Published online before print May 20, 2004
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Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, United Kingdom
1Correspondence: Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London, SW3 6LY, UK. E-mail: l.donnelly{at}imperial.ac.uk
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and neutrophil-activating peptide (NAP)-2 but not toward monocyte chemoattractant protein, interleukin-8, or epithelial-derived NAP(ENA)-78. The enhanced chemotactic response toward GRO and NAP-2 was not mediated by differences in expression of their cellular receptors, CXCR1 or CXCR2. Receptor expression studies using flow cytometry indicated that in COPD, monocyte expression of CXCR2 is regulated differently from nonsmokers and smokers, which may account for the enhanced migration toward GRO and NAP-2. The results highlight the potential of CXCR2 antagonists as therapy for COPD and demonstrate that an enhanced PBMC/monocyte response to specific CXC chemokines in these patients may contribute to increased recruitment and activation of macrophages in the lungs.
Key Words: GRO • NAP-2 • macrophages • chemotaxis
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Induced sputum from patients with COPD contains elevated levels of interleukin (IL)-8 [8
], growth-related oncogene (GRO), and monocyte chemoattractant protein (MCP)-1 [9
] when compared with the induced sputum from nonsmokers. These chemokines are also present in the BAL fluid of patients with COPD [9
, 10
]. Therefore, the increased levels of these chemokines may contribute to greater numbers of inflammatory cells in the airways of patients with COPD.
MCP-1 is a CC chemokine that mediates its effects via the CCR2 receptor, which is the specific receptor for MCP-1 and is expressed by monocytes, macrophages, and T lymphocytes [4 ] and is thought to mediate MCP-1 cellular effects [11 ], including recruitment of macrophages into the airway epithelium in COPD [4 ]. MCP-1 has been implicated in respiratory disease. BAL fluid from smokers with or without chronic bronchitis contained increased concentrations of MCP-1 when compared with BAL fluid from nonsmokers [12 ], suggesting that this chemokine might play a role in inflammatory cell recruitment associated with cigarette smoking. MCP-1 is an activating factor for monocytes and T lymphocytes and can act as a chemoattractant [4 ]. Moreover, it has been reported that MCP-1 can play a key role in monocyte migration [13 ], as monocytes are selectively attracted by specific chemokines that belong predominantly to the CC subfamily [14 ]. Therefore, increased levels of MCP-1 may contribute to the increase in macrophages, the product of monocyte differentiation, observed in the airways of patients with COPD.
IL-8 and GRO- are CXC chemokines that have broadly similar effects on leukocytes. IL-8 activates neutrophils via CXCR1 and CXCR2, and the latter appears to be the predominant receptor mediating its chemotactic response [15
]. Similarly, GRO, a structurally related chemokine, is a powerful activator of neutrophils with chemotactic ability for neutrophils and basophils [16
]. Other CXC chemokines include neutrophil-activating peptide (NAP)-2 and epithelial-derived NAP (ENA)-78. IL-8, NAP-2, GRO, and ENA-78 bind CXCR2 with high affinity, whereas IL-8 exhibits high affinity for CXCR1 and CXCR2 [17
, 18
]. NAP-2 and GRO have 
100-fold lower affinity for CXCR1 [17
]. This suggests that the effects of these chemokines are most likely mediated via CXCR2. IL-8 and ENA-78 are chemotactic for monocytes, and IL-8 exhibits significantly more monocyte chemotactic ability than ENA-78 [19
]. The presence of CXCR1 and CXCR2 receptors on monocytes [20
] suggests they are responsible for the observed chemotaxis toward IL-8 [19
].
The aim of this study was to determine whether there were any differences in expression of CXCR1, CXCR2, or CCR2 expression on leukocytes from patients with COPD compared with smokers and nonsmoking subjects and also to determine whether an increase in the chemotactic response of peripheral blood mononuclear cells (PBMC)/monocytes could contribute to the increase in macrophages observed in the airways of patients with COPD.
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, IL-8, NAP-2, ENA-78, and MCP-1 were purchased from R&D Systems (Abingdon, UK). TRI [receptor (R)-PE-Cy5] color-labeled anti-human CD8 and mouse IgG2a negative control were purchased from Dako (Ely, UK). Pharmlyse and FACSFlow were purchased from Becton Dickinson (Oxford, UK). Hydroxyethyl starch (eloHAES; 6% w/v) was purchased from Fresenius Ltd. (Basingstoke, UK). Percoll was purchased from Amersham Pharmacia Biotech (Little Chalfont, UK). Magnetic cell sorter (MACS) monocyte isolation kit and magnetic depletion columns were purchased from Miltenyi Biotec (Bisley, UK). Cellulose nitrate filters (8 µm pore size) were purchased from Neuroprobe (Receptor Technologies, Oxon, UK). Cy3-conjugated AffiniPure F(ab')2 fragment rabbit anti-mouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). SB468477 (combined CXCR1 and CXCR2 antagonist) and SB332235 (CXCR2 antagonist) were kind gifts from GlaxoSmithKline (King of Prussia, PA).
Subject selection
Patients with stable COPD were recruited from the outpatient department of the Royal Brompton Hospital (London, UK) and from local general practices. COPD subjects had a smoking history of at least 10 pack years. Inclusion criteria for entry were forced expiry volume in 1 s (FEV1)/forced vital capacity (FVC), ratio <0.7, and FEV1 percent predicted <70% reversibility with inhaled bronchodilators of <15% of predicted FEV1. All criteria were required for entrance into the study. Patients who had taken inhaled or oral steroids or who had suffered an exacerbation of their airway disease in the previous 6 weeks were excluded. Nonsmokers and smokers without evidence of airflow limitation (FEV1/FVC ratio, >70%; FEV1, >80% predicted normal) were recruited from within the department of Thoracic Medicine at the National Heart and Lung Institute (Imperial College London, UK). Inclusion criteria for nonsmokers included no history of respiratory or allergic disease, normal baseline spirometry as predicted for age, sex, and height, normal bronchial reactivity as evidenced by a provocation concentration(20) to methacholine of >64 mg/ml, no evidence of atopy on skin-prick testing to common aeroallergens (grass pollen, cat hair, house dust mite, or Aspergillus fumigatus), no smoking history, no history of upper respiratory tract infection in the preceding 6 weeks, and not taking any regular medication. Inclusion criteria for smokers were similar to nonsmokers, but they were required to have a smoking history of at least 10 pack years. All subjects gave written, informed consent, and the Royal Brompton and Harefield Hospital Ethics Committee (London, UK) approved the study.
Determination of chemokine receptor expression by flow cytometry
Leukocytes in whole blood from all three subject groups were analyzed for the expression of CXCR1, CXCR2, and CCR2. Individual cell types were identified using flurochrome-conjugated antibodies directed against specific phenotypic makers, i.e., monocytes; FITC-labeled anti-human CD14, neutrophils; FITC-labeled anti-human CD16, T lymphocytes; FITC-labeled anti-human CD4 and TRI (R-PE-Cy5) color-labeled anti-human CD8, B lymphocytes; and FITC-labeled anti-human CD19. Briefly, whole blood was incubated with FITC-labeled anti-human CD16, FITC-labeled anti-human CD19, or FITC-labeled anti-human CD4 and TRI-labeled CD8 and PE-labeled anti-human CXCR1, PE-labeled anti-human CXCR2, PE-labeled CCR2, or PE-labeled IgG2a isotype control for 30 min at 4°C in the dark. Cells where then incubated with Pharmlyse for 10 min at room temperature and washed twice with HBSS. The supernatant was removed, and the cells were resuspended in fluorescein-activated cell sorter (FACS) fix [FACSFlow supplemented with 0.5% (v/v) formaldehyde] for the samples used to determine neutrophil and T and B lymphocyte chemokine receptor expression. The samples used to determine monocyte chemokine receptor expression were then incubated with FITC-labeled anti-human CD14 for 20 min at 4°C in the dark. After incubation, the cells were washed and resuspended in FACS fix. Samples were analyzed on a FACScan (Becton Dickinson). For each determination, a minimum of 4000 specific events was analyzed. The data are expressed as relative fluorescence, which was calculated by the fluorescence values (mean channel) of the cells stained with CXCR1, CXCR2, or CCR2, divided by the fluorescence values (mean channel) for the IgG2a isotype control.
Isolation of PBMC
PBMC were isolated from whole blood from all subject groups by sedimentation on 6% (w/v) eloHAES, followed by centrifugation on a discontinuous Percoll gradient. After separation, the cells were counted and resuspended at 3 x 106 cells/ml in suspension buffer [RPMI-1640 medium supplemented with 0.5% (w/v) BSA].
Isolation of monocytes
Monocytes were isolated from the PBMC fraction, using a MACS monocyte isolation kit and magnetic depletion columns according to the manufacturer’s instructions. This yielded >90% monocytes, as determined by flow cytometry following labeling with FITC-labeled anti-CD14. After separation, the cells were resuspended at 3 x 106 cells/ml in suspension buffer.
Measurement of chemotaxis
Chemotaxis of PBMC/monocytes was measured using a 48-well micro-chemotaxis chamber by a modification of the methods of Wilkinson [21
] and Matsushima et al. [22
]. Chemokine diluted in suspension buffer was loaded in the lower compartment of the chemotaxis chamber. PBMC/monocyte suspension (3x106/ml) was loaded into the upper chamber with a cellulose nitrate filter (8 µm pore size) to separate the two compartments. The chamber was incubated at 37°C for 90 min, then the filter was removed, and the cells in the filter were fixed in 70% (v/v) ethanol. The cell nuclei were stained with hematoxylin, and the filter dehydrated with increasing concentrations of ethanol. The filters were left in xylene overnight and mounted onto glass slides. The leading front method of counting [21
] was used to determine cell migration into the filter.
Measurement of chemokinesis
PBMC were isolated from whole blood as described previously. Half of the PBMC were migrated in the presence of 1, 10, or 100 ng/ml of the chemoattractant of interest and the remainder in suspension buffer. PBMC resuspended in chemoattractant were allowed to migrate toward the same concentration of chemoattractant as that in which they were resuspended. PBMC that had been incubated with suspension buffer were allowed to migrate to suspension buffer, 1, 10, or 100 ng/ml chemoattractant and chemotaxis, performed as outlined previously.
Measurement of chemotaxis in the presence of antagonists
The monocytes were incubated with the combined CXCR1 and CXCR2 antagonist SB468477 [N-(2-hydroxy-3-dimethylsulfonylamido-4-chlorophenyl)-N'-(2-bromophenyl)-N''-cyanoguanidine], the CXCR2 antagonist SB332235 [N-(2-hydroxy-3-sulfamyl-4-chlorophenyl)-N'-(2,3-dichlorophenyl)urea], or dimethyl sulfoxide (DMSO), the vehicle control, for 1 h before chemotaxis. The monocytes were then allowed to migrate toward suspension buffer or the following chemokines: GRO, 10 ng/ml; IL-8, 10 ng/ml; NAP-2, 5 ng/ml; and ENA-78, 10 ng/ml.
CXCR2 receptor expression upon exposure to chemokines as analyzed by flow cytometry
Cell-surface expression of CXCR2 following agonist exposure was a modification of a previously published method of Feniger-Barish et al. [23
]. PBMC were resuspended at 4 x 106 cells/ml and kept on ice until required. The PBMC were incubated with no chemokine (unstimulated), vehicle (suspension buffer), or CXC chemokines (50 ng/ml) at 37°C for the times indicated. Following incubation, PBMC were placed on ice for 10 min and then washed with PAB [phosphate-buffered saline, pH 7.4, supplemented with 0.5% (w/v) BSA and 0.3% (w/v) sodium azide]. The cells were resuspended in PAB supplemented with 100-fold excess mouse IgG2a negative control. The PBMC were then incubated with monoclonal PE-labeled anti-human CXCR2 or PE-labeled mouse IgG2a isotype control for 30 min on ice in the dark. PBMC were then washed as before with PAB. The supernatant was removed, and the PBMC were incubated with FITC-labeled anti-human CD14 for 20 min on ice in the dark. After incubation, the PBMC were washed as before with PAB. The supernatant was removed and FACS fix added. The PBMC were kept at 4°C in the dark until required. Samples were analyzed as described previously. The results are expressed as receptor expression in arbitrary units (AU), calculated as the ratio of the fluorescence values (mean channel) of cells treated with ligand, divided by the fluorescence (mean channel) for the IgG2a isotype control, and the fluorescence values (mean channel) of nonstimulated cells divided by the mean channel fluorescence for the IgG2a isotype control. Receptor expression at 0 min is considered 100%.
Statistical analysis
GraphPad Prism (GraphPad Software, San Diego, CA) was used to perform all statistical tests. When the data were analyzed, nonparametric distribution was assumed; therefore, the Kruskal-Wallis test was used initially with Dunns’ post-test for ANOVA analysis. If this analysis was not feasible, nonparametric t-tests (Mann-Whitney test) were performed. Correlation coefficients were obtained using Spearman’s rank correlation. Results were considered significant when P < 0.05 (*P<0.05; **P<0.0l; ***P<0.001).
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Table 1. Demographic Data of Subjects
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Table 2. CXCR1, CXCR2, and CCR2 Expression on Inflammatory Cells
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and IL-8 (Fig. 1a
and 1c
). There was no difference in the effective concentration (EC)50 concentrations for GRO among the three subject groups, but the concentration of chemokine required to produce a maximal response was significantly increased in patients with COPD when compared with nonsmoking controls (Table 3
). There was no significant correlation between age and the concentration of GRO required to produce a maximal chemotactic response. There was no similar increase in the efficacy of IL-8 in patients with COPD; however, there was a significant increase in the EC50 concentration between smokers and patients with COPD (Table 3)
. Migration toward NAP-2 and ENA-78 was concentration-dependent (Fig. 1b
and 1d)
. There were no significant differences in the potency or efficacy of ENA-78 on PBMC migration among any of the subject groups (Table 3)
. Similar to GRO, there was a significant increase in the concentration of NAP-2 required to elicit a maximal response in PBMC from patients with COPD (Table 3)
. In addition, significantly more cells from patients with COPD migrated toward GRO when compared with nonsmokers at 0.1, 5, 50, and 100 ng/ml GRO (Fig. 1a ) and NAP-2 at 5, 10, 50, and 100 ng/ml NAP-2 when compared with smokers and 50 ng/ml when compared with nonsmokers and smokers (Fig. 1b)
. There was no difference in migration toward IL-8 and ENA-78 among the three subject groups (Fig. 1c
and 1d) . Also, there was no difference in baseline migration among the three subject groups. Similar results for GRO and ENA-78 were obtained with purified monocytes, confirming that the observed migration toward these chemokines is a result of monocytes and not T and B lymphocytes (Fig. 2
and Table 3
). Again, there was no correlation between age and the maximal concentration of GRO required to elicit a maximal chemotactic response. In contrast to the PBMC responses, there was no significant increase in the EC50 value for IL-8 when using purified monocytes (Table 3) . However, the enhanced migration toward NAP-2 of PBMC from patients with COPD was not seen when using purified monocytes (Fig. 2b)
; nevertheless. there was a significant increase in the maximal concentration of NAP-2 required for chemotaxis of monocytes from smokers and COPD patients compared with nonsmokers (Table 3)
. In contrast to GRO, there was a positive correlation between age and the maximal concentration of NAP-2 required to elicit a maximal chemotactic response of PBMC (r=0.4; P<0.01). However, this correlation was lost when chemotaxis experiments were performed using purified monocytes. This suggests that CD8-positive T lymphocytes may be contributing to the enhanced PBMC migration observed toward NAP-2.
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Figure 1. Concentration response of migrating PBMC to CXC chemokines. PBMC from nonsmokers, smokers, and patients with COPD migrated for 90 min toward (a) GRO, (b) NAP-2, (c) IL-8, and (d) ENA-78 or suspension buffer control. The number of cells present in the filter from 20 µm onward was counted. n = 8 nonsmokers ( ), n = 8 smokers (•), and n = 11 patients with COPD ( ; a and c), and n = 8–16 nonsmokers (), n = 8–16 smokers (•), and n = 10–21 patients with COPD (; b and d). Each point is expressed as the mean ± SEM. *, P < 0.05, COPD versus nonsmokers;  , P < 0.05, COPD versus smokers.
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Table 3. A Comparison of EC50 and Efficacy
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Figure 2. Concentration response of migrating monocytes to CXC chemokines. Monocytes from nonsmokers, smokers, and patients with COPD migrated for 90 min toward (a) GRO, (b) NAP-2, (c) IL-8, and (d) ENA-78 or suspension buffer control. The number of cells present in the filter from 20 µm onward was counted. n = 4 nonsmokers (), n = 3 smokers (•), and n = 3 patients with COPD (; a and c), and n = 3–8 nonsmokers (), n = 3–8 smokers (•), and n = 3–8 patients with COPD (; b and d). Each point is expressed as the mean ± SEM. *, P < 0.05, COPD versus nonsmokers; , P < 0.05, COPD versus smokers; #, P < 0.05, smokers versus nonsmokers.
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Figure 3. Concentration response of migrating PBMC and monocytes to MCP-1. (a) PBMC migrated for 90 min through a cellulose nitrate filter toward MCP-1 or suspension buffer control. The number of cells present in the filter from 20 µm onward was counted. n = 6 nonsmokers (), n = 6 smokers (•), and n = 3 patients with COPD (); each point is expressed as the mean ± SEM. (b) Monocytes were migrated in the same way as the PBMC. n = 8 nonsmokers (), n = 8 smokers (•), and n = 10 patients with COPD (); each point is expressed as the mean ± SEM.
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or IL-8 did not migrate toward GRO or IL-8 (Fig. 4a
and 4b
). In contrast, PBMC that had been incubated with suspension buffer migrated toward GRO and IL-8 (Fig. 4a
and 4b)
. Similarly, PBMC that were migrated in the presence of MCP-1 did not migrate toward MCP-1 (Fig. 4c)
. In contrast, cells that had been incubated in suspension buffer migrated toward MCP-1 (Fig. 4c)
. These data indicate that chemotaxis rather than chemokinesis is occurring and that the cells exhibit directed movement along a gradient of chemoattractant.
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Figure 4. Determination of chemokinesis or chemotaxis. Half of the PBMC was migrated in the presence of 1, 10, or 100 ng/ml of the chemoattractant of interest (), and the remainder was in suspension buffer (). PBMC resuspended in chemokine were allowed to migrate toward the same concentration of chemokine as that in which they were resuspended. PBMC were then allowed to migrate through a cellulose nitrate filter for 90 min toward (a) GRO, (b) IL-8, or (c) MCP-1 or suspension buffer control. The number of cells present in the filter from 20 µm onward was counted. Cells exposed to suspension buffer migrated in significantly higher numbers than those exposed to chemokine at all concentrations examined; P < 0.05. Each point is the mean ± SEM; n = 4.
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- and IL-8-mediated chemotaxis (Fig. 5a
and 5c)
but not NAP-2 and ENA-78 chemotaxis followed a biphasic response curve (Fig. 5b
and 5d)
. SB468477, 200 ng/ml, maximally inhibited migration to GRO, NAP-2, and ENA-78, with inhibition of 75%, 84%, and 84%, respectively. Maximal inhibition of 87% for IL-8 was achieved at 0.5 ng/ml SB468477. The vehicle [DMSO 0.04% (v/v)] had no effect on migration toward any chemokine investigated (Fig. 5a
5b
5c
5d)
. SB468477 had no effect on monocyte viability at any concentration used in this study, as measured by trypan blue exclusion (data not shown).
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Figure 5. Effect of a combined CXCR1 and CXCR2 antagonist SB468477 on CXC chemokine-directed monocyte migration. Monocytes that had been incubated with the indicated concentration of SB468477 or DMSO vehicle control for 1 h before chemotaxis migrated toward (a) GRO, 10 ng/ml (Control); (b) NAP-2, 5 ng/ml (Control); (c) IL-8, 10 ng/ml (Control); or (d) ENA-78, 10 ng/ml (Control). The number of cells present in the filter from 20 µm onward was counted. Each point is expressed as the mean ± SEM; n = 6–9. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Migration toward suspension buffer is represented by the dashed line (—).
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, IL-8, and ENA-78, with inhibition of 92%, 50%, and 73%, respectively. Maximal inhibition of 88% for NAP-2 was observed with SB332235, 1 ng/ml. The vehicle [DMSO 0.02% (v/v)] had no effect on migration toward any chemokine investigated (Fig. 6a
6b
6c
6d)
. SB332235 had no effect on monocyte viability at any concentration used in this study, as measured by trypan blue exclusion (data not shown).
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Figure 6. Effect of a CXCR2 antagonist SB332235 on CXC chemokine-directed monocyte migration. Monocytes that had been incubated with the indicated concentration of SB332235 or DMSO vehicle control for 1 h before chemotaxis migrated toward (a) GRO, 10 ng/ml (Control); (b) NAP-2, 5 ng/ml (Control); (c) IL-8, 10 ng/ml (Control); or (d) ENA-78, 10 ng/ml (Control). The number of cells present in the filter from 20 µm onward was counted. Each point is expressed as the mean ± SEM; n = 6–9. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Migration toward suspension buffer is represented by the dashed line (—).
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for up to 90 min did not alter CXCR2 expression significantly at any time-point examined (Fig. 7a
and 7b
). In contrast, monocytes from patients with COPD showed a significant decrease in CXCR2 expression at 10 min, followed by an increase at 60 min and another decrease at 90 min (Fig. 7c)
. There was also significantly less receptor expression on monocytes from patients with COPD when compared with expression on monocytes from nonsmokers and smokers at 90 min (Fig. 7d)
. Monocytes from all three subject groups did not show any difference in their chemotactic response toward IL-8. Similarly, there was no difference in the levels of CXCR2 expression on the cell surface of monocytes incubated in buffer from these three subject groups over the time-course examined (Fig. 8a
8b
8c
). Furthermore, there were no differences in the pattern of receptor expression over this time-course among the three subject groups (Fig. 8d)
.
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Figure 7. CXCR2 cell-surface expression following GRO exposure. Receptor expression on monocytes from (a) nonsmokers and (b) smokers did not change over time when the cells were stimulated with GRO. Receptor expression on monocytes from (c) patients with COPD significantly decreased at 10 min, increased at 60 min, and then decreased at 90 min. (d) There was significantly less receptor expression on monocytes from patients with COPD when compared with expression on monocytes from nonsmokers and smokers at 90 min. Each point is expressed as mean receptor expression ± SEM; n = 6 nonsmokers (), n = 6 smokers (•), and n = 6 patients with COPD (). *, P < 0.05, versus receptor expression at 0 min; ##, P < 0.01, versus receptor expression at 10 min; , P < 0.05, versus receptor expression at 60 min;  , P < 0.05, versus receptor expression on monocytes from nonsmokers and smokers.
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Figure 8. CXCR2 cell-surface expression following IL-8 exposure. Receptor expression on monocytes from (a) nonsmokers, (b) smokers, and (c) patients with COPD did not change over time when the cells were stimulated with IL-8. (d) There was no difference in receptor expression among the three subject groups. Each point is expressed as mean receptor expression ± SEM; n = 6 nonsmokers (), n = 6 smokers (•), and n = 6 patients with COPD ().
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Migration of PBMC and monocytes toward MCP-1 was similar from all three subject groups. There was a tendency of PBMC from patients with COPD to migrate in higher numbers than PBMC from control subjects; however, this failed to reach significance. Similarly, there were no concomitant differences in the potency or efficacy of MCP-1 to modulate chemotaxis, with the exception of an increased EC50 for monocytes from smokers. A comparison of the chemotactic response of PBMC and monocytes toward MCP-1 indicates that PBMC are more responsive than monocytes. This suggests that T and B lymphocytes may be contributing to the response of PBMC. One possibility is that lymphocytes may also be migrating toward MCP-1 and thus increasing the chemotactic response, although expression of CCR2 on these cells is low. Alternatively, lymphocytes might facilitate monocytic migration toward MCP-1 by priming monocytes, although the mechanism of action is unknown. It is possible that such priming of monocytes may render the cells more responsive to lower concentrations of MCP-1 and thereby shift the concentration of MCP-1 required for maximum migration to the left compared with purified monocytes. Elevated levels of MCP-1 are found in induced sputum from patients with COPD when compared with sputum from smokers and nonsmokers; however, the concentration of MCP-1 is 1 ng/ml [9
]. This concentration is lower than that required to elicit a maximum chemotactic response in monocytes and PBMC. MCP-1 and related CCR2 agonists are considered the dominant chemoattractant for monocytes, as a greater number of CCR2 receptors are expressed on the cell surface. It is possible that MCP-1 may be more important in activating the monocytes once they have entered the airways, thus only having a minor role in recruiting the monocytes into the airways; however, there is currently no evidence of this in COPD.
PBMC and monocytes from all three subject groups migrate toward the CXC chemokines, GRO, NAP-2, IL-8, and ENA-78. Other workers [19
] have reported migration of monocytes toward IL-8 and ENA-78. However, the observation that GRO and NAP-2 are also chemotactic for PBMC/monocytes conflicts with other studies that indicated that these chemokines are not chemotactic for monocytes [27
, 28
]. The conflict within the literature regarding monocyte migration toward CXC chemokines is unexpected, as the receptors for these chemokines are present on monocytes; however, this discrepancy may be a result of differences in the techniques used to measure chemotaxis.
PBMC from patients with COPD migrate in higher numbers toward GRO and NAP-2 but not toward IL-8 or ENA-78 when compared with nonsmokers and smokers. Similar data were obtained with purified monocytes, with the exception of migration toward NAP-2. Although PBMC from patients with COPD migrated in higher numbers toward NAP-2, purified monocytes did not. As the patients with COPD are significantly older than either of the two control groups, it is possible that age rather than disease status may contribute to altered migratory capacity of monocytes. However, this is unlikely, as there is no correlation of chemokine receptor expression with age or of responsiveness to GRO of PBMC or purified monocytes. There was a positive correlation between age of subject and the response to NAP-2 of PBMC but not monocytes. Taken together, these results indicate that the enhanced migration observed with PBMC toward NAP-2 may be a result of migration of T lymphocytes, as CXCR2 is expressed on the surface of 10% of T lymphocytes. Again, it is possible that the lymphocytes might facilitate monocytic migration toward NAP-2 by priming monocytes. The reason why these differences occur with NAP-2 and not other CXCR2 chemokines is unknown. Previous studies have shown that neutrophils from patients with COPD migrate in higher numbers toward IL-8 when compared with nonsmokers [29
] and that neutrophils from patients with emphysema show enhanced migration toward N-formyl-Met-Leu-Phe [30
]. In contrast to the neutrophil studies [29
, 30
], the levels of baseline migration for PBMC/monocytes were similar among the three subject groups, indicating that PBMC/monocytes are not already activated in COPD but need to be actively recruited into the airways. The study presented herein suggests that there are several mechanisms involved in recruitment of different leukocyte populations in COPD. In particular, the lack of enhanced migration by PBMC/monocytes toward MCP-1 together with no change in baseline migration indicate that the effect of GRO and NAP-2 is receptor-agonist interaction-dependent rather than cell type-dependent.
The present study indicates that in COPD PBMC/monocytes can be recruited in higher numbers in response to GRO and NAP-2, even if they are present at low concentrations. Both chemokines elicited significant increases in the concentration required to produce the maximal chemotactic response for cells derived from COPD patients. This was not apparent with IL-8 or ENA-78. This suggests that at higher concentrations of GRO and NAP-2, which may be refractory for cells derived from nonsmokers and smokers, the cells from patients with COPD continue to migrate and in greater numbers. This has relevance to the disease, as GRO is elevated in the induced sputum from these patients [9
], and the levels of GRO present in induced sputum from patients with COPD (30 ng/ml) [9
] are chemotactic for monocytes. Therefore, this chemokine may be important for the accumulation of macrophages and hence, the pathogenesis of COPD, as monocytes are the precursor cells for macrophages [31
]. Taken together, these data indicate that inflammatory cells in COPD have a heightened chemotactic response with elevated levels of GRO and IL-8 in the airways of patients with COPD leading to increased macrophage and neutrophil numbers in the lungs of these patients. Chemokines such as MCP-1 contribute to the priming and increased expression of CD43 and CD11b receptors on monocytes found in peripheral blood of patients with COPD. This priming of the monocytes results in their migration to inflammatory loci such as the lung. However, the risk of irregular migration of monocytes to tissue sites outside of the inflammatory loci is also increased, and monocytes then participate in pathogenic tissue responses, leading to systemic effects such as the muscle wasting and weight loss associated with COPD [32
]. Monocytes have been shown to have a role in heart disease and atherosclerosis, which are increased in patients with COPD when compared with control subjects [32
], indicating that the cellular inflammation associated with COPD is not confined to the lungs. A systemic effect of low-grade bronchitis, which is associated with COPD, might also prime chemokine receptors, leading to enhanced responses. However, there was no up-regulation of chemokine receptors on the surface of any of the cell types examined from patients with COPD when compared with the control groups. It is also possible that some of the subjects in the smoking control group could be suffering from low-grade bronchitis; therefore, if this condition resulted in "priming" of the receptor responses, cells from these subjects should also demonstrate enhanced migration toward the CXC chemokines.
Monocyte migration toward the CXC chemokines GRO, NAP-2, IL-8, and ENA-78 is mediated via CXCR1 and CXCR2. When using a combined CXCR1 and CXCR2 antagonist, a biphasic response curve was observed for GRO and IL-8 chemotaxis, indicating that one receptor is inhibited at low concentrations of the antagonist and the other or both, at higher concentrations of the antagonist. As the inhibitory concentration (IC)50 values for the antagonist at CXCR2 (IC50, 12 nM, 
5.7 ng/ml) and CXCR1 (IC50, 67 nM, 31.7 ng/ml; Mary Barnette, GlaxoSmithKline, King of Prussia, PA, personal communication), it would indicate that at low concentrations of the antagonist, CXCR2-mediated chemotaxis is inhibited, and higher concentrations of the antagonist inhibited CXCR1 or both receptors. The specific CXCR2 antagonist (IC50, 19 nM, 8.2 ng/ml; Barnette, personal communication) proved more effective at inhibiting migration toward GRO and NAP-2, suggesting that migration toward these chemokines is mediated predominantly via CXCR2. This concurs with the current literature, indicating that these chemokines bind CXCR2 with high affinity and CXCR1 with low affinity [17
]. These studies also demonstrated that NAP-2 induced two optima of neutrophil chemotaxis [17
]. The first was elicited within a low concentration range (7.6–100 ng/ml), as used in the present study; whereas the second occurred at concentrations more than 200-fold higher (760–7600 ng/ml). Their results indicate that CXCR1 and CXCR2 are involved in NAP-2-induced neutrophil chemotaxis, CXCR2 renders the cells responsive to low concentrations of NAP-2, and CXCR1 extends their responsiveness to much higher concentrations [17
].
The increased migration of PBMC/monocytes from patients with COPD toward GRO and NAP-2 is not a result of an increase in CXCR1 or CXCR2 expression. However, there is a difference in CXCR2 expression on monocytes from patients with COPD but not cells from smokers and nonsmokers when exposed to GRO. The most noticeable difference was the decrease in expression of CXCR2 within 10 min of GRO exposure followed by a significant cell-surface increase. Moreover, such changes in CXCR2 expression could not be observed following exposure to IL-8. Discrepancies in the response of CXCR2 to different agonists have been suggested by the observation that IL-8, NAP-2, and GRO bind to different amino acid residues within the receptor, leading to intracellular Ca2+ responses [33
]. Moreover, interaction of IL-8 and NAP-2 with CXCR2 leads to differential phosphorylation of the receptor [34
]. This may be important, as attenuation of chemotaxis is mediated by chemokine-induced receptor phosphorylation [34
], and phosphorylation/dephosphorylation of G protein-coupled receptors may allow receptor recycling and/or desensitization [23
]. It is possible, therefore, that in cells from patients with COPD, the enhanced migratory response to GRO and NAP-2 is mediated via an alteration in the desensitization of the cell-surface receptor. It is unlikely that new receptors are being synthesized and transported to the cell surface, as cyclohexamide has been shown to have no effect on CXCR1 or CXCR2 recycling in human embryonic kidney 293 cells transfected with the receptors after exposure to agonists for 2 h [23
].
In conclusion, this study demonstrates that specific CXC chemokines result in enhanced migration PBMC/monocytes from patients with COPD, which is mediated via CXCR1 and CXCR2. This increase in migration toward GRO and NAP-2 but not IL-8 and ENA-78 may be a result of differences in CXCR2 recycling. This study demonstrates clearly that despite binding to the same receptor, different agonists can elicit quite different responses. However, it may be a result of divergent intercellular signaling pathways, resulting from different binding sites on the receptor for the chemokines, or alternatively, the manner in which the receptors dimerize upon activation might also be responsible for the observations. At present, there are no treatments that can halt the relentless progression of COPD [7
], and current thinking is that more efficient therapies for the treatment of inflammatory diseases would involve the prevention of excessive recruitment of specific leukocyte populations into the airways by antagonism of specific chemokine receptors [24
]. The antagonists used herein could therefore be considered as potential therapeutic agents. This would allow the "correct" level of inflammatory cells to migrate into the airways so that controlled trafficking of cells into the airways could be resumed in COPD.
Received October 21, 2003; revised April 16, 2004; accepted April 17, 2004.
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in induced sputum from patients with chronic obstructive pulmonary disease or asthma Am. J. Respir. Crit. Care Med. 153,530-534[Abstract] induces T lymphocyte chemotaxis. A process regulated via IL-8 receptors by IFN-
, TNF-, IL-4, IL-10, and IL-13 J. Immunol. 155,5359-5368[Abstract], GRO ß, and GRO activate human neutrophil and basophil leukocytes J. Biol. Chem. 268,15419-15424 bind to and elicit cell activation via specific and different amino acid residues of CXCR2 Cytokine 12,1480-1488[CrossRef][Medline]This article has been cited by other articles:
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|
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|
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|
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|
|
|
|
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|
|
|
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|
|
|
|
 |
|
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|
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|
|
|
|
 |
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|
|
|
|
 |
|
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|
|
|
|
 |
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
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