A) and MPR 5 years. These and subsequent functional data included the retest- donor (B) purified eosinophils to CCL3/MIP-1␣ (E) and CCL11/eotaxin f ␣ ing of three previously characterized donors (two MPRs and one ( ). C, CCL3/MIP-1 (1Ð100 nM) and CCL11/eotaxin (10 nM) induced p Ⅺ MHR) referred to in our previous study (13), included to determine up-regulation of MHR donor ( ) and MPR donor ( ) eosinophil CD11b expression. Chemotactic migration responses to CCL3/MIP-1␣ and the stability of the phenotype and the consistency of results be- by guest on September 27, 2021 CCL11/eotaxin are expressed as the ratio of migrated cells relative to buffer tween independent observers. alone (mean Ϯ SEM; MHR donors, n ϭ 3; MPR donors, n ϭ 4); up- Agonist-induced eosinophil shape change occurs in response to regulation of eosinophil CD11b is expressed as mean Ϯ SEM relative to all tested eosinophil chemoattractants, and we hypothesized that buffer-stimulated cells (MHR donors, n ϭ 5; MPR donors, n ϭ 8). MHR eosinophils from MHR individuals would also undergo chemo- donors showed significantly greater CCL3-induced eosinophil CD11b up- taxis to this ligand. Fig. 2A shows that eosinophils from MHR regulation compared with that of MPR donors, as calculated by two-way subjects, identified in assays of eosinophil shape change to CCL3, ANOVA (p Ͻ 0.005). were also highly responsive to CCL3 in assays of leukocyte chemotaxis, whereas eosinophils from the majority of individuals (MPR individuals) were not (Fig. 2B). Leukocyte recruitment in- MHR donor eosinophils express higher levels of CCR1 than volves not just changes in cytoskeletal arrangement as indicated by MPR donor eosinophils measurement of cell shape, but also typically changes in adhesion To investigate whether increased CCL3 responsiveness was asso- molecule expression. Consistent with this, CCL3 induced up-reg- ciated with increased CCR1 expression, we studied eosinophil che- ulation of the integrin subunit CD11b on the surface of purified mokine receptor expression in donors characterized as having the eosinophils from this group of blood donors (Fig. 2C), but not MHR or MPR phenotype in assays of eosinophil shape change. from the MPR majority. Analysis of representative (Fig. 3, A and B) and mean (C) eosinophil CCR1 expression data in mixed PMNL preparations from 7 MHR and 21 MPR donors is shown, with significantly higher levels of CCR1 found on eosinophils from MHR donors ( p ϭ 0.0001; Table I. Numbers of MHR and MPR donors characterized in determined using the Mann-Whitney U test). There was no differ- eosinophil shape change assays ence in eosinophil CCR3 expression levels between the two groups in the mean data (not shown). Donorsa Number of Analysis of eosinophil CCR3 and CCR1 expression levels in Blood Preparation Analysed Donors MHR MPR atopic vs nonatopic subjects Mixed PMNL only 17 2 15 All MHR individuals studied in the assays of eosinophil functional Whole blood only 37 8 29 Whole blood ϩ mixed PMNL 19 4 15 responses above gave a clinical history of one or more atopy- associated diseases (asthma, eczema, or hay fever). Despite this, Total 73 14 59 many donors who gave a history of atopic disease fell into the a Seventy-three donors in total were classed as MHR or MPR in shape change MPR group. Therefore, we prospectively examined expression of assays on either PMNL, whole-blood preparations, or both types of preparation. the chemokine receptors CCR1 and CCR3 on peripheral blood 6194 CCR1-MEDIATED EOSINOPHIL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Expression of CCR1 by eosinophils. Illustrative eosinophil expression profiles of CCR1 (A) and CCR3 (B) from an MHR donor (solid line) and an MPR donor (shaded histogram). Dotted lines in A and B represent background fluorescence. C, Anti-CCR1 mean fluorescence as measured by FACS in eosinophils from MPR donors (n ϭ 21) and MHR donors (n ϭ 7).

FIGURE 4. Expression of eosinophil chemokine receptors in atopic vs nonatopic subjects. A, CCR3 and CCR1 expression as measured by FACS in 109 atopic and nonatopic donors. B, CCR1 expression in 34 nonatopic eosinophils, neutrophils, basophils, and monocytes to study the and 62 atopic donors. C, Comparison of CCR1 expression (mean fluores- relationship between leukocyte CCR expression and clinical phe- cence of anti-CCR1 staining) with serum IgE levels (U/ml) in 107 donors. notype. A large panel of donors (n ϭ 110) was recruited, and Individual points represent expression levels in individual donors; solid leukocyte chemokine receptor expression was measured by flow lines represent median receptor expression. cytometry by an investigator blinded to the clinical history of the donor. Atopy was determined by the measurement of allergen- specific IgE Abs as described in Materials and Methods. Receptor expressed higher levels of CCR1, known to be closely linked with expression level data available for 109 donors are shown in Fig. MHR status as shown in Figs. 1Ð3, were more likely to be atopic 4A. All donor eosinophils exhibited high binding of anti-CCR3 and than nonatopic. The data in Fig. 4B show that eosinophil CCR1 much lower binding of anti-CCR1 Abs, although absolute numbers expression was normally distributed in nonatopic subjects, but not of CCR1 and CCR3 receptors on leukocyte subsets were not as- normally distributed in atopic subjects ( p ϭ 0.0008 by the Sha- sessed. In accordance with our flow cytometry data, previous stud- piro-Wilkes test). There were more donors displaying high values ies using radioligand binding techniques have shown that eosino- for CCR1 expression in the atopic group compared with the non- phils typically express high levels of CCR3 and low levels of atopic group, and so discriminate analysis was used to ascertain CCR1 (18). We investigated whether donors whose eosinophils whether the level of CCR1 expression could be used to allocate the The Journal of Immunology 6195 subjects to the two groups. Stepwise discriminate analysis did not sion of CCR1 appeared to be regulated in a more independent identify CCR1 expression as having predictive value for a diag- manner with a poor correlation between eosinophil and basophil nosis of atopy. Further analysis of CCR1 expression on eosinophils CCR1 expression (r2 ϭ 0.2; p Ͻ 0.0001; Fig. 5C) and no corre- from symptomatic atopic (49 donors) vs nonatopic individuals lations between CCR1 expression levels on other populations of again showed no significant differences between groups (data not leukocytes analyzed (data not shown). shown). In keeping with this, we observed no correlation between serum IgE and eosinophil CCR1 expression across all donors (Fig. Identification of the CCR1 chemokine receptor promoter region 4C). No differences in the mean levels of CCR3 expression in To determine the basic mechanisms that regulate the expression of atopic vs nonatopic donors were observed (data not shown). Sim- CCR1, and also to identify contributing factors leading to in- ilarly, there was no correlation between IgE and CCR3 expression creased CCR1 expression by a subgroup of donor eosinophils, we levels in all donors (data not shown). identified and characterized the CCR1 promoter. The CCR1 gene transcription start site was identified by 5Ј RACE analysis, using Characterization of CCR1 and CCR3 expression in multiple CCR1 gene-specific primers and RNA templates from PBMCs, leukocyte subsets THP-1 cells, and butyric acid-treated HL-60 clone 15 cells (which In addition to the analysis of chemokine receptor expression by have the potential to become eosinophilic in phenotype (19)). We eosinophils from atopic or nonatopic donors, the expression of also attempted RACE analysis of CCR1 mRNA from primary eo- CCR1 and CCR3 on neutrophils, monocytes, and basophils was sinophils, but these cells contained only very low levels of CCR1 also determined. Eosinophils and neutrophils expressed relatively mRNA, making RACE impractical. Nested PCR of 5Ј RACE low levels of CCR1 in comparison with monocyte and basophil products from all cells typically identified two similar-sized bands Downloaded from CCR1 expression levels (Fig. 5A). Eosinophils and basophils ex- of ϳ200 and 300 bp (Fig. 6A), and 5Ð12 clones from each band pressed higher levels of CCR3 than did neutrophils and monocytes from each cell type sequenced. Alignment of the longest 5Ј RACE (Fig. 5B). Comparison of CCR3 receptor levels between leukocyte products identified in the three cell types with the genomic se- populations showed a correlation between eosinophil and basophil quence of human chromosome 3 (accession no. NT_034534) is CCR3 expression levels (r2 ϭ 0.37; p Ͻ 0.0001; Fig. 5D) and also shown in Fig. 6B. Our proposed genomic organization of CCR1 is a good correlation between monocyte and neutrophil CCR3 ex- shown in Fig. 7A.5Ј RACE analysis showed that exon 1 is longer http://www.jimmunol.org/ pression levels (r2 ϭ 0.73; p Ͻ 0.0001; data not shown). Expres- (127 bp) than previously described (12); however, comparison of by guest on September 27, 2021

FIGURE 5. Leukocyte subset expression of CCR1 and CCR3 in 109 donors. Expression of CCR1 (A) and CCR3 (B) measured by FACS in eosinophils, neutrophils, monocytes, and basophils. Data in A and B are shown as box-and-whiskers plots, with box extending from the 25th to the 75th percentile, a horizontal line at the median (50th percentile), and whiskers showing the range of the data. Correlations between basophil and eosinophil CCR1 (C), and basophil and eosinophil CCR3 expression (D) in individual donors are shown. 6196 CCR1-MEDIATED EOSINOPHIL RESPONSES Downloaded from FIGURE 6. 5Ј RACE products. A, DNA products (bands of ϳ200 and 300 bp) obtained by nested PCR using a THP-1 cDNA template. B, Alignment of 5Ј RACE products obtained from differentiated HL-60 clone 15 cells (H), THP-1 cells (T), and PBMC (M) cDNA templates. RACE sequences are aligned with human chromosome 3 genomic sequence (accession no. NT_034534), and exons 1 and 2 are indicated. The start of the CCR1 coding region sequence is indicated in lower case; the noncoding sequence is in uppercase. http://www.jimmunol.org/ RACE products with genomic DNA did not identify exons up- ϩ122), indicates that the major functional regions of the CCR1 stream of exon 1. Therefore, we analyzed a region including the promoter lie in a region Ϫ177/Ϫ51 bp relative to the putative first 122-bp region of the first exon of CCR1 and 1582 bp upstream transcriptional start site (ϩ1), because the Ϫ51/ϩ122 bp sequence of this for promoter activity (Fig. 7B). Sequencing analysis of this had weaker promoter activity in HL-60 clone 15, RAW 264.7, and region of genomic DNA identified a triple base polymorphic de- HEK 293 cells (Fig. 9 and data not shown). Binding sites for the letion (GAA), Ϫ49/Ϫ46 bp relative to the proposed transcription transcription factors GATA-1, GATA-2, and Sp1 were found in start. Using a luciferase reporter gene assay, we identified a CCR1 the CCR1 promoter region using the TRANSFAC search engine promoter region Ϫ177/ϩ122 bp relative to the transcription start (http://www.cbrc.jp/research/db/TFSEARCH.html) (20). Compar- site, active in human eosinophilic HL-60 clone 15 cells, mouse ison of the CCR1 promoter region with other chemokine receptor by guest on September 27, 2021 monocytic RAW 264.7 cells, human HEK 293 cells (Fig. 8), and promoters using the multiple alignment general interface search human myeloid K562 cells (see Fig. 10). In differentiated HL-60 tool (http://www.hgmp.mrc.ac.uk) showed 25Ð31% identity with clone 15 cells, minimal promoter activity was enhanced by the the CCR2 (accession no. AF068265), CCR3 (accession no. presence of a 1405-bp upstream region (pGL3 Ϫ1582/ϩ122 bp), AF237380), and CCR5 (accession no. AF032132) promoters (21Ð which alone (pGL3 Ϫ1582/Ϫ177 bp) had no promoter activity 23). Comparisons of the CCR1 promoter region with other eosi- (Figs. 8A and 9). This 1405-bp sequence also caused an enhance- nophil-selective promoters showed 26Ð27% identity with the hu- ment of promoter activity in myeloid K562 cells (Fig. 10) and a man eosinophil cationic protein promoter (accession no. D86343), nonsignificant enhancement of promoter activity in murine mono- human major basic protein promoter (accession no. AF304354), cytic RAW 264.7 cells, but no enhancement in the nonleukocyte and the IL-5R␣ chain promoter (accession no. U18373) (24Ð26). HEK 293 cells (Fig. 8, B and C). Treatment of K562 cells with Preliminary sequencing analyses of the GAA polymorphism (Ϫ49/ IL-3, TNF-␣, or IFN-␥ for 24 h had no effect on the relative ac- Ϫ46 bp) in five MPR and three MHR donors did not identify an tivity of the CCR1 promoter (Fig. 10), although TNF-␣ treatment association between this polymorphism and MHR status. Single resulted in an increase in the activity of all promoters, including nucleotide polymorphisms in the open reading frame (ORF) and 3Ј the SV40 promoter of the internal control and positive control region of CCR1 (described in the database at http://www.ncbi. vectors (data not shown). When compared with the positive control nlm.nih.gov/entrez/query.fcgi?db ϭ snp) are indicated in Fig. 7A. vector in which the luciferase gene was constitutively active under No differences in the sequences of the Ϫ177/ϩ122 bp promoter re- the control of the viral SV40 promoter, CCR1 promoter (Ϫ177/ gion, the first intron, and exon 2 of the CCR1 gene were observed ϩ122 bp) activity was greatest in differentiated HL-60 clone 15 between two MHR and two MPR donors, suggesting that polymor- cells (35.0 Ϯ 5.8% of SV40 promoter activity; n ϭ 12 Ϯ SEM). phisms within these regions do not confer the MHR phenotype. These differentiated HL-60 clone 15 cells were responsive to CCL3 in assays of calcium mobilization (data not shown). Pro- moter activity (Ϫ177/ϩ122 bp) in K562 cells was ϳ26% of the Discussion SV40 promoter activity; in RAW 264.7 and HEK 293 cells, pro- CCR1-mediated signaling may contribute to the pathology of moter activity was ϳ18% of SV40 promoter activity (data not asthma (14, 27Ð29) in part by mediating the recruitment of lym- shown). Although CCR1 promoter activity appeared to be greatest phocytes and monocytes to the lungs (30, 31). Contradictory re- in HL-60 clone 15 cells, we cannot exclude the possibility that the ports existed over the ability of CCL3 to induce eosinophil recruit- apparently greater activity of the promoter in these human eosin- ment (18, 27, 32), which were partially reconciled by our studies ophilic cells could be due to differences in SV40 promoter activity demonstrating in a small donor population that eosinophils from between cell lines. Analysis of a 5Ј truncated region of the pro- some individuals showed increased responsiveness to CCL3 in as- moter region, consisting primarily of exon 1 sequence (pGL3 Ϫ51/ says of chemoattractant-induced eosinophil shape change (13). In The Journal of Immunology 6197 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Proposed genomic organization of the CCR1 gene and promoter region. A, Genomic organization of the CCR1 gene with exons 1 and 2, separated by a 3958-bp intronic sequence represented by open boxes. The shaded box in exon 2 represents the 1067-bp ORF of CCR1. Single nucleotide polymorphisms (SNPs) indicated are listed in the human chromosome 3 genomic contig (accession no. NT_034534). B, Genomic sequence analyzed for CCR1 promoter activity (Ϫ1582/ϩ122 bp relative to transcription start site). The site of a 3-base polymorphic deletion Ϫ46/Ϫ49 bp relative to the .present in the genomic sequence analyzed for promoter activity, is indicated in A and B ,(ءءء) transcription start this study, the initial aim was to gain a better estimate of the fre- in CCL3 efficacy. We previously showed that calcium deprivation quency and importance of this phenotype. up-regulates CCL3 responses in eosinophils (13) (and for this rea- Using assays of eosinophil shape change in mixed granulocyte son, characterization of MHR/MPR phenotype, particularly in preparations, we found that CCL3 and CCL11 induced equipotent whole blood, needs to be done rapidly after venesection), and it eosinophil responses in ϳ19% of a large panel of donors. As pre- may be that the difference in efficacy, and possibly some of the viously described (13), we designated these donors as MHR, in differences in potency, between whole blood and mixed PMNL contrast to the majority of individuals who showed poor or absent reflects some up-regulation of CCL3 responses during preparation. eosinophil responses to this ligand (MPR). We found similar re- However, in MHR donors, higher eosinophil responses to CCL3 sults when responses were assayed in whole blood. We have pre- were observed in freshly venesected whole blood, mixed PMNL, viously shown that the apparent CCL11 potency in assays of eo- and also highly purified eosinophil preparations, indicating that, in sinophil shape change was lower in whole blood than PMNL MHR donors, eosinophil CCR1 is likely to be functional in vivo. preparations because of CCL11 binding to the red cell promiscu- In keeping with our original smaller study (13), donors showing ous chemokine receptor Duffy (15). In the data of this study, ap- the MHR phenotype exhibited eosinophil CCR1 expression that parent CCL3 potency was similarly reduced in whole blood, pos- was greater than in MPR individuals, with three donors showing sibly through interactions of chemokine with whole-blood particularly high CCR1 expression. constituents, although we have not specifically investigated this. In donors phenotyped as MHR in assays of eosinophil shape However, the characteristic phenotype of equivalent CCL3 and change, we found that CCL3 induced chemotaxis of eosinophils CCL11 potency in MHR donors was maintained in whole-blood and up-regulation of eosinophil expression of the integrin subunit assays of eosinophil responsiveness, although with some reduction CD11b. Together, these data indicate important potential roles for 6198 CCR1-MEDIATED EOSINOPHIL RESPONSES

FIGURE 8. CCR1 promoter activity in differentiated HL-60 clone 15 cells (A), monocytic RAW cells (B), and HEK 293 cells (C). Figures show promoter activity of the Ϫ177/ ϩ122 bp sequence (ࡗ) and Ϫ1582/ ϩ122 bp sequence (F). Relative luciferase activity in cells transfected with promoterless pGL3.enhancer vector is also shown (Œ). Promoter activity is expressed as firefly luciferase activity relative to the internal control (pRL.SV40 Renilla lucif- Downloaded from erase activity) (arbitrary relative luminescence units (RLU)). Individual symbols represent separate experiments; solid lines represent mean RLU values. Significance values of promoter activity with respect to promoterless pGL3.enhancer vector http://www.jimmunol.org/ are indicated, as calculated by one- way ANOVA and Tukey’s multiple comparisons posttest. by guest on September 27, 2021

CCR1 signaling in MHR donors, contributing to the multistep pro- and atopic) and excluded those taking systemic medication. Al- cess of eosinophil recruitment from the microcirculation to the though not formally quantiﬁed, because no donors altered pheno- lungs. In accordance with our ﬁndings, Struyf et al. (33) recently type with time, these data supported an underlying genetic predis- reported that eosinophils from ϳ20% of their donors showed che- position to higher CCR1 expression and the MHR phenotype. motactic responses to CCR1 ligands. However, all donors exhibiting the MHR phenotype gave a history The MHR phenotype is stable over time. All donors were tested of a potentially atopy-associated disease (asthma, eczema, or hay more than once, and some were tested many times, over intervals fever) in a routine prephlebotomy questionnaire. Because all MHR ranging from a few months to several years, in carefully standard- donors showed increased levels of eosinophil CCR1 expression, ized assays. The donor pool comprised healthy volunteers (normal we sought to investigate whether eosinophil CCR1 expression, as

FIGURE 9. Further characterization of the CCR1 promoter. Comparison of promoter activity of fragments: Ϫ177/ ϩ122 bp (ࡗ), Ϫ1582/ϩ122 bp (F), Ϫ51/ϩ122 bp (Ⅺ), and Ϫ1582/Ϫ177 bp (”) in differentiated HL-60 clone 15 cells. Promoter activity is expressed as fold increase over background relative luminescence unit (RLU) values (promoterless pGL3.enhancer). Individual symbols represent four separate experiments; solid lines represent mean fold increase in promoter activity. The Journal of Immunology 6199

a marker of the MHR phenotype, correlated with a diagnosis of atopy in a formal prospective study. We hypothesized that, in MHR donors, CCR1 expression might also be elevated on other leukocyte types. We also investigated the possibility that individual variations in CCR1 expression on eosinophils might correlate with interindividual variations in expression of the major eosinophil chemokine receptor CCR3. We divided donors into atopics and nonatopics, and subdivided atopics into asymptomatic (defined by a positive RAST but no clinical history of disease) or symptomatic (defined by a clinical history of asthma, eczema, or hay fever with perennial or seasonal disease, showing correlation with appropriate RAST results). In this large prospective study, CCR1 distribution was nongaussian in atopic (including both asymptomatic and symptomatic subjects), but not nonatopic donors, although we did not identify any subjects with very high CCR1 expression, as had been observed in the first part of this work. Mean fluorescence levels for CCR1 in whole blood were lower in this clinical study than in the nonprospective

study, probably because we used a different anti-CCR1 mAb that Downloaded from was available commercially in a directly conjugated format. To determine whether those subjects whose eosinophils expressed higher levels of CCR1 were more likely to be atopic, we used statistical discriminate analysis. CCR1 failed inclusion in this mathematical model, showing that, in this sample, higher eosino-

phil CCR1 expression was not associated with an increased like- http://www.jimmunol.org/ lihood of a diagnosis of atopy. Similarly, higher eosinophil CCR1 expression failed to be significantly associated with symptomatic atopic disease, and eosinophil CCR1 expression throughout the whole sample population did not correlate with serum IgE levels, which are frequently elevated in atopy. CCR1 expression showed only very poor correlation between leukocyte subtypes, suggesting that the MHR phenotype was un- likely to be associated with altered CCL3 responsiveness of peripheral blood leukocytes other than eosinophils. We were also by guest on September 27, 2021 unable to reliably detect significant plasma levels of CCL3 or CCL11 in our donor population, nor did we observe a correlation between plasma IL-5 and levels of eosinophil chemokine receptor expression (data not shown). The lack of association of phenotype with IL-5 levels suggested that CCR1 expression was not a reflec- tion of disease activity. To address this further, we cultured purified eosinophils for 24 h with IL-5, IL-3, TNF-␣, or IFN-␥,as described in Materials and Methods, and at the doses tested, we did not observe any increase in eosinophil CCR1 expression in the presence of these cytokines (data not shown). These data again favor an underlying genetic explanation for the phenotype. Thus, although high levels of eosinophil CCR1 expression were not associated with an increased likelihood of a diagnosis of atopy in this study, ϳ15Ð20% of atopic individuals are likely to be MHR individuals, and this remains an important therapeutic consideration (13, 34). The nonnormal distribution of CCR1 expression on eosinophils from atopic donors raises the possibility that larger stud- FIGURE 10. Effects of cytokines on CCR1 promoter function. Pro- ies of MHR donors, perhaps with the inclusion of analysis of sub- moter activity of fragments: Ϫ177/ϩ122 bp (o) and Ϫ1582/ϩ122 bp (f) groups such as severe asthmatics, may yet identify associations of in K562 cells treated with 10Ð1000 pM IL-3 (A), 10Ð100 ng/ml TNF-␣ this phenotype with clinical disease. (B), and 10Ð100 ng/ml IFN-␥ (C) for 24 h before assay. Activities of In comparison to CCR1, we sought evidence of interindividual promoter fragments in K562 cells incubated in the absence of cytokine variations in expression levels of the major eosinophil chemokine (buffer) are shown. Relative luciferase activity in cells transfected with receptor CCR3 on leukocyte subsets. Again, variations in eosino- Ⅺ promoterless pGL3.enhancer vector is also shown ( ). Promoter activity is phil CCR3 expression between individuals did not correlate with expressed as firefly luciferase activity relative to the internal control atopy or serum IgE levels. In keeping with published data, we (pRL.SV40 Renilla luciferase activity) (arbitrary relative luminescence units (RLU)). Mean RLU values Ϯ SEM of four independent experiments detected high levels of CCR3 on basophils (7, 35, 36) and much performed in triplicate are shown. Significance values of promoter activity lower levels on neutrophils (37) and monocytes. MHR status was p Ͻ 0.001) and between promoter not associated with a general increase in chemoattractant receptor ,ء) with respect to pGL3.enhancer vector -p Ͻ 0.001) are indicated, as calculated by one-way ANOVA expression, and there was very little correlation between eosino ,#ء) fragments and Tukey’s multiple comparisons posttest. phil CCR1 and CCR3 expression. 6200 CCR1-MEDIATED EOSINOPHIL RESPONSES

We observed significant correlations between levels of CCR3 polymorphic variants that could define responsiveness to CCL3/ expression on eosinophils, basophils, neutrophils, and monocytes. MIP-1␣; however, only a small number of subjects have been in- These data suggest that, potentially, a single underlying common vestigated thus far. Polymorphisms affecting other pathways such factor plays a role in controlling CCR3 expression by leukocytes, as G protein expression could also underlie the MHR phenotype. which is either enhanced or suppressed to regulate CCR3 expres- Collectively, these data show that CCR1 expression is donor- sion on eosinophils and basophils vs neutrophils and monocytes, and cell-specific, giving rise to additional pathways that may reg- respectively. A recent study by Menzies-Gow et al. (37) showed ulate leukocyte recruitment in some individuals. Our data also neutrophil recruitment in response to intradermal CCR3 ligands; raise the possibility that expression of other chemokine receptors however, neutrophils from these donors did not respond to CCL11 may show similar phenotypes. These are important points to con- in assays of neutrophil shape change (37), and we did not detect sider when developing therapies aimed at modulating leukocyte neutrophil shape change in response to CCL11/eotaxin in 56 recruitment in inflammatory disease. donors. However, our data demonstrating a spectrum of CCR3 expression raise the possibility that there may be yet-unidentified Acknowledgments donors in whom, akin to the MHR phenomenon regulating eosin- We are grateful to Mr. Jonathan Aldis and Dr. William Egner (Immunol- ophil CCL3 responsiveness, a threshold of CCR3 expression may ogy Department, Northern General Hospital, Sheffield, U.K.) for assistance be reached permitting neutrophil or monocyte responses to CCR3 with RAST and IgE analyses. We are also grateful to Miss Elizabeth Jones ligands. (Division of Genomic Medicine, University of Sheffield) for assistance The CCR1, CCR2, CCR3, and CCR5 genes are clustered on a with eosinophil culture experiments. We thank the following for helpful

350-kb region of chromosome 3 (3p21.3), which suggests that discussions: Dr. Colin Bingle (Division of Genomic Medicine, University Downloaded from these chemokine receptors are derived from a common primitive of Sheffield), Dr. Endre Kiss-Toth (Clinical Sciences Division, University of Sheffield), and Drs. Peter J. Jose, James N. Francis, and Adele Hartnell originator whose expression may thus be regulated by similar path- (Imperial College, London). We are also grateful to Jackie Turner of Turn- ways (38). To investigate mechanisms that might regulate CCR1 Stat for statistical analyses of clinical study data. We also thank the many expression, we identified the CCR1 promoter, with a view also to volunteer donors who made this study possible. identify any polymorphic variants that could account for variabil- ity in CCR1 expression levels between donors. Aligning the References http://www.jimmunol.org/ mRNA for CCR1 (accession no. NM_001295) (12) with chromo- 1. Gleich, G. J., C. R. Adolphson, and K. M. Leiferman. 1993. The biology of the some 3, we identified an exon containing 5Ј-untranslated sequence, eosinophilic leukocyte. Annu. Rev. Med. 44:85. ϳ 2. Kroegel, C., J. C. Virchow, Jr., W. Luttmann, C. Walker, and J. A. Warner. 1994. located 4 kb upstream of exon 2, which contained the full ORF. Pulmonary immune cells in health and disease: the eosinophil leucocyte (part I). Database searching also identified a sequence that was listed as the Eur. Respir. J. 7:519. CCR1 promoter (accession no. AF051305), which highlighted the 3. MacKenzie, J. R., J. Mattes, L. A. Dent, and P. S. Foster. 2001. Eosinophils promote allergic disease of the lung by regulating CD4ϩ Th2 lymphocyte func- same upstream exon and had extensive homology to chromosome tion. J. Immunol. 167:3146. 3 in the region of this putative promoter site. However, the up- 4. Humbles, A. A., D. M. Conroy, S. Marleau, S. M. Rankin, R. T. Palframan, stream homology of AF051305 to chromosome 3 became erratic, A. E. Proudfoot, T. N. Wells, D. Li, P. K. Jeffery, D. A. Griffiths-Johnson, et al. 1997. Kinetics of eotaxin generation and its relationship to eosinophil accumu- by guest on September 27, 2021 and the whole sequence had been misattributed as belonging to lation in allergic airways disease: analysis in a guinea pig model in vivo. J. Exp. chromosome 6. Using 5Ј RACE, we found that CCR1 mRNA from Med. 186:601. 5. Ponath, P. D., S. Qin, D. J. Ringler, I. Clark-Lewis, J. Wang, N. Kassam, monocytes, monocytic cell lines (THP-1), and a cell line with eo- H. Smith, X. Shi, J. A. Gonzalo, W. Newman, et al. 1996. Cloning of the human sinophilic characteristics (HL-60 clone 15) contained this single eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional upstream nontranslated exon. According to our longest RACE properties suggest a mechanism for the selective recruitment of eosinophils. J. Clin. 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