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Original Research: COUGH |

Chemokine Concentrations and Mast Cell Chemotactic Activity in BAL Fluid in Patients With Eosinophilic Bronchitis and Asthma, and in Normal Control Subjects* FREE TO VIEW

Lucy Woodman, BSc; Amanda Sutcliffe, BSc; Davinder Kaur, PhD; Mike Berry, MD; Peter Bradding, DM; Ian D. Pavord, DM; Christopher E. Brightling, PhD, FCCP
Author and Funding Information

*From the Institute for Lung Health (Ms. Woodman, Ms. Sutcliffe, Drs. Kaur, Bradding, Brightling, and Berry), University of Leicester, Leicester, UK; and Department of Respiratory Medicine (Dr. Pavord), University Hospitals of Leicester, Leicester, UK.

Correspondence to: Christopher E. Brightling, PhD, Insitute for Lung Health, University of Leicester, Glenfield Hospital, Groby Rd, Leicester LE3 9QP, UK; e-mail: ceb17@le.ac.uk



Chest. 2006;130(2):371-378. doi:10.1378/chest.130.2.371
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Background: Asthma and eosinophilic bronchitis share many immunopathologic features including increased numbers of eosinophils and mast cells in the superficial airway. The mast cell chemotactic activity of airway secretions has not been assessed in patients with eosinophilic bronchitis.

Objectives: To investigate the concentration of chemokines in bronchial wash samples and BAL fluid, and the mast cell chemotactic activity in BAL fluid from subjects with asthma and eosinophilic bronchitis, and from healthy control subjects.

Methods: We measured the concentrations of CCL11, CXCL8, and CXCL10 in bronchial wash samples and BAL fluid from 14 subjects with eosinophilic bronchitis, 14 subjects with asthma, and 15 healthy control subjects. Mast cell chemotaxis to BAL fluid from these subjects was examined using the human mast cell line HMC-1.

Results: The bronchial wash sample and BAL fluid concentrations of CXCL10 and CXCL8 was increased in subjects with eosinophilic bronchitis compared to those in subjects with asthma and healthy control subjects (p < 0.05). The CCL11 concentration was below the limit of detection in most subjects. BAL fluid from subjects with eosinophilic bronchitis was chemotactic for mast cells (1.4-fold migration compared to a control, 95% confidence interval, 1.1 to 1.9; p = 0.04) and was inhibited by blocking CXCR1 (45% inhibition; p = 0.002), CXCR3 (38% inhibition; p = 0.034), or both (65% inhibition; p = 0.01). BAL fluid from the subjects with asthma and healthy control subjects was not chemotactic for mast cells. Mast cell migration to BAL fluid was correlated with the concentration of CXCL8 (r = 0.42; p = 0.031) and CXCL10 (r = 0.52; p = 0.007).

Conclusion: In subjects with eosinophilic bronchitis, CXCL8 and CXCL10 concentrations were elevated in airway secretions. These chemokines may play a key role in mast cell recruitment to the superficial airway in this condition.

Figures in this Article

Nonasthmatic eosinophilic bronchitis presents with chronic cough and is characterized by sputum eosinophilia similar to that seen in asthma, but, unlike the situation in patients with asthma, the patients with nonasthmatic eosinophilic bronchitis have no symptoms or objective evidence of variable airflow obstruction or airway hyperresponsiveness.1We2and others3 have shown that eosinophilic bronchitis is a common cause of cough in patients presenting to a respiratory specialist.

The immunopathology of eosinophilic bronchitis is very similar to that of asthma. Both conditions are associated with eosinophilic airway inflammation,1,45 increased interleukin (IL)-5 expression,68 and reticular lamina and basement membrane thickening.4 Asthma can be distinguished from eosinophilic bronchitis by increased mast cell infiltration into the airway smooth muscle4 and by increased IL-13 expression.7,9 The number of sputum mast cell is increased in subjects with both conditions, but, interestingly, the number of mast cells in bronchial brushing samples and the sputum concentration of mast cell mediators were greater in subjects with eosinophilic bronchitis than in those with asthma,8,10 suggesting that mast cell infiltration into the superficial airway may be a particular feature of this condition.

The recruitment of mast cells into the bronchial epithelium in subjects with asthma and eosinophilic bronchitis is likely to be mediated by a chemotactic signal arising from the superficial airway. The C-C and C-X-C chemokines in particular are attractive candidates as inflammatory cell chemoattractants. Several chemokine receptors and their respective ligands have been implicated in mediating mast cell migration.11The chemokine receptors that are most highly expressed by human lung mast cells are CCR3, CXCR1, CXCR3, and CXCR4.1213 The concentration of chemokines in airway secretions in subjects with eosinophilic bronchitis has not been reported; thus, whether there are important differences between eosinophilic bronchitis and asthma is unknown.

Our hypothesis is that that the concentration of chemokines that mediate mast cell migration is increased in airway secretions in subjects with asthma and eosinophilic bronchitis. To test our hypothesis, we have measured the concentrations of the chemokines CCL11 (eotaxin), CXCL8 (IL-8), and CXCL10 (IP-10) in bronchial wash samples and BAL fluid from subjects with eosinophilic bronchitis and asthma, and healthy control subjects, and investigated whether the BAL fluid from these subjects was chemotactic for mast cells.

Subjects

Patients with eosinophilic bronchitis and asthma, and healthy volunteers were recruited from respiratory outpatient clinics and from staff at the Glenfield Hospital. Subjects with asthma (n = 14) gave a suggestive history and had objective evidence of variable airflow obstruction as indicated by one or more of the following: (1) methacholine airway hyperresponsiveness (provocative concentration of methacholine causing a 20% fall in FEV1 [PC20], < 8 mg/mL); (2) > 15% improvement in FEV1 10 min after inhaling 200 μg of salbutamol; or (3) peak expiratory flow (PEF) [> 20% maximum within day amplitude from twice-daily PEF measurements over 14 days]. The subjects with eosinophilic bronchitis (n = 14) had an isolated cough, no symptoms suggesting variable airflow obstruction, normal spirometric values, normal PEF variability, normal airway responsiveness (PC20, > 16 mg/mL), and sputum eosinophilia (> 3% nonsquamous cells). None of the subjects had been treated with inhaled or oral corticosteroids, long-acting bronchodilators, or leukotriene antagonists for at least 1 month before entry into the study. The healthy subjects (n = 15) gave no history of respiratory diseases, had negative allergen skin prick test results, normal spirometry findings, and normal airway responsiveness. Thirteen subjects with eosinophilic bronchitis, 7 subjects with asthma, and 7 healthy control subjects had been included in a previous study.5 All subjects gave informed written consent to participate in this study, and the Leicestershire Research and Ethics Committee approved the study.

Protocol and Clinical Measurements

Subjects underwent spirometry, allergen skin prick tests for Dermatophagoides pteronyssinus, cat fur, grass pollen, and Aspergillus fumigatus, a methacholine inhalation test,14and sputum induction.15One week later, patients underwent bronchoscopy conducted according to the current British Thoracic Society guidelines.16 A 20-mL bronchial wash into the bronchus intermedius and a 180-mL BAL into the right lower lobe was performed. Bronchial wash and BAL fluid samples were centrifuged at 790g for 10 min, and the supernatant was stored at –80°C for later analysis.

Mediator Measurements

The concentrations of CXCL8 (IL-8), CXCL10 (interferon [IFN]-inducible protein-10), and CCL11 (eotaxin) were all measured by a commercial enzyme immunoassay (BD Biosciences; Cowley, Oxfordshire, UK). The concentration of CCL11 in 59% of the BAL fluid samples was below the limit of detection, so these samples were concentrated ×30 (Vivascience AG; Hanover, Germany) and the assay was repeated. An insufficient volume of bronchial wash was available to concentrate the samples. The interassay and intraassay variabilities were between 5% and 10%. The limit of detection was 3.1 pg/mL in BAL fluid or bronchial wash sample for CXCL8, 15.6 pg/mL in BAL fluid or bronchial wash sample for CXCL10, and 0.21 pg/mL in BAL fluid or 6.25 pg/mL in bronchial wash sample for CCL11. There was an insufficient volume of the bronchial wash sample to measure any chemokines in one subject from each group.

HMC-1 Chemotaxis

Mast cell migration toward recombinant CCL11, CXCL8, and CXCL10 (100 ng/mL) [R&D Systems; Abingdon, Oxfordshire, UK], and BAL fluid was investigated in 10 subjects with eosinophilic bronchitis, 10 subjects with asthma, and 8 healthy control subjects who had sufficient BAL fluid samples. Insufficient bronchial wash samples were available to assess HMC-1 migration toward these samples. The HMC-1 cell line was a generous gift from Dr. J. Butterfield (Mayo Clinic; Rochester, MN). The cells were maintained in Iscove modified Dulbecco modified Eagle medium.17 Chemotaxis assays were performed using transwells with 8-μm fibronectin-coated inserts in 24-well plates (BD Biosciences). We placed 2 × 105 HMC-1 cells in 100 μL of culture medium with 2% fetal calf serum into the top well, and 450 μL of BAL fluid diluted 1:2 with culture medium with 2% fetal calf serum or a negative control into the bottom well. After incubating the cells for 4 h at 37°C, we counted the number of HMC-1 cells in the bottom well using Kimura stain in a hemocytometer. Checkerboard analysis was used to distinguish chemotactic activity from chemokinetic activity. To assess the role of CXCR1 and CXCR3 in HMC-1 migration to BAL fluid, we preincubated the cells with receptor blocking antibodies (R&D Systems) alone or in combination with appropriate isotype controls (Dako; Ely, Cambridgeshire, UK) for 1 h prior to the chemotaxis assay, as previously described.,12,18

HMC-1 Expression of CXCR1 and CXCR3

HMC-1 (105 cells/well) were seeded onto fibronectin-coated chamber slides and cultured for 24 h. The HMC-1 cells were labeled with CXCR3 monoclonal antibody (MoAb), indirectly labeled with fluorescein isothiocyanate and CXCR1-PE MoAb (R&D Systems) or appropriate isotype controls. Cells were counterstained with 4′,6′-diamidine-2-phenylindole (Sigma; Dorset, UK), and the proportion of positively stained cells was identified by immunofluorescence. We had identified technical problems with the indirectly labeled CXCR1 MoAb that was used in our earlier report,12 leading us to believe that we had previously underestimated the CXCR1 expression by the HMC-1. Therefore, we reanalyzed the expression of CXCR1 by HMC-1 using a conjugated CXCR1-PE MoAb (R&D Systems) with an appropriate isotype control by single-color flow cytometry. HMC-1 expression of the other chemokine receptors identified previously by flow cytometry was consistent and is not rereported here.

The HMC-1 functional response to CXCL8 and CXCL10 (100 ng/mL) was assessed by measuring changes in the cytosolic free Ca2+ concentration ([Ca2+]i) by ratiometric Ca2+ imaging on FURA-2-loaded cells using appropriate software (Openlab; Improvision; Coventry, UK). This was converted to a [Ca2+]i concentration using a commercially available calibration kit (Molecular Probes; Invitrogen; Paisley, UK).

Statistical Analysis

Bronchial wash sample and BAL fluid cell characteristics were described as the median and interquartile range (IQR). Bronchial wash and BAL fluid sample CXCL8 and CXCL10 concentrations were log-normally distributed and were described as geometric means (log SEM). Bronchial wash and BAL fluid sample CCL11 concentration was described as the median and IQR. Supernatants with undetectable levels of cytokine were assigned a concentration of zero. Mast cell migration was expressed as the mean (SEM) fold difference in migration compared with controls. HMC-1 cells were defined as responders to ligand activation if the increase in [Ca2+]i was > 2 SEMs above the baseline variation. The increase in [Ca2+]i of the responding cells was described as the mean (SEM). Cytokine concentrations were compared across all groups by analysis of variance, those between disease groups were compared by unpaired t test for parametric data, and those across groups were compared by the Kruskal-Wallis test and between disease groups by Mann-Whitney test for nonparametric data. CCL11 was measurable in about half of the samples, so the number of subjects who had measurable CCL11 concentrations was compared across groups by χ2 test. Correlations between BAL fluid chemokine concentration and HMC-1 migration toward BAL fluid were analyzed by the Spearman rank correlation coefficient. Significance was accepted at the level of 95%.

The clinical characteristics, bronchial wash sample and BAL fluid differential cell counts, and chemokine concentrations were as shown (Table 1 ). The bronchial wash sample CXCL8 and CXCL10 concentrations were increased in those subjects with eosinophilic bronchitis (p < 0.05) [Table 1]. The individual BAL fluid concentrations of CXCL10 and CXCL8 for each subject were as shown (Fig 1 ).

The BAL fluid CXCL10 concentration was significantly different between groups (p = 0.003). The BAL fluid CXCL10 concentration was greater in those subjects with eosinophilic bronchitis than in those with mild asthma (p = 0.002) and in healthy control subjects (p = 0.041) [Table 1, Fig 1, top, A]. There was no difference in the BAL fluid CXCL10 concentration between subjects with asthma and healthy control subjects.

Similarly, the BAL fluid CXCL8 concentration was significantly different between groups (p = 0.038) and was greater in those subjects with eosinophilic bronchitis than in healthy control subjects (p = 0.02) [Table 1, Fig 1, bottom, B]. The BAL fluid CXCL8 concentration was not different in subjects with asthma compared to those with eosinophilic bronchitis or healthy control subjects.

There were no between-group differences in the CCL11 concentrations in BAL fluid (p = 0.43). The number of subjects with a measurable BAL fluid CCL11 concentration was not significantly greater in those with eosinophilic bronchitis (6 of 14 subjects), compared to those with asthma (7 of 14 subjects) and healthy control subjects (9 of 15 subjects; p = 0.65).

HMC-1 cells highly expressed CXCR3 (mean, 95%; SEM, 0.8%) by immunofluorescence and CXCR1 (by immunofluorescence: mean, 89%; SEM, 2.3%; by flow cytometry: mean, 84%; SEM, 5%; n = 4) [Fig 2] . HMC-1 [Ca2+]i increased in response to activation by CXCL8 (geometric mean, 267 nmol/L; log SEM, 30 nmol/L; 81% of cells responded; n = 27) and CXCL10 (geometric mean 80 nmol/L; log SEM, 11 nmol/L; 96% of cells responded; n = 45).

HMC-1 cells migrated significantly toward recombinant CXCL8, CXCL10, and CCL11 (n = 6) [Fig 3 ]. This migration was inhibited by the chemokine receptor blocking antibodies CXCR1 (mean inhibition, 83%; SEM, 17%; p = 0.004) CXCR3 (mean inhibition, 73%; SEM, 13%; p < 0.0001), but not CCR3 (mean inhibition, 50%; SEM, 22%; p = 0.08) compared to the appropriate isotype controls.

The BAL fluid from subjects with eosinophilic bronchitis (n = 10) exhibited chemotactic activity for HMC-1 (1.4-fold increase compared to the control; 95% confidence interval [CI], 1.1 to 1.9; p = 0.04) [Fig 4 , top, A]. However, there was no significant HMC-1 migration toward BAL fluid from subjects with asthma (1.1-fold increase; 95% CI, 0.67 to 1.98; p = 0.6; n = 10) or healthy control subjects (1.1-fold increase; 95% CI, 0.77 to 1.52; p = 0.6; n = 8). Checkerboard analysis confirmed that HMC-1 migration toward BAL fluid from subjects with eosinophilic bronchitis was due to chemotaxis rather than chemokinesis (n = 7) [Fig 4, middle, B].

The clinical characteristics and BAL fluid chemokine concentrations were not significantly different in the subgroup of patients whose BAL fluid was used for the chemotaxis assays compared to those for the whole group (Fig 1). In this subgroup, the BAL fluid CXCL10 concentration was increased in the subjects with eosinophilic bronchitis (geometric mean, 517 pg/mL; log SEM, 0.1 pg/mL) compared to those with asthma (geometric mean, 209 pg/mL; log SEM, 0.1 pg/mL) and healthy control subjects (geometric mean, 270 pg/mL; log SEM, 0.1 pg/mL; p = 0.02). The CXCL8 BAL fluid concentration was increased in subjects with eosinophilic bronchitis (geometric mean, 224 pg/mL; log SEM, 0.1 pg/mL) compared to that in healthy control subjects (geometric mean, 109 pg/mL; log SEM, 0.06 pg/mL; p = 0.05), but not compared to that in asthmatic subjects (geometric mean, 137 pg/mL; log SEM, 0.05 pg/mL; p = 0.17).

As significant HMC-1 migration was only observed toward BAL fluid from subjects with eosinophilic bronchitis, BAL fluid from five of those subjects was used to assess the effect of chemokine receptor blockade on HMC-1 migration. The chemotactic effect of the BAL fluid was markedly reduced by CXCR1 blockade (45% inhibition; 95% CI, 28 to 62%; p = 0.002) and CXCR3 blockade (38% inhibition; 95% CI, 5 to 72%; p = 0.034) alone and in combination (65% inhibition; 95% CI, 46 to 85%; p = 0.01) compared to the isotype control (Fig 4, bottom, C). There was a positive correlation between HMC-1 migration toward the BAL fluid and the BAL fluid concentration of CXCL8 (r = 0.42; p = 0.031) and CXCL10 (r = 0.52; p = 0.007).

We describe for the first time that BAL fluid from subjects with eosinophilic bronchitis is chemotactic for mast cells. In contrast, BAL fluid from subjects with mild asthma and healthy control subjects did not demonstrate chemotactic activity for mast cells. Mast cell migration was significantly correlated with the BAL fluid concentrations of CXCL8 and CXCL10. The BAL fluid concentrations of CXCL10 and CXCL8 were increased in subjects with eosinophilic bronchitis, and blocking CXCR1 and CXCR3, the receptors for these chemokines, inhibited mast cell migration. The concentrations of CXCL8 and CXCL10 were similarly elevated in bronchial wash samples compared to those subjects with eosinophilic bronchitis. This suggests that CXCL8 and CXCL10 may promote mast cell migration into the airway lumen in subjects with eosinophilic bronchitis.

Mast cell localization to the superficial airway is a feature of asthma and eosinophilic bronchitis. In both conditions, there were increased numbers of mast cells in bronchial brushing and induced sputum samples.1,8,19We found that BAL fluid from subjects with eosinophilic bronchitis was chemotactic for mast cells. However, we were unable to demonstrate mast cell chemotactic activity in the BAL fluid from asthmatic subjects. Similarly, Olsson and colleagues20 found that BAL fluid from allergic asthmatic subjects outside of the pollen season had chemotactic activity for mast cells in only 30% of cases compared to 100% of cases from the same subjects during the pollen season. This may provide a plausible explanation for our inability to show mast cell migration toward BAL fluid from asthmatic subjects as we recruited subjects with mild asthma who had minimal symptoms and perhaps BAL fluid from asthmatic subjects may be chemotactic for mast cells only when the subjects have more active disease. Of note, the mast cell chemotactic activity of the asthmatic BAL fluid obtained during the pollen season in this previous report20 was increased 1.5-fold over that of the control subjects, which was similar to that observed in our subjects with eosinophilic bronchitis, suggesting that this degree of chemotaxis is likely to be biologically relevant. An alternative explanation for the differential effect on mast cell chemotaxis toward BAL fluid from subjects with asthma and eosinophilic bronchitis is that mast cell migration to the superficial airway may be a more prominent feature of eosinophilic bronchitis. In support of this view, the number of mast cells in bronchial brushing samples and the sputum concentration of mast cell mediators were greater in subjects with eosinophilic bronchitis than in those with asthma.8,10

Several chemotaxins have been described for mast cells. Olsson et al20 reported that mast cell migration to allergic asthmatic BAL fluid was mediated mainly by the activation of Gi-protein-coupled receptors, and to a lesser extent by stem cell factor and transforming growth factor-β, suggesting that chemokines contribute to the majority of the mast cell chemotactic activity of the BAL fluid from these subjects. A number of chemokine receptors have been identified on human blood-derived mast cells and mast cells present in tissues, namely, CCR1, CCR2, CCR3, CCR4, CXCR1, CXCR2, CXCR3, CXCR4, and CX3CR1 (reviewed in the study by Brightling et al,11). We have previously described the fact that CXCR3 was the most highly expressed chemokine receptor by mast cells in bronchial biopsy specimens and by HMC-1 cells, and that CXCL10 was chemotactic for mast cells.12,18 In contrast to our earlier report,12 we found that CXCR1 was highly expressed by HMC-1 cells. This discrepancy in CXCR1 expression by HMC-1 was due to different receptor identification by the directly and indirectly labeled monoclonal antibodies used and the different techniques employed. The chemokine receptor profiles of HMC-1 and primary lung mast cells were similar, supporting the view that HMC-1 cells are a suitable model for lung mast cell migration. We are confident that HMC-1 CCR3, CXCR1, and CXCR3 are functional as the activation of HMC-1 cells by CXCL8 and CXCL10 resulted in a transient rise in [Ca2+]i in a percentage of cells similar to the proportion that expressed CXCR1 and CXCR3, and that these ligands and CCL11 were chemotactic for HMC-1 cells.

All of these ligands are expressed by the airway epithelium.2123 Therefore, all of the chemokines studied here have the potential to mediate mast cell migration to the superficial airway. We found that the BAL fluid concentrations of CXCL10 and CXCL8 were increased in subjects with eosinophilic bronchitis. BAL fluid from these subjects was chemotactic for mast cells, and blocking CXCR1, CXCR3, or both inhibited mast cell migration. This inhibition of migration was incomplete, and it is likely that other chemokines, such as CXCL12, the ligand for CXCR4, may also play a part in mast cell migration toward the superficial airway. Mast cell migration toward BAL fluid from the subjects with eosinophilic bronchitis was reduced compared to migration toward recombinant CXCL8 and CXCL10. This probably reflects the lower concentration of the chemokines in the BAL fluid. We did not find an increase in the BAL fluid chemokine concentrations in subjects with asthma, which may provide an explanation for the absence of mast cell chemotactic activity in these subjects. In addition, mast cell migration was significantly correlated with the BAL fluid concentration of CXCL8 and CXCL10.

One shortcoming of our study design was that BAL fluid cytospins were not stained appropriately to be able to enumerate the number of mast cells in BAL fluid across the groups, and therefore the concentration of these chemokines and BAL fluid mast cell numbers cannot be correlated. Importantly, CXCL8 and CXCL10 concentrations were also elevated in the bronchial wash samples from those subjects with nonasthmatic eosinophilic bronchitis, suggesting that these chemokines may play a role in mast cell migration toward the airway lumen in both large and small airways. In contrast to CXCL8 and CXCL10 concentrations, the BAL fluid and bronchial wash sample CCL11 concentration was detectable in less than half of the subjects, and no differences were observed between subjects with disease and healthy control subjects. This is in contrast with some previous reports that found that BAL fluid CCL11 concentration was increased in stable subjects with disease2425 and after allergen challenge.25However, it is consistent with a report26 that sputum CCL11 concentrations were not increased in subjects with mild asthma. The subjects with asthma in our study had mild disease, and it is likely that CCL11 concentrations increase with severity of disease.26 Thus, our findings suggest that CXCL8 and CXCL10, but not CCL11, promote mast cell migration into the superficial airway in subjects with eosinophilic bronchitis.

The distribution of mast cells within particular airway structures is important in the development of specific features of airway diseases. In patients with eosinophilic bronchitis and idiopathic cough, there was an increased sputum histamine level,27supporting the view that superficial mast cells may play a role in the genesis of chronic cough perhaps due to interactions with airway nerves.28 In mast cells from subjects with asthma, microlocalization within the airway smooth muscle4 and mucosal glands29 were associated with increased airway responsiveness and mucous plugging, respectively. We have recently found18 that in subjects with asthma there was up-regulation of CXCL10 released by the airway smooth muscle together with an enhanced expression of CXCR3 by mast cells within the airway smooth muscle bundle, suggesting that the CXCR3/CXCL10 axis may be critical in the localization of mast cells to the airway smooth muscle in subjects with asthma. Therefore, it is likely that there are common mechanisms that control the microlocalization of mast cells to different parts of the airway wall.

Eosinophilic bronchitis, like asthma, is generally regarded as a T helper (Th) type 2-driven disease,6 although there is sufficient evidence to suggest that the immunology in subjects with asthma is far more complex and that Th-1 cytokines play a role.30Indeed, we report here that the BAL fluid concentration of the Th-1 chemokine CXCL10 was increased in subjects with eosinophilic bronchitis in contrast to very low concentrations of the Th2 chemokine CCL11. Tumor necrosis factor-α and IFN-γ induce the expression of CXCL10.31Tumor necrosis factor-α expression is up-regulated in subjects with asthma,32and IFN-γ is increased by several stimuli, including viral infection.33Interestingly, in one report34CXCL10 expression by BAL fluid cells was increased in subjects with asthma, illustrating that this chemokine can be up-regulated in subjects with mild disease. In addition, CXCL10 production was increased markedly following segmental allergen challenge in subjects with asthma.3537 Therefore, CXCL10 may be an important chemokine in mast cell recruitment to the airway in response to a variety of stimuli.

In summary, we found that the bronchial wash sample and BAL fluid concentrations of CXCL8 and CXCL10 were increased in subjects with eosinophilic bronchitis. BAL fluid from these subjects had chemotactic activity for mast cells that was inhibited by blocking CXCR1 and CXCR3. This suggests that the chemokines CXCL8 and CXCL10 may be important in mast cell localization to the superficial airway in subjects with eosinophilic bronchitis.

Abbreviations: [Ca2+]i = cytosolic free Ca2+ concentration; CI = confidence interval; IFN = interferon; IL = interleukin; IQR = interquartile range; MoAb = monoclonal antibody; PC20 = provocative concentration of methacholine causing a 20% fall in FEV1; PEF = peak expiratory flow; Th = T helper

This research was supported by Asthma UK and a Department of Health Clinician Scientist Award.

The authors have no conflicts of interest to disclose.

Table Graphic Jump Location
Table 1. Clinical and BAL Characteristics*
* 

BW = bronchial wash sample.

 

Values are given as the mean (SEM).

 

Values are given as the geometric mean (log SEM).

§ 

Values are given as the median (IQR).

 

p < 0.05 (by analysis of variance).

Figure Jump LinkFigure 1. BAL fluid concentration of (top, A) CXCL10 and (bottom, B) CXCL8 in each subject with eosinophilic bronchitis and asthma, and in healthy control subjects. ▴ = subjects who were included in the chemotaxis assays. Δ = subjects not included in chemotaxis assays. Horizontal bars represent the geometric mean.Grahic Jump Location
Figure Jump LinkFigure 2. CXCR1 and CXCR3 expression by HMC-1 cells was confirmed by immunofluorescence. Top left, A: IgG2a isotype control. Top right, B: red CXCR1+ cells. Bottom left, C: IgG1 isotype control. Bottom right, D: green CXCR3+ cells. Blue nuclear counterstain (original × 400).Grahic Jump Location
Figure Jump LinkFigure 3. The recombinant chemokines CXCL10, CXCL8, and CCL11 (100 ng/mL) were chemotactic for HMC-1 cells (n = 6). Error bars represent the mean ± SEM of HMC-1 migration compared to controls. * = p < 0.05.Grahic Jump Location
Figure Jump LinkFigure 4. Top, A: HMC-1 chemotaxis to BAL fluid from subjects with eosinophilic bronchitis (n = 10), subjects with asthma (n = 10), and healthy control subjects (n = 8). Errors bars represent the mean ± SEM HMC-1 migration compared to controls. Middle, B: HMC-1 cells were analyzed for chemotactic and chemokinetic activity toward BAL fluid from subjects with eosinophilic bronchitis (n = 7) by the addition of BAL fluid to the upper chamber (where the cells were added) or lower chamber, as indicated. Errors bars represent the mean ± SEM HMC-1 migration compared to controls. Bottom, C: the mean ± SEM percentage inhibition of HMC-1 migration to BAL fluid from subjects with eosinophilic bronchitis by blocking MoAbs to CXCR1 and CXCR3 alone or in combination (n = 5) compared to appropriate isotype control. * = p < 0.05.Grahic Jump Location
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Lamkhioued, B, Renzi, PM, Abi-Younes, S, et al Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation.J Immunol1997;159,4593-4601. [PubMed]
 
Lilly, CM, Nakamura, HIDE, Belostotsky, OI, et al Eotaxin expression after segmental allergen challenge in subjects with atopic asthma.Am J Respir Crit Care Med2001;163,1669-1675. [PubMed]
 
Dent, G, Hadjicharalambous, C, Yoshikawa, T, et al Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum.Am J Respir Crit Care Med2004;169,1110-1117. [CrossRef] [PubMed]
 
Birring, SS, Parker, D, Brightling, CE, et al Induced sputum inflammatory mediator concentrations in chronic cough.Am J Respir Crit Care Med2004;169,15-19. [PubMed]
 
Gibson, PG Cough is an airway itch?Am J Respir Crit Care Med2004;169,1-2. [PubMed]
 
Carroll, NG, Mutavdzic, S, James, AL Distribution and degranulation of airway mast cells in normal and asthmatic subjects.Eur Respir J2002;19,879-885. [CrossRef] [PubMed]
 
Salvi, SS, Suresh, BK, Holgate, ST Is asthma really due to a polarized T cell response toward a helper T cell type 2 phenotype?Am J Respir Crit Care Med2001;164,1343-1346. [PubMed]
 
Hardaker, EL, Bacon, AM, Carlson, KARE, et al Regulation of TNF-α- and IFN-γ-induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease.FASEB J2004;18,191-193. [PubMed]
 
Bradding, P, Roberts, JA, Britten, KM, et al Interleukin-4, -5, and -6 and tumor necrosis factor-α in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines.Am J Respir Cell Mol Biol1994;10,471-480. [PubMed]
 
Dahl, ME, Dabbagh, K, Liggitt, D, et al Viral-induced T helper type 1 responses enhance allergic disease by effects on lung dendritic cells.Nat Immunol2004;5,337-343. [PubMed]
 
Miotto, D, Christodoulopoulos, P, Olivenstein, R, et al Expression of IFN-γ-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in T(H)1- and T(H)2-mediated lung diseases.J Allergy Clin Immunol2001;107,664-670. [CrossRef] [PubMed]
 
Bochner, B, Hudson, S, Xiao, H, et al Release of both CCR4-active and CXCR3-active chemokines during human allergic pulmonary late-phase reactions.J Allergy Clin Immunol2003;112,930-934. [CrossRef] [PubMed]
 
Liu, L, Jarjour, NN, Busse, WW, et al Enhanced generation of helper T type 1 and 2 chemokines in allergen-induced asthma.Am J Respir Crit Care Med2004;169,1118-1124. [CrossRef] [PubMed]
 
Pilette, C, Francis, JN, Till, SJ, et al CCR4 ligands are up-regulated in the airways of atopic asthmatics after segmental allergen challenge.Eur Respir J2004;23,876-884. [CrossRef] [PubMed]
 

Figures

Figure Jump LinkFigure 1. BAL fluid concentration of (top, A) CXCL10 and (bottom, B) CXCL8 in each subject with eosinophilic bronchitis and asthma, and in healthy control subjects. ▴ = subjects who were included in the chemotaxis assays. Δ = subjects not included in chemotaxis assays. Horizontal bars represent the geometric mean.Grahic Jump Location
Figure Jump LinkFigure 2. CXCR1 and CXCR3 expression by HMC-1 cells was confirmed by immunofluorescence. Top left, A: IgG2a isotype control. Top right, B: red CXCR1+ cells. Bottom left, C: IgG1 isotype control. Bottom right, D: green CXCR3+ cells. Blue nuclear counterstain (original × 400).Grahic Jump Location
Figure Jump LinkFigure 3. The recombinant chemokines CXCL10, CXCL8, and CCL11 (100 ng/mL) were chemotactic for HMC-1 cells (n = 6). Error bars represent the mean ± SEM of HMC-1 migration compared to controls. * = p < 0.05.Grahic Jump Location
Figure Jump LinkFigure 4. Top, A: HMC-1 chemotaxis to BAL fluid from subjects with eosinophilic bronchitis (n = 10), subjects with asthma (n = 10), and healthy control subjects (n = 8). Errors bars represent the mean ± SEM HMC-1 migration compared to controls. Middle, B: HMC-1 cells were analyzed for chemotactic and chemokinetic activity toward BAL fluid from subjects with eosinophilic bronchitis (n = 7) by the addition of BAL fluid to the upper chamber (where the cells were added) or lower chamber, as indicated. Errors bars represent the mean ± SEM HMC-1 migration compared to controls. Bottom, C: the mean ± SEM percentage inhibition of HMC-1 migration to BAL fluid from subjects with eosinophilic bronchitis by blocking MoAbs to CXCR1 and CXCR3 alone or in combination (n = 5) compared to appropriate isotype control. * = p < 0.05.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1. Clinical and BAL Characteristics*
* 

BW = bronchial wash sample.

 

Values are given as the mean (SEM).

 

Values are given as the geometric mean (log SEM).

§ 

Values are given as the median (IQR).

 

p < 0.05 (by analysis of variance).

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Lamkhioued, B, Renzi, PM, Abi-Younes, S, et al Increased expression of eotaxin in bronchoalveolar lavage and airways of asthmatics contributes to the chemotaxis of eosinophils to the site of inflammation.J Immunol1997;159,4593-4601. [PubMed]
 
Lilly, CM, Nakamura, HIDE, Belostotsky, OI, et al Eotaxin expression after segmental allergen challenge in subjects with atopic asthma.Am J Respir Crit Care Med2001;163,1669-1675. [PubMed]
 
Dent, G, Hadjicharalambous, C, Yoshikawa, T, et al Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum.Am J Respir Crit Care Med2004;169,1110-1117. [CrossRef] [PubMed]
 
Birring, SS, Parker, D, Brightling, CE, et al Induced sputum inflammatory mediator concentrations in chronic cough.Am J Respir Crit Care Med2004;169,15-19. [PubMed]
 
Gibson, PG Cough is an airway itch?Am J Respir Crit Care Med2004;169,1-2. [PubMed]
 
Carroll, NG, Mutavdzic, S, James, AL Distribution and degranulation of airway mast cells in normal and asthmatic subjects.Eur Respir J2002;19,879-885. [CrossRef] [PubMed]
 
Salvi, SS, Suresh, BK, Holgate, ST Is asthma really due to a polarized T cell response toward a helper T cell type 2 phenotype?Am J Respir Crit Care Med2001;164,1343-1346. [PubMed]
 
Hardaker, EL, Bacon, AM, Carlson, KARE, et al Regulation of TNF-α- and IFN-γ-induced CXCL10 expression: participation of the airway smooth muscle in the pulmonary inflammatory response in chronic obstructive pulmonary disease.FASEB J2004;18,191-193. [PubMed]
 
Bradding, P, Roberts, JA, Britten, KM, et al Interleukin-4, -5, and -6 and tumor necrosis factor-α in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines.Am J Respir Cell Mol Biol1994;10,471-480. [PubMed]
 
Dahl, ME, Dabbagh, K, Liggitt, D, et al Viral-induced T helper type 1 responses enhance allergic disease by effects on lung dendritic cells.Nat Immunol2004;5,337-343. [PubMed]
 
Miotto, D, Christodoulopoulos, P, Olivenstein, R, et al Expression of IFN-γ-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in T(H)1- and T(H)2-mediated lung diseases.J Allergy Clin Immunol2001;107,664-670. [CrossRef] [PubMed]
 
Bochner, B, Hudson, S, Xiao, H, et al Release of both CCR4-active and CXCR3-active chemokines during human allergic pulmonary late-phase reactions.J Allergy Clin Immunol2003;112,930-934. [CrossRef] [PubMed]
 
Liu, L, Jarjour, NN, Busse, WW, et al Enhanced generation of helper T type 1 and 2 chemokines in allergen-induced asthma.Am J Respir Crit Care Med2004;169,1118-1124. [CrossRef] [PubMed]
 
Pilette, C, Francis, JN, Till, SJ, et al CCR4 ligands are up-regulated in the airways of atopic asthmatics after segmental allergen challenge.Eur Respir J2004;23,876-884. [CrossRef] [PubMed]
 
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