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* Leukocyte Biology Section, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London, United Kingdom; and
Division of Genomic Medicine, University of Sheffield, Royal Hallamshire Hospital, Sheffield, United Kingdom
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Abstract |
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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 
19% 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 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 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. |
Introduction |
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The closely related receptor, CCR1, which is thought to share a common ancestry with CCR3, is expressed by basophils, monocytes, and memory T cells (11, 12). We have previously shown that high levels of CCR1 are expressed by eosinophils from a proportion of individuals (15–20% of donors) (13). Eosinophils from these donors are highly responsive to the CCR1 ligand CCL3/macrophage-inflammatory protein (MIP)-1 in functional 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 lung (14) and may contribute significantly to eosinophil recruitment in MHR individuals, and thus, this variation in donor responsiveness to CCL3 is an important consideration when developing small-molecule antagonists of chemokine receptors for the treatment of eosinophil-mediated pathologies.
In this study, we investigated the ability of CCL3 to induce responses in eosinophils from a large panel of 73 donors. In a further separate cohort of 110 individuals, we investigated expression levels of CCR1 and CCR3 on eosinophils, as well as neutrophils, basophils, and monocytes. We also examined possible correlations between chemokine receptor expression levels and the diagnosis of atopy, and identified a functional CCR1 promoter.
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Materials and Methods |
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General laboratory reagents were from Sigma-Aldrich (Poole, U.K.) unless otherwise specified. Cell culture reagents were from Invitrogen (Paisley, U.K.). CellFix was from BD Immunocytometry Systems (San Jose, CA). Chemokines and cytokines were from PeproTech (London, U.K.). The mAbs mouse anti-human CCR1 (2D4; IgG1) and mouse anti-human CCR3 (7B11; IgG2a) were generous gifts from Dr. S. Qin (Millennium Pharmaceuticals, Cambridge, MA). Abs to HLA-DR (FITC conjugate) were from Sigma-Aldrich. Biotinylated anti-CD123, FITC-conjugated anti-CD14, and streptavidin-conjugated APC were purchased from eBioscience (San Diego, CA). PE-conjugated anti-CCR1 (clone 53504.111) and anti-CCR3 (clone 61828.111) Abs were obtained from R&D Systems (Abingdon, U.K.). PE-conjugated anti-CD123 was obtained from BD Biosciences (Mountain View, CA), and Abs to CD16 (FITC) and CD11b (PE) were from DAKO (Ely, U.K.). Relevant isotype controls were purchased from the manufacturer or Sigma-Aldrich.
Leukocyte gated autofluorescence/forward scatter (FSC) assays
Venous blood was sampled according to a St. Mary’s Hospital Local Research Ethics Committee-approved protocol, and was anti-coagulated with 3.8% trisodium citrate. Polymorphonuclear leukocytes (PMNLs) were separated from mononuclear cells over a discontinuous plasma-Percoll gradient as previously described (13). Cells were rested at 37°C for 30 min in assay buffer (PBS containing Ca2+/Mg2+ (pH 7.4) with 10 mM HEPES, 10 mM glucose, and 0.1% BSA). The cells were centrifuged and resuspended in assay buffer as described previously (13), and aliquots of 5 x 105 cells were incubated with buffer or chemokine for 4 min at 37°C, transferred to ice, and fixed with an optimized fixative. Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences), as previously described (13), and eosinophils were separated from neutrophils by their autofluorescence. A total of 500 eosinophils per sample were acquired. In some experiments, eosinophils were further purified from granulocyte populations by negative magnetic selection using a human eosinophil enrichment mixture containing mAbs to CD2, CD14, CD16, CD19, CD56, and glycophorin A (StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions. Resulting populations of eosinophils were typically >97% pure, with the majority of contaminating cells identified as lymphocytes by flow cytometry FSC/side-scatter (SSC) plots. Whole-blood gated autofluorescence/FSC assays were performed using a modification of methods described by Bryan et al. (15). Citrate-anticoagulated whole blood was added to aliquots of buffer or chemokine and incubated at 37°C for 4 min in a shaking water bath. Tubes were placed on ice, and 250 µl of optimized ice-cold fixative was added. After 1 min, samples were added to 2 ml of cold lysis solution (0.15 M NH4Cl and 0.01 M KHCO3), vortexed and left on ice until lysis of RBCs had been achieved. Samples were centrifuged, and the leukocytes were resuspended in optimized ice-cold fixative and analyzed by flow cytometry as described previously (15).
Chemotaxis assays
Eosinophils were purified by negative magnetic selection. Cells (1 x 105; in HBSS containing Ca2+ and Mg2+, 0.25% BSA, and 30 mM HEPES (pH 7.4)) were placed onto the top of a porous filter (5-µm pore size) of a micro-Boyden chamber (NeuroProbe, Gaithersburg, MD), with chemokines in the bottom well of the plate. Plates were incubated at 37°C in a humidified CO2 incubator for 1 h. Cells in the lower chamber were counted by flow cytometry as previously described (5). Migration in response to chemokine was expressed as a ratio of the migration observed in response to buffer alone (chemotactic index).
Analysis of CD11b expression
Purified eosinophils were incubated at 5 x 106 cells/ml in assay buffer (PBS containing Ca2+ and Mg2+, 0.1% BSA, 10 mM glucose, and 10 mM HEPES (pH 7.4)) containing chemokine/buffer alone for 30 min. Following stimulation, cells were washed in PBS without Ca2+/Mg2+, containing 0.25% BSA and 10 mM HEPES (pH 7.4), and stained with PE-conjugated anti-CD11b. CD11b expression by eosinophils was determined by flow cytometry. Data are expressed as the percentage increase in fluorescence compared with samples incubated in buffer alone.
Chemokine receptor expression in a defined population
A total of 110 volunteers were recruited in a study approved by the South Sheffield Local Research Ethics Committee. Subjects were aged 18–60 and either had a history of atopic disease or were healthy, nonatopic controls. Atopic donors were not taking systemic immunosuppressive therapy, but requirement of inhaled and topical corticosteroids was not a contraindication to taking part in this study. A clinical history was taken for atopic disease (asthma, hay fever, eczema), and blood was taken for measurement of serum IgE and specific IgE Abs to Timothy grass pollen, cat dander, and house dust mite by radioallergosorbent test (RAST). For investigation of the correlation of receptor expression on different leukocyte types and vs serum IgE, data were available on 109 subjects. For investigation of the correlation between atopy and chemokine receptor expression, data were available on 96 subjects. We aimed to recruit 50% symptomatic atopics and 50% normal controls. Subjects were subsequently classified as nonatopic if RASTs were negative and there was no clinical history of atopic disease (34 subjects). Subjects were classified as atopic if one or more RASTs were positive (62 subjects). Of the 62 subjects, 13 did not have a history of atopic disease (asymptomatic atopic). We were unable to reliably classify 14 subjects as atopic or nonatopic, e.g., those with a clinical history of hay fever in the spring season alone (for which specific RAST analysis was not performed).
Analysis of chemokine receptor expression
In studies correlating leukocyte chemokine receptor expression with function, expression was measured in mixed PMNL populations. Cells (5 x 106/ml) were incubated with unconjugated primary Abs (anti-CCR1 at 10 µg/ml; anti-CCR3 at 3 µg/ml) in staining buffer (PBS without Ca2+ and Mg2+ containing 10 mM HEPES and 0.25% BSA (pH 7.3–7.4)) on ice. Cells were washed and resuspended in secondary Ab (PE-conjugated goat-anti-mouse F(ab')2) for 30 min on ice. Nonspecific binding sites were blocked by incubation with mouse IgG (50 µg/ml), followed by incubation with FITC-conjugated anti-CD16 to distinguish eosinophils from neutrophils. Samples were analyzed vs the appropriate matched isotype controls by flow cytometry. To determine chemokine receptor expression in whole blood in the prospective study of leukocyte receptor expression patterns, whole blood was anticoagulated with EDTA (10 mM), and aliquots were stained with 1) anti-CCR1-PE plus anti-CD16-FITC, 2) anti-CCR3-PE plus anti-CD16-FITC, 3) anti-CCR1-PE plus anti-HLA-DR-FITC plus anti-CD123-biotin, and 4) anti-CCR3-PE plus anti-HLA-DR-FITC plus anti-CD123-biotin. Cells were then washed and labeled with streptavidin-APC (3 and 4). RBCs were lysed using Optilyse B (Beckman Coulter, Fullerton, CA) according to the manufacturer’s instructions. Chemokine receptor expression was determined by flow cytometry on eosinophils as CD16-negative cells in the granulocyte FSC/SSC region, neutrophils as CD16-positive cells in the granulocyte region, and basophils as CD123-positive, HLA-DR-negative cells in the PBMC FSC/SSC gates, and monocytes were identified by FSC/SSC gating. Data are expressed as the geometric mean fluorescence after subtraction of isotype control values for all cells except monocytes, for which data are expressed as mean fluorescence. To assess chemokine receptor expression levels following cytokine stimulation, purified eosinophils were incubated with IL-3 (10–1000 pM), IL-5 (10–1000 pM), TNF- (100 ng/ml), or IFN-
(100 ng/ml) for 24 h at 37°C in a humidified CO2 incubator, before staining with anti-CCR1-PE.
5' Rapid amplification of cDNA ends (RACE)
Total RNA was extracted from human PBMCs, butyric acid-treated (0.5 mM; 5 days) HL-60 clone 15 cells, and THP-1 cells using the RNeasy RNA extraction kit (Qiagen, Crawley, U.K.). One microgram of each of the RNAs were used as templates for 5' RACE, using the FirstChoice RLM-RACE kit (Ambion, Austin, TX). The sequences of the CCR1 gene-specific primers were as follows: primary 5'-caggatgtttccaaccaggc-3' and nested 5'-gttcaccttctggcacggagttgc-3'. The 5' RACE products were subcloned by TA cloning into pCR2.1 (Invitrogen) and sequenced in their entirety (MWG-Biotech, Milton Keynes, U.K.).
Cell culture and transfections
Human embryonic kidney (HEK) 293 cells were a generous gift from Prof. A. Magee (Cell and Molecular Biology Section, Faculty of Medicine, Imperial College London). HL-60 clone 15 cells were a generous gift from Drs. L. Tiffany and P. Murphy (Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). RAW 264.7 cells, K562 cells, and THP-1 cells were obtained from the American Type Culture Collection (Manassas, VA). HEK 293 cells were transfected in 96-well tissue culture plates using the Polyfect reagent (Qiagen). HL-60 clone 15 cells were treated with 0.5 mM butyric acid 5 days before transfection for maximal up-regulation of CCR1 gene expression (16), and transfected by electroporation as described (17). RAW 264.7 cell transfection was conducted in 96-well tissue culture plates using FuGENE6 transfection reagent (Roche Molecular Biochemicals, Lewes, U.K.). K562 cells were transfected in 96-well tissue culture plates using DMRIE-C transfection reagent (Invitrogen). Buffer, IL-3 (10–1000 pM), TNF- (10–100 ng/ml), or IFN- (10–100 ng/ml) was added to the cells 24 h after the initial 5-h incubation with DMRIE-C/DNA complexes, and luciferase activity was determined a further 24 h after cytokine addition. Each assay was performed in triplicate.
Reporter gene constructs
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 (Sigma-Aldrich) by PCR using XhoI-flanked primers: 5'-ATATCTCTCGAGaggtcatccctcttgctgggt and 3'-ATATCTCTCGAGggttccaagggactttgtccg (MWG-Biotech; XhoI sites and linker sequence for optimal enzymatic cleavage are indicated in capitals). Internal NheI and SacI sites in the chromosomal sequence at positions -1582 and -177 bp, respectively, were used to generate NheI-XhoI-digested (-1582/+122 bp) and 5'-truncated SacI-XhoI-digested (-177/+122 bp) fragments of the 2-kb PCR product, which were cloned into the appropriate restriction sites of the promoterless pGL3.enhancer vector (Promega, Southampton, U.K.), upstream of the firefly luciferase gene (pGL3 -1582/+122 and pGL3 -177/+122, respectively). A 3' truncated -1582/-177 bp fragment was obtained by PCR using the original 5' primer and a XhoI-flanked 3' primer: ATATCTCTCGAGctcttgagtcttggccctggg. A 5' truncated fragment of -51/+122 bp was obtained by PCR using a NheI-flanked 5' primer, ATATCTGCTAGCgaactttgtccctttcttgtc, and the original 3' primer. These fragments were also cloned into pGL3.enhancer (pGL3 -1582/-177 and pGL3 -51/+122). HEK 293, K562, and RAW 264.7 cells were cotransfected with 400 ng of pGL3 vector plus 50 ng of internal control vector (pRL.SV40 containing the Renilla luciferase gene; Promega); differentiated HL-60 clone 15 cells were cotransfected with 10 µg of pGL3 vector plus 5 µg of pRL.SV40. Transfection efficiency was normalized by calculating firefly luciferase activity relative to activity of the Renilla luciferase, which was under the control of the SV40 promoter.
Dual-Luciferase reporter gene assay
HEK 293 and RAW 264.7 cells were lysed in 1x passive lysis buffer (Promega) 24 h posttransfection; HL-60 clone 15 cells and K562 cells were lysed 48 h posttransfection. The Dual-Luciferase reporter gene assay (DLR assay; Promega) was performed on cell lysates according to the manufacturer’s instructions. Firefly and Renilla luciferase activities were detected using an Anthos Lucy 1.0 luminescence plate reader with dual injectors (Labtech International, East Sussex, U.K.).
Statistical analysis
Distributions of leukocyte CCR1 expression in atopic and nonatopic subject groups were tested for deviation from normality using a Shapiro-Wilkes test. Correlations between chemokine receptor expression on different leukocyte types and between chemokine receptor expression and markers of atopy such as serum IgE were determined using nonparametric analysis by calculating the Spearman correlation coefficient and calculating r2 values. Other data originating from two or more groups were analyzed by Mann-Whitney U test, one-way ANOVA with Tukey’s posttest, or two-way ANOVA, as appropriate. Analyses were performed using the GraphPad Prism program (GraphPad Software, San Diego, CA). Correlations, analysis of distribution normality, and discriminate analysis of data from the prospective study of chemokine receptor expression and atopy were performed by TurnStat (Reading, U.K.).
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Results |
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induces eosinophil shape change, chemotaxis, and CD11b up-regulation in 19% 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 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 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 over a period of many months, with some donors tested for up to 5 years. These and subsequent functional data included the retesting of three previously characterized donors (two MPRs and one MHR) referred to in our previous study (13), included to determine the stability of the phenotype and the consistency of results between independent observers.
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To investigate whether increased CCL3 responsiveness was associated with increased CCR1 expression, we studied eosinophil chemokine receptor expression in donors characterized as having the MHR or MPR phenotype in assays of eosinophil shape change. 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 = 0.0001; determined using the Mann-Whitney U test). There was no difference in eosinophil CCR3 expression levels between the two groups in the mean data (not shown).
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All MHR individuals studied in the assays of eosinophil functional responses above gave a clinical history of one or more atopy-associated diseases (asthma, eczema, or hay fever). Despite this, many donors who gave a history of atopic disease fell into the MPR group. Therefore, we prospectively examined expression of the chemokine receptors CCR1 and CCR3 on peripheral blood eosinophils, neutrophils, basophils, and monocytes to study the relationship between leukocyte CCR expression and clinical phenotype. A large panel of donors (n = 110) was recruited, and leukocyte chemokine receptor expression was measured by flow 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 expression level data available for 109 donors are shown in Fig. 4A. All donor eosinophils exhibited high binding of anti-CCR3 and much lower binding of anti-CCR1 Abs, although absolute numbers of CCR1 and CCR3 receptors on leukocyte subsets were not assessed. In accordance with our flow cytometry data, previous studies using radioligand binding techniques have shown that eosinophils typically express high levels of CCR3 and low levels of CCR1 (18). We investigated whether donors whose eosinophils expressed higher levels of CCR1, known to be closely linked with MHR status as shown in Figs. 1–3, were more likely to be atopic than nonatopic. The data in Fig. 4B show that eosinophil CCR1 expression was normally distributed in nonatopic subjects, but not normally distributed in atopic subjects (p = 0.0008 by the Shapiro-Wilkes test). There were more donors displaying high values for CCR1 expression in the atopic group compared with the nonatopic group, and so discriminate analysis was used to ascertain whether the level of CCR1 expression could be used to allocate the subjects to the two groups. Stepwise discriminate analysis did not identify CCR1 expression as having predictive value for a diagnosis of atopy. Further analysis of CCR1 expression on eosinophils from symptomatic atopic (49 donors) vs nonatopic individuals again showed no significant differences between groups (data not shown). In keeping with this, we observed no correlation between serum IgE and eosinophil CCR1 expression across all donors (Fig. 4C). No differences in the mean levels of CCR3 expression in atopic vs nonatopic donors were observed (data not shown). Similarly, there was no correlation between IgE and CCR3 expression levels in all donors (data not shown).
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In addition to the analysis of chemokine receptor expression by eosinophils from atopic or nonatopic donors, the expression of CCR1 and CCR3 on neutrophils, monocytes, and basophils was also determined. Eosinophils and neutrophils expressed relatively low levels of CCR1 in comparison with monocyte and basophil CCR1 expression levels (Fig. 5A). Eosinophils and basophils expressed higher levels of CCR3 than did neutrophils and monocytes (Fig. 5B). Comparison of CCR3 receptor levels between leukocyte populations showed a correlation between eosinophil and basophil CCR3 expression levels (r2 = 0.37; p < 0.0001; Fig. 5D) and also a good correlation between monocyte and neutrophil CCR3 expression levels (r2 = 0.73; p < 0.0001; data not shown). Expression of CCR1 appeared to be regulated in a more independent manner with a poor correlation between eosinophil and basophil CCR1 expression (r2 = 0.2; p < 0.0001; Fig. 5C) and no correlations between CCR1 expression levels on other populations of leukocytes analyzed (data not shown).
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To determine the basic mechanisms that regulate the expression of CCR1, and also to identify contributing factors leading to increased CCR1 expression by a subgroup of donor eosinophils, we identified and characterized the CCR1 promoter. The CCR1 gene transcription start site was identified by 5' RACE analysis, using CCR1 gene-specific primers and RNA templates from PBMCs, THP-1 cells, and butyric acid-treated HL-60 clone 15 cells (which have the potential to become eosinophilic in phenotype (19)). We also attempted RACE analysis of CCR1 mRNA from primary eosinophils, but these cells contained only very low levels of CCR1 mRNA, making RACE impractical. Nested PCR of 5' RACE products from all cells typically identified two similar-sized bands of 200 and 300 bp (Fig. 6A), and 5–12 clones from each band from each cell type sequenced. Alignment of the longest 5' RACE products identified in the three cell types with the genomic sequence of human chromosome 3 (accession no. NT_034534) is shown in Fig. 6B. Our proposed genomic organization of CCR1 is shown in Fig. 7A. 5' RACE analysis showed that exon 1 is longer (127 bp) than previously described (12); however, comparison of RACE products with genomic DNA did not identify exons upstream of exon 1. Therefore, we analyzed a region including the first 122-bp region of the first exon of CCR1 and 1582 bp upstream of this for promoter activity (Fig. 7B). Sequencing analysis of this region of genomic DNA identified a triple base polymorphic deletion (GAA), -49/-46 bp relative to the proposed transcription start. Using a luciferase reporter gene assay, we identified a CCR1 promoter region -177/+122 bp relative to the transcription start site, active in human eosinophilic HL-60 clone 15 cells, mouse monocytic RAW 264.7 cells, human HEK 293 cells (Fig. 8), and human myeloid K562 cells (see Fig. 10). In differentiated HL-60 clone 15 cells, minimal promoter activity was enhanced by the presence of a 1405-bp upstream region (pGL3 -1582/+122 bp), which alone (pGL3 -1582/-177 bp) had no promoter activity (Figs. 8A and 9). This 1405-bp sequence also caused an enhancement of promoter activity in myeloid K562 cells (Fig. 10) and a nonsignificant enhancement of promoter activity in murine monocytic RAW 264.7 cells, but no enhancement in the nonleukocyte HEK 293 cells (Fig. 8, B and C). Treatment of K562 cells with IL-3, TNF-, or IFN- for 24 h had no effect on the relative activity of the CCR1 promoter (Fig. 10), although TNF- treatment resulted in an increase in the activity of all promoters, including the SV40 promoter of the internal control and positive control vectors (data not shown). When compared with the positive control vector in which the luciferase gene was constitutively active under the control of the viral SV40 promoter, CCR1 promoter (-177/+122 bp) activity was greatest in differentiated HL-60 clone 15 cells (35.0 ± 5.8% of SV40 promoter activity; n = 12 ± SEM). These differentiated HL-60 clone 15 cells were responsive to CCL3 in assays of calcium mobilization (data not shown). Promoter activity (-177/+122 bp) in K562 cells was 26% of the SV40 promoter activity; in RAW 264.7 and HEK 293 cells, promoter activity was 18% of SV40 promoter activity (data not shown). Although CCR1 promoter activity appeared to be greatest in HL-60 clone 15 cells, we cannot exclude the possibility that the apparently greater activity of the promoter in these human eosinophilic cells could be due to differences in SV40 promoter activity between cell lines. Analysis of a 5' truncated region of the promoter region, consisting primarily of exon 1 sequence (pGL3 -51/+122), indicates that the major functional regions of the CCR1 promoter lie in a region -177/-51 bp relative to the putative transcriptional start site (+1), because the -51/+122 bp sequence had weaker promoter activity in HL-60 clone 15, RAW 264.7, and HEK 293 cells (Fig. 9 and data not shown). Binding sites for the transcription factors GATA-1, GATA-2, and Sp1 were found in the CCR1 promoter region using the TRANSFAC search engine (http://www.cbrc.jp/research/db/TFSEARCH.html) (20). Comparison of the CCR1 promoter region with other chemokine receptor promoters using the multiple alignment general interface search tool (http://www.hgmp.mrc.ac.uk) showed 25–31% identity with the CCR2 (accession no. AF068265), CCR3 (accession no. AF237380), and CCR5 (accession no. AF032132) promoters (21, 22, 23). Comparisons of the CCR1 promoter region with other eosinophil-selective promoters showed 26–27% identity with the human eosinophil cationic protein promoter (accession no. D86343), human major basic protein promoter (accession no. AF304354), and the IL-5R chain promoter (accession no. U18373) (24, 25, 26). Preliminary sequencing analyses of the GAA polymorphism (-49/-46 bp) in five MPR and three MHR donors did not identify an association between this polymorphism and MHR status. Single nucleotide polymorphisms in the open reading frame (ORF) and 3' region of CCR1 (described in the database at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db = snp) are indicated in Fig. 7A. No differences in the sequences of the -177/+122 bp promoter region, the first intron, and exon 2 of the CCR1 gene were observed between two MHR and two MPR donors, suggesting that polymorphisms within these regions do not confer the MHR phenotype.
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Discussion |
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Using assays of eosinophil shape change in mixed granulocyte preparations, we found that CCL3 and CCL11 induced equipotent eosinophil responses in 19% of a large panel of donors. As previously described (13), we designated these donors as MHR, in contrast to the majority of individuals who showed poor or absent eosinophil responses to this ligand (MPR). We found similar results when responses were assayed in whole blood. We have previously shown that the apparent CCL11 potency in assays of eosinophil shape change was lower in whole blood than PMNL preparations because of CCL11 binding to the red cell promiscuous chemokine receptor Duffy (15). In the data of this study, apparent CCL3 potency was similarly reduced in whole blood, possibly through interactions of chemokine with whole-blood constituents, although we have not specifically investigated this. However, the characteristic phenotype of equivalent CCL3 and CCL11 potency in MHR donors was maintained in whole-blood assays of eosinophil responsiveness, although with some reduction in CCL3 efficacy. We previously showed that calcium deprivation up-regulates CCL3 responses in eosinophils (13) (and for this reason, characterization of MHR/MPR phenotype, particularly in whole blood, needs to be done rapidly after venesection), and it may be that the difference in efficacy, and possibly some of the differences in potency, between whole blood and mixed PMNL reflects some up-regulation of CCL3 responses during preparation. However, in MHR donors, higher eosinophil responses to CCL3 were observed in freshly venesected whole blood, mixed PMNL, and also highly purified eosinophil preparations, indicating that, in MHR donors, eosinophil CCR1 is likely to be functional in vivo. In keeping with our original smaller study (13), donors showing the MHR phenotype exhibited eosinophil CCR1 expression that was greater than in MPR individuals, with three donors showing particularly high CCR1 expression.
In donors phenotyped as MHR in assays of eosinophil shape change, we found that CCL3 induced chemotaxis of eosinophils and up-regulation of eosinophil expression of the integrin subunit CD11b. Together, these data indicate important potential roles for CCR1 signaling in MHR donors, contributing to the multistep process of eosinophil recruitment from the microcirculation to the lungs. In accordance with our findings, Struyf et al. (33) recently reported that eosinophils from 20% of their donors showed chemotactic responses to CCR1 ligands.
The MHR phenotype is stable over time. All donors were tested more than once, and some were tested many times, over intervals ranging from a few months to several years, in carefully standardized assays. The donor pool comprised healthy volunteers (normal and atopic) and excluded those taking systemic medication. Although not formally quantified, because no donors altered phenotype with time, these data supported an underlying genetic predisposition to higher CCR1 expression and the MHR phenotype. However, all donors exhibiting the MHR phenotype gave a history of a potentially atopy-associated disease (asthma, eczema, or hay fever) in a routine prephlebotomy questionnaire. Because all MHR donors showed increased levels of eosinophil CCR1 expression, we sought to investigate whether eosinophil CCR1 expression, as 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 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 eosinophil CCR1 expression was not associated with an increased likelihood 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 unlikely to be associated with altered CCL3 responsiveness of peripheral blood leukocytes other than eosinophils. We were also 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 reflection 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, 15–20% 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 studies of MHR donors, perhaps with the inclusion of analysis of subgroups such as severe asthmatics, may yet identify associations of this phenotype with clinical disease.
In comparison to CCR1, we sought evidence of interindividual variations in expression levels of the major eosinophil chemokine receptor CCR3 on leukocyte subsets. Again, variations in eosinophil CCR3 expression between individuals did not correlate with atopy or serum IgE levels. In keeping with published data, we detected high levels of CCR3 on basophils (7, 35, 36) and much lower levels on neutrophils (37) and monocytes. MHR status was not associated with a general increase in chemoattractant receptor expression, and there was very little correlation between eosinophil CCR1 and CCR3 expression.
We observed significant correlations between levels of CCR3 expression on eosinophils, basophils, neutrophils, and monocytes. These data suggest that, potentially, a single underlying common factor plays a role in controlling CCR3 expression by leukocytes, which is either enhanced or suppressed to regulate CCR3 expression on eosinophils and basophils vs neutrophils and monocytes, respectively. A recent study by Menzies-Gow et al. (37) showed neutrophil recruitment in response to intradermal CCR3 ligands; however, neutrophils from these donors did not respond to CCL11 in assays of neutrophil shape change (37), and we did not detect neutrophil shape change in response to CCL11/eotaxin in 56 donors. However, our data demonstrating a spectrum of CCR3 expression raise the possibility that there may be yet-unidentified donors in whom, akin to the MHR phenomenon regulating eosinophil CCL3 responsiveness, a threshold of CCR3 expression may be reached permitting neutrophil or monocyte responses to CCR3 ligands.
The CCR1, CCR2, CCR3, and CCR5 genes are clustered on a 350-kb region of chromosome 3 (3p21.3), which suggests that these chemokine receptors are derived from a common primitive originator whose expression may thus be regulated by similar pathways (38). To investigate mechanisms that might regulate CCR1 expression, we identified the CCR1 promoter, with a view also to identify any polymorphic variants that could account for variability in CCR1 expression levels between donors. Aligning the mRNA for CCR1 (accession no. NM_001295) (12) with chromosome 3, we identified an exon containing 5'-untranslated sequence, located 4 kb upstream of exon 2, which contained the full ORF. Database searching also identified a sequence that was listed as the CCR1 promoter (accession no. AF051305), which highlighted the same upstream exon and had extensive homology to chromosome 3 in the region of this putative promoter site. However, the upstream homology of AF051305 to chromosome 3 became erratic, and the whole sequence had been misattributed as belonging to chromosome 6. Using 5' RACE, we found that CCR1 mRNA from monocytes, monocytic cell lines (THP-1), and a cell line with eosinophilic characteristics (HL-60 clone 15) contained this single upstream nontranslated exon. According to our longest RACE product, we found exon 1 to be 127 bases in length, 76 bases longer than the original deposited mRNA sequence (12). We identified an active minimal promoter upstream of exon 1 causing gene transcription in HL-60 clone 15 cells, myeloid K562 cells, the mouse monocytic RAW 264.7 cell line, and the nonleukocyte HEK 293 line. We also identified an enhancer region upstream of the minimal promoter whose ability to increase promoter activity was predominantly evident in the eosinophilic HL-60 clone 15 cells and myeloid leukemia K562 cells (both of human origin), identifying a relatively leukocyte-selective regulatory region. These data show similarity to those obtained in analysis of the CCR3 promoter, which also demonstrated enhancer regions favoring leukocyte expression, although organization of the CCR3 gene itself appears to be more complex than CCR1 gene organization (22, 39). IFN- has been shown to increase neutrophil CCR1 expression (40), but in keeping with their lack of effect on eosinophil CCR1 expression, neither IL-3, TNF-, or IFN- had any effect on the relative activity of the CCR1 promoter constructs in K562 cells, although these cytokines have previously been shown to induce or modulate expression of other genes in this cell line (41, 42). Analysis using the transcription factor (TRANSFAC) search tool identified putative binding sites for several leukocyte-expressed transcription factors including GATA-1 and GATA-2, which are known to be important regulators of eosinophil development (43). Sequence analysis of the CCR1 promoter region (-177/+122 bp), intron 1, and exon 2 from two MHR and two MPR donors did not identify polymorphic variants that could define responsiveness to CCL3/MIP-1; however, only a small number of subjects have been investigated thus far. Polymorphisms affecting other pathways such as G protein expression could also underlie the MHR phenotype.
Collectively, these data show that CCR1 expression is donor- and cell-specific, giving rise to additional pathways that may regulate leukocyte recruitment in some individuals. Our data also raise the possibility that expression of other chemokine receptors may show similar phenotypes. These are important points to consider when developing therapies aimed at modulating leukocyte recruitment in inflammatory disease.
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Acknowledgments |
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Footnotes |
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2 R.M.P. and V.E.L.S. contributed equally to this study.
3 Address correspondence and reprint requests to Dr. James Pease, Leukocyte Biology Section, Division of Biomedical Sciences, Imperial College London, Sir Alexander Fleming Building, South Kensington Campus, London, U.K., SW7 2AZ. E-mail address: j.pease{at}imperial.ac.uk
4 J.E.P. and I.S. contributed equally to this study as principal investigators.
5 Abbreviations used in this paper: CCL, CC chemokine ligand; MIP, macrophage-inflammatory protein; MHR, CCL3/MIP-1 highly responsive; MPR, CCL3/MIP-1 poorly responsive; PMNL, polymorphonuclear leukocyte; FSC, forward scatter; SSC, side scatter; RAST, radioallergosorbent test; RACE, rapid amplification of cDNA ends; HEK, human embryonic kidney; ORF, open reading frame.
Received for publication October 21, 2002. Accepted for publication April 3, 2003.
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) or CCL3/MIP-1
). 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 = 6 (A), 30 (B), 12 (C), and 44 (D)) ± SEM.
) and MPR donor (
) eosinophil CD11b expression. Chemotactic migration responses to CCL3/MIP-1
) and -1582/+122 bp sequence (•). Relative luciferase activity in cells transfected with promoterless pGL3.enhancer vector is also shown (
). Promoter activity is expressed as firefly luciferase activity relative to the internal control (pRL.SV40 Renilla luciferase 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 are indicated, as calculated by one-way ANOVA and Tukey’s multiple comparisons posttest.
) and -1582/+122 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.