Abstract
We have applied immunohistology and in situ hybridization to bronchial biopsies of patients with chronic obstructive pulmonary disease (COPD) to examine neutrophil recruitment and to determine neutrophil chemoattractant and CXC receptor (CXCR) 1 and CXCR2 gene expression associated with acute severe exacerbations. Cells were counted in endobronchial biopsies of (1) patients with COPD intubated for exacerbations (E-COPD; n = 15), (2) those with COPD in a stable phase of their disease (S-COPD; n = 7), and (3) nonsmoker surgical control subjects intubated for a nonrespiratory surgical procedure (n = 15). In comparison with the nonrespiratory surgical procedure and S-COPD groups, neutrophilia and gene expression for epithelial-derived neutrophil attractant-78 (CXCL5), interleukin-8 (CXCL8), CXCR1, and CXCR2 were each upregulated in the E-COPD group (p < 0.01); compared with the S-COPD group, by 97-, 6-, 6-, 3-, and 7-fold, respectively (p < 0.01). In E-COPD, there was a significant positive association between the number of neutrophils and CXCR2 mRNA–positive cells (r = 0.79; p < 0.01) but not between the number of neutrophils and CXCR1 mRNA–positive cells. At the time of sampling of the mucosa, there was no association between neutrophil number and either the length of intubation or viral infection. Thus, in COPD, in addition to CXCL8 and CXCR1, CXCL5 and CXCR2 appear to play important roles in the airway neutrophilia characteristic of severe exacerbations.
It has been estimated that 44 million people currently suffer from chronic obstructive pulmonary disease (COPD) worldwide, and it is predicted that by 2020 it will become the third leading cause of death, up from its current sixth place (1). As lung function worsens, patients with COPD become increasingly prone to exacerbations, a major reason for hospital admission and a relatively large component of health care cost (2).
Pathologically, COPD is characterized by chronic inflammation and remodeling throughout the conducting airways, parenchyma, and pulmonary vasculature (3–5). In stable COPD, there is a characteristic infiltration of the bronchial mucosa with increased numbers of CD8+ T lymphocytes and macrophages (6–9), but not neutrophils (6). However, in an exacerbation in mild COPD and in severe disease there is an increase in the number of neutrophils and their markers in the airways (10–18). Also, in exacerbations in mild COPD, there is an accumulation of eosinophils in the mucosa (10), likely due to the reported upregulation of the eosinophil chemoattractant regulated upon activation, normal T cell expressed and secreted, whose effects may be mediated through chemokine receptor (CCR) 3 (19).
The chemoattractants and receptors influencing the recruitment and accumulation of tissue neutrophils in exacerbations of COPD, and how these changes relate to infection and symptoms, are poorly understood (16, 17, 20).
Chemoattraction of neutrophils is thought to occur via the production of a number of neutrophil-selective chemokines including growth-related protein-α, -β, and -γ (CXCL1–3), epithelial-derived neutrophil attractant-78 (CXCL5), human granulocyte chemotactic protein-2 (CXCL6), neutrophil-activating peptide-2 (CXCL7), and interleukin-8 (CXCL8) (20–25). Each of the aforementioned belongs to the same CXC chemokine family, characterized by separation of the conserved cysteines by a single amino acid and containing the Glu-Leu-Arg amino acid motif of glutamic acid-leucine-arginine and the designated gene SCYb (small secreted cytokine) (23, 26). Among them, CXCL8 has been relatively well characterized as a neutrophil chemoattractant and activating cytokine for neutrophils and to a lesser extent for eosinophils (20, 24, 26). Epithelial cells, endothelial cells, fibroblasts, alveolar macrophages, and neutrophils themselves are able to release CXCL8 in response to proinflammatory molecules such as tumor necrosis factor, IL-1, and endotoxin (20, 22).
In contrast, the roles of other CXC chemokines in airway inflammation have been less well studied. Imaizumi and coworkers examined CXCL5 expression in cultured human endothelial cells stimulated with IL-1α: these authors reported that CXCL5 and CXCL8 share certain features of regulation and have overlapping biological activities, including the capacity to induce neutrophil adhesiveness (25). There are no published biopsy data on the relative expression and upregulation of these two neutrophil chemoattractants in the bronchial mucosa of patients with COPD, and none in exacerbations. Moreover, although data have been published on CXCR3 expression (27), there has been no previous report in COPD of tissue CXCR1 and CXCR2 expression, the relevant receptors on which these neutrophil chemoattractant ligands act (21).
Our premise for the present study was that an improved understanding of the mechanisms responsible for initiating and maintaining the increased neutrophilia in acute exacerbations may lead to more effective control and prevention of such exacerbations, the frequency of which is associated with accelerated FEV1 decline (16, 28, 29). Accordingly we present the results of localization and quantification of neutrophils recruited to the airway mucosa, and the numbers of cells expressing genes for CXCL5, CXCL8, CXCR1, and CXCR2 in critically ill patients requiring intubation for the management of acute severe exacerbations of COPD. We have compared the counts with those of patients intubated in the course of nonrespiratory elective surgery and with bronchial biopsies of patients with mild stable COPD. Some of the results of this study have been reported previously in abstract form (30, 31).
Three groups of subjects (see Table 1)
NSC | S-COPD | E-COPD | |
|---|---|---|---|
| n | 15 | 7 | 15 |
| Age, yr* | 47 ± 1.7 | 55 ± 3.9 | 62 ± 2.4† |
| Sex, male/female | 2/13 | 2/5 | 7/8 |
| FEV1% predicted | 98 ± 1.6 | 51 ± 3.3 | 36 ± 2.4‡ |
| FEV1/FVC, % | 79 ± 0.8 | 53 ± 3.7 | 35 ± 3.3‡ |
Biopsies from third- to fifth-order bronchi were fixed immediately in 10% formaldehyde and processed to paraffin wax.
Haemophilus influenzae in bronchial biopsies was detected by immunohistostaining and hybridization techniques described in a previous publication (32). The methods for detection and identification of respiratory tract viruses have been previously described by Atmar and coworkers (33).
Mouse monoclonal antibodies, raised against human neutrophil intracellular elastase (Ne) (M0725; Dako, Ely, UK), were applied to the tissue sections and the results were visualized by a validated EnVision–alkaline phosphatase technique (Dako).
Riboprobes were applied to detect intracytoplasmic mRNA for chemokines CXCL5 and CXCL8 and for receptors CXCR1 and CXCR2 (19, 34). The sizes (base pairs [bp]) and vectors of the probes were as follows: CXCL5 (220 bp; PBSIISK-), CXCL8 (246 bp; PGEM-3Z), CXCR1 (1.1 kbp; PBSIISK-), and CXCR2 (1.1 kbp; PBSIISK-). Sense probes were used as appropriate negative control subjects.
The areas of subepithelium, excluding muscle, gland, and large vessel, were measured. Immunopositive and in situ hybridization-positive cells were counted at ×200 magnification. An eyepiece graticule was used to “point count” and assess the percentage of epithelium expressing mRNA for CXCL5 (35).
The Mann–Whitney U test was applied to nonnormally distributed data to test for differences in area profile cell counts between E-COPD, S-COPD, and NSC groups and between the E-COPD subgroups with and without viral infection. The Student t test was used for the analysis of point count data and epithelial CXCL5 expression. The Spearman rank correlation test was used to determine the association between parameters and to examine that between length of intubation and number of tissue neutrophils in the E-COPD group. A p value less than 0.05 was accepted as statistically significant in the Mann–Whitney U test and t test. For the Spearman rank correlation, a Bonferroni correction for multiple comparisons was applied and a p value less than 0.02 was used as the threshold for statistical significance. See the online supplement for additional detail on the methods used in this study.
The characteristics of the subjects and patients examined are shown in Table 1. There was no significant difference in age between the two COPD groups. However, the ages of the patients in both COPD groups were greater than those of patients in the NSC group (p < 0.05). Both the FEV1% of predicted and FEV1/FVC values of the COPD groups was significantly lower than those of the NSC group (p < 0.01). As expected, the lowest FEV1% of predicted and FEV1/FVC values were found among the patients with E-COPD. The most recently obtained outpatient results of pulmonary function tests, obtained within 120 days of the exacerbation, were used for these data. Thirteen of the 15 biopsies tested from patients with acute severe exacerbations of COPD, but none from the control subjects, were positive for nontypeable Haemophilus influenzae detected by monoclonal antibody (32). Seven of the patients with E-COPD (i.e., 47%) were positive for acute viral infections, as demonstrated by viral cultures or polymerase chain reaction. No virus was isolated from either the NSC or S-COPD group.
Ne-positive cells were seen in both the epithelium and subepithelial tissue (Figure 1)
Figure 1. Neutrophil elastase positivity in the airway mucosa of a patient with a severe exacerbation of chronic obstructive pulmonary disease (COPD). Original magnification, ×200.
[More] [Minimize]Group | Ne-Positive* | Ne-Positive† | CXCL8-Positive† | CXCL5-Positive† | CXCR1-Positive† | CXCR2-Positive† |
|---|---|---|---|---|---|---|
| NSC (n = 15) | 0 (0–0.1) | 0 (0–7) | 7 (0–45) | 54 (7–151) | 22 (0–83) | 14 (0.4–95) |
| S-COPD (n = 7) | 0 (0–0.1) | 1 (0–7) | 10 (0–60) | 42 (14–165) | 33 (5–253) | 12 (0–29) |
| E-COPD (n = 15) | 0.3 (0–8)‡ | 97 (1–594)‡ | 62 (0–242)§ | 250 (76–940)‡ | 92 (12–344)‡ | 84 (27–591)‡ |
In dividing the E-COPD group into those with and without detectable virus infection, there appeared to be more neutrophils in those with virus (n = 6, median = 196/mm2 subepithelium and 0.36/0.1 mm2 epithelium) than without (n = 7, 68/mm2 subepithelium and 0.25/0.1 mm2 epithelium), but this was due to a particularly high count in one patient and the between-group differences were not significant statistically (p = 0.20).
Whereas the sense probes for CXCL5, CXCL8, CXCR1, and CXCR2 mRNA were negative (Figure 2)
Figure 2. Sense control for the CXC receptor (CXCR) 2 gene probe, showing the absence of gene expression. Original magnification, ×200.
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Figure 3. Cells positive for CXCL8 messenger RNA (mRNA) in the airway mucosa of (A) a patient with stable COPD (S-COPD) and (B) a patient with a severe exacerbation of COPD (E-COPD). There are more CXCL8-positive cells and the intensity of expression is greater in (B). Original magnification, ×200.
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Figure 4. Cells positive for CXCL5 mRNA in the airway mucosa of (A) a patient with S-COPD and (B) a patient with a severe exacerbation. The number of positive cells and the intensity of expression are far greater, relative to CXCL8, in both the epithelium and subepithelium and greater than that seen in S-COPD. Original magnification, ×200.
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Figure 5. Cells positive for CXCR2 mRNA in the airway mucosa of (A) a patient with S-COPD and (B) a patient with an exacerbation. The latter shows more positive cells and greater intensity of gene expression than the former. Original magnification, ×200.
[More] [Minimize]In the subepithelium, the counts for CXCL5, CXCL8, CXCR1, and CXCR2 mRNA-positive cells are shown in Table 2 and Figures 6A–6D
Figure 6. (A) Counts of CXCL8 mRNA–positive cells in the subepithelium of biopsies taken from intubated nonsmoker surgical control subjects (NSC), patients with S-COPD, and those intubated for severe E-COPD. Median values are indicated. (B) Counts of CXCL5 mRNA–positive cells in the subepithelium of NSC, patients with S-COPD, and those with severe E-COPD. (C) Counts of CXCR1 mRNA–positive cells in the subepithelium: NSC, patients with S-COPD, and those with severe E-COPD. (D) Counts of subepithelial CXCR2 mRNA–positive cells: NSC, patients biopsied with S-COPD, and those with severe E-COPD.
In comparing the numbers of positive cells for CXCL5, CXCL8, CXCR1, and CXCR2 mRNA in each of the three study groups, the number of CXCL5 mRNA-positive cells was greater than any other. In the NSC group, the number of CXCL5-positive cells was about eight-, three-, and fourfold higher than those of CXCL8-, CXCR1-, and CXCR2-positive cells (p < 0.05). In S-COPD this pattern remained but the differences were not significant statistically. In the E-COPD group the number of CXCL5-positive cells was approximately three times higher than any other parameter (p < 0.05).
The numbers of subepithelial CXCL5, CXCL8, and CXCR2 mRNA-positive cells were similar in the patients with E-COPD with and without evidence of viral infection. There were, however, significantly fewer CXCR1 mRNA-positive cells in the virally infected subjects than in those without infection (median, 53 versus 222 cells per mm2 subepithelium, respectively; p < 0.05).
There were no significant associations between the numbers of Ne-positive cells and the numbers of cells expressing CXCL5, CXCL8, CXCR1, and CXCR2 in either the NSC or S-COPD group. However, among the patients with E-COPD there were significant and positive associations between Ne-positive cells and cells with mRNA positive for CXCL5, CXCL8, and CXCR2 (Spearman rank correlation: r = 0.61, 0.59, and 0.79, respectively; p < 0.02 for each). There was also a significant correlation between CXCL8-positive cells and CXCR1-positive cells (r = 0.69, p < 0.01; Figure 7)
Figure 7. Correlation between CXCL8 mRNA–positive cells and CXCR1 mRNA–positive cells in the subepithelium of patients intubated for severe E-COPD.
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Figure 8. Correlation between CXCL5 mRNA–positive and CXCR2 mRNA–positive cells in the subepithelium of patients with severe E-COPD.
[More] [Minimize]We have used endobronchial biopsies and counted neutrophils and cells expressing the genes for neutrophil chemoattractants and their complementary receptors in patients with COPD hospitalized for severe exacerbations. The results show that compared with patients with stable COPD (S-COPD) and also with nonsmoker control subjects (NSC), patients with COPD with severe exacerbations (E-COPD) have significantly increased numbers of neutrophils and that this is associated with increases in CXCL5, CXCL8, and CXCR2 but not in CXCR1 expression. Moreover, the increased numbers of neutrophils in E-COPD were not associated with length of intubation. To our knowledge, this is the first study of these tissue parameters in COPD in relation to an acute severe exacerbation.
Previously published studies demonstrate that neutrophils form a major component of the inflammatory infiltrate in exacerbations of COPD (10, 13, 15) and when disease is severe (11, 18). It has been hypothesized that neutrophils are stimulated, activated, and recruited through upregulation and binding of a number of CXC chemokines to their complementary receptors, notably CXCR1 and CXCR2 (21, 36–38). Until now, this has not been tested by direct examination of airway tissues in exacerbations of COPD and our results provide supportive evidence of a role for these two neutrophil chemoattractants and major receptors in exacerbation.
It has been considered, in neutrophil chemotaxis, that neutrophil cell surface CXCR1 is highly selectively bound and activated by CXCL8 whereas CXCR2 responds not only to CXCL8 but also to other chemoattractants including CXCL1, CXCL5, and CXCL7 (36, 38). In a previous study of the intrapulmonary airway mucosa in lungs resected for tumor, smokers with stable COPD showed upregulation of epithelial CXCL8 (39). Our study, of subepithelial CXCL8, in stable disease, does not. However, we confirm the previously reported lack of correlation between neutrophil numbers and CXCL8 expression. We report additional data to show that, in stable disease, there is also no significant correlation between neutrophilia and subepithelial CXCL5 expression, another putative neutrophil chemoattractant. In contrast in the biopsies of patients with a severe exacerbation of COPD, we do demonstrate positive correlations between neutrophils and cells positive for both CXCL8 mRNA (Spearman rank correlation: r = 0.59; p = 0.02) and CXCL5 mRNA (r = 0.61; p = 0.02). Moreover, our findings in the bronchial mucosa of all groups demonstrate that CXCL5 rather than CXCL8 is the dominant CXC chemoattractant.
CXCL5 is a 78-residue chemokine originally isolated from the supernatant of a lung Type II alveolar epithelial cell line stimulated by IL-1, tumor necrosis factor-α, and by both viable and heat-killed Mycoplasma hominis (40–42). Our results demonstrate that CXCL5 is upregulated in exacerbations of COPD. In addition to epithelial cells, monocytes, neutrophils, fibroblasts, endothelial cells, platelets, and tumor cells have also been demonstrated to be a source for CXCL5 (26). The intensity of expression of epithelial CXCL5 mRNA indicates the epithelium is clearly one important source of this chemokine in exacerbations of COPD and, once released, CXCL5 may target neutrophils, likely stimulating their epithelial chemotaxis by binding CXCR2 (43). Several studies support a role for CXCL5 in neutrophil chemotaxis (44, 45). Other roles for CXCL5 (e.g., as an angiogenic factor in pulmonary fibrosis or non-small cell lung cancer) are likely to emerge in future (46, 47).
The receptor CXCR1 is thought to be activated by CXCL8 and is a major receptor responsible for chemotaxis, superoxide production, and phospholipase D activation in response to CXCL8 (26, 37). We show that the numbers of CXCR1 and CXCR2 mRNA–positive cells in the bronchial mucosa of patients with stable COPD were similar. However, the extent of upregulation and the intensity of CXCR2 mRNA expression in E-COPD are considerably greater than that of CXCR1. Moreover, the increase in the number of neutrophils is significantly and positively associated with increased expression of CXCR2 (r = 0.79; p < 0.02) and not with CXCR1. As CXCR2 may also be expressed by activated T lymphocytes, mast cells, dendritic cells, macrophages, basophils, and eosinophils, these cells may also be recruited to the mucosa via binding of CXCR2. Overall, the data we report are supportive of major roles for both CXCL5 and CXCR2 in controlling tissue neutrophilia in severe exacerbations of COPD. Our results provide data additional to that published for CXCR3 and CXCL10 in the inflammation of COPD (27).
Finally, we faced several difficulties in the interpretation of our results, for example: (1) age, (2) the undetermined extent of the contribution to neutrophilia made by intubation and mechanical ventilation per se, (3) the effect of steroid treatment, (4) the role of infection, and (5) the numbers of neutrophils in the S-COPD group. Taking each in turn: (1) The patients with E-COPD were significantly older in comparison with the NSC group. We attempted to reduce this difference by exclusion of all patients below the age of 40 years. Also, we consider that the differences in inflammatory indices for the exacerbated group are likely not due to age alone, as they are controlled for by the S-COPD group, which was of similar age to the E-COPD group; (2) the lag periods between initiation of the intubation procedure and biopsy differed between the NSC and E-COPD groups: in the former, biopsies were taken within minutes and in the latter at times up to 26 hours after the start of intubation. There have been no studies that investigate the effect of intubation time on neutrophil numbers in COPD. However, in asthma there have been some weak and variable associations reported between time of intubation and neutrophilia and these have been debated (48–50). We found no association between the length of intubation and the increase of neutrophil numbers, either in the entire E-COPD group or in a subset with relatively high numbers of neutrophils; (3) it has been reported that there are increased numbers of neutrophils in bronchial biopsies of patients receiving long-term high-dose treatment with glucocorticoids (at least in severe asthma) and that the effect is mediated via reduction of neutrophil apoptosis (51–53). In our study of COPD, only three patients in the E-COPD group had received treatment with long-term low-dose inhaled steroids. The counts of neutrophils in their biopsies showed that the numbers, although increased with respect to S-COPD and NSC control subjects, were less than those of subjects who had not received steroid treatment. It is, therefore, unlikely that the increased number of neutrophils we observed was related to steroid treatment; (4) infection is considered a major determinant of airway neutrophilia in exacerbations of COPD and asthma (13, 29, 54). In the present study, 13 of the 15 subjects in the E-COPD group were positive for nontypeable Haemophilus influenzae and 7 (47%) were positive for acute viral infections. The study by Seemungal and co-workers demonstrates that 40% of acute exacerbations in COPD are associated with respiratory viral infection (29). However, we compared neutrophil numbers in the E-COPD group with and without positive viral culture and found no significant differences. These findings agree with a study of 14 patients by Aaron and co-workers (13), who reported that markers of neutrophilic inflammation increase in the airways of patients with an acute exacerbation of COPD, yet the response occurs independently of a demonstrable viral or bacterial infection. However, our own data do not exclude the influence of bacterial infection and viral infection (such as rhinovirus) that may have been present before the time of sampling; and (5) finally, the number of neutrophils present in our S-COPD group was low (range, 0–7 neutrophils per mm2) compared with several European studies of patients with stable COPD reported in the literature, including our own (e.g., range = 0–241 neutrophils per mm2) (55). However, we confirmed the present results by restaining sections. As both the U.S. and European patients we have previously studied had COPD of similar severity, the reason for the differences in neutrophil counts is presently unclear. This encourages us to look more closely for an explanation of such regional differences in future.
In conclusion, the present study of bronchial biopsies taken in a critical care setting demonstrates that in severe exacerbations of COPD there is increased neutrophil recruitment and that this is associated with upregulation of both CXCL5 and CXCL8. Upregulation of the gene for CXCL8 correlates well with that for CXCR1 whereas CXCL5 is better associated with CXCR2 gene expression. We have highlighted putative novel roles for CXCL5 and CXCR2 in neutrophil recruitment in severe exacerbations of COPD, improving our understanding of the mechanisms responsible for the associated tissue neutrophilia.
Y.Q. has no declared conflict of interest; J.Z. has no declared conflict of interest; V.B. has no declared conflict of interest; R.L.A. has no declared conflict of interest; K.H. has no declared conflict of interest; K.K.G. has no declared conflict of interest. P.K.J. has been reimbursed by GlaxoSmithKline (GSK), Astra-Zeneca (A-Z) and Merck, Sharpe & Dohme (Merck) for attending many conferences, has participated as a paid speaker in scientific meetings or courses organized and financed by various pharmaceutical companies (such as GSK, A-Z, Merck and Boehringer Ingelheim), has served as a consultant to GSK and Novartis, and has received research grants from several pharmaceutical companies over many years and currently holds research grants from GSK (approximately $750,000), Merck ($120,000), and A-Z ($140,000), the first of which includes a grant for a multicenter clinical trial, and his institution has received unrestricted grants from a wide variety of pharmaceutical companies.
The authors thank GSK, UK and the NIH for their generous support; Mr. D. Wang for statistical advice; Drs. Tim Wells and Christine Powers (previously of Glaxo, Geneva), who provided the gene probes; and the technicians at the Baylor Influenza Research Center for viral PCR and culture studies.
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