Plasma Chemokine signature correlates with lung goblet cell hyperplasia in smokers with and without chronic obstructive pulmonary disease

As previously shown, we found that GCH was greatest in smokers without airflow obstruction compared to COPD subjects and nonsmokers, and that this effect was primarily driven by current smoking [13]. In this small cohort, we demonstrated that plasma chemokines CXCL8, CCL22, and CCL4 were also greatest in the healthy smoker group, following the same pattern as the degree of GCH. These cytokines were also greater in those currently smoking, suggesting a causal relationship between smoking and these cytokines which result in GCH. Interestingly, we found that CCL7 was greatest in the COPD group, but found no other significant differences in other cytokines between groups. Finally, we showed that there was a moderate correlation with plasma CXCL8 concentrations with MVD. These findings suggest that smoking upregulates these plasma neutrophil and macrophage chemokines which result in the development of lung GCH.

Several cytokines and biomarkers have been examined in COPD subjects in various compartments, including plasma, sputum, and BAL. The most well characterized ones include C-reactive protein, fibrinogen, surfactant protein-D, IL-6, TNF-α, and CXCL8 [14‐17]. However, prior literature has emphasized levels in COPD subjects in the chronic stable state compared to controls, compared to periods of exacerbation, or in response to therapy [14, 15, 17‐19]. Hurst et al. found that systemic levels of IL-6 correlated with sputum concentrations of CXCL8 during exacerbations compared to the chronic stable state [15]. Sin et al. reported that inhaled fluticasone and fluticasone/salmeterol combination reduced systemic levels of surfactant protein-D but not C-reactive protein or IL-6 [19]. Few studies have addressed the role of cytokines in relationship to GCH in smokers with and without airflow obstruction. Interestingly, we found that the levels of certain chemokines, particularly CXCL8, were significantly correlated with GCH. Another interesting finding was that smokers without airflow obstruction had greater MVD compared to COPD subjects, a novel finding compared to prior studies [4, 5].

CXCL8 has been the subject of many prior investigations in COPD. CXCL8 is a potent neutrophil chemoattractant [20], which is a known stimulant of mucin production and degranulation of mucin stores [21]. CXCL8 also regulates mucin gene expression at the posttranscriptional level [22]. A bronchoscopic study of 39 COPD subjects and 18 healthy controls found that CXCL8 in BAL was significantly higher in frequent exacerbators [23]. Furthermore, recent studies have shown that CXCL8 levels are significantly elevated in the blood in COPD patients, and that CXCL8 (as well as CCL4) levels correlate with mortality, exacerbation rate, and BODE scores, and inversely correlate with FEV₁ and DLCO [24‐26]. Moreover, analysis of the levels of CXCL8 in BAL and sputum found higher levels of sputum CXCL8 in COPD subjects compared to healthy smokers and nonsmoking controls but no difference in BAL CXCL8 [27]. However, one study found BAL CXCL8 levels to be greater in smokers and COPD subjects compared to normal controls [28], and another study showed that BAL CXCL8 levels could distinguish current smokers with emphysema from those without emphysema [29]. In contradistinction to prior studies, we found that plasma CXCL8 levels to be highest in the healthy smoker group and was highly dependent on current smoking. Moreover, We found a moderate correlation between plasma CXCL8 and MVD, a novel finding in comparison with current literature, and as the greatest levels of each were found in current smokers, this association suggests a relationship with plasma neutrophils and the development of GCH in the lung. It is known that cigarette smoke causes influx of neutrophils and macrophages to the lung. Cigarette smoke extract has been shown to increase CXCL8 release from bronchial epithelial cells in a concentration- and time-dependent manner [30]. Cigarette smoking acutely increases plasma neutrophil activation as well in young smokers susceptible to the development of COPD (defined as those with familial aggregation) [31]. Our findings support this phenomenon by demonstrating the upregulation of the plasma levels of the chemokines CXCL8, CCL4, and CCL22 in current smokers.

Some studies have tried to relate cytokines with the presence of chronic bronchitis. A bronchoscopic study of 42 subjects with chronic bronchitis (with and without airflow obstruction) and 13 healthy controls found increased activity of CXCL8, myeloperoxidase, hyaluronan, and eosinophil cationic protein [32]. Sputum CCL2 levels have recently been shown to be increased in COPD subjects with chronic bronchitis compared to COPD subjects without chronic bronchitis [33]. In this study, sputum neutrophil and eosinophil counts were also higher in the chronic bronchitic group. Moreover, Monzon et al. described a CCL2/CCR2B dependent loop which appeared to upregulate mucin gene expression in human airway epithelial cells [34]. In contrast, de Moraes et al. found an association with serum IL-6 levels in ex-smoker COPD subjects with chronic bronchitis, but were unable to demonstrate a relationship of IL-6 or CXCL8 with disease severity [35].

There is growing evidence that CCL22 may be involved in the pathogenesis of chronic lung inflammation. CCL22 is produced predominantly by monocytes, macrophages, and dendritic cells, and is a potent chemoattractant for macrophages, NK cells, and some T cells [36]. It is apparent that the levels of CCL22 mRNA and protein are elevated in both lung tissue and lavage fluid in COPD [37]. The levels of CCL22 are also elevated in the lavage fluid obtained from patients with idiopathic pulmonary fibrosis [38]. Furthermore, Frankenberger et al. recently reported a 10-fold increase in the expression of CCL22 by macrophages obtained from BAL of either COPD patients or smokers [39]. Finally, Belperio et al. have shown that the levels of CCL22 are overexpressed in a bleomycin model of murine pulmonary fibrosis [40]. Similarly, the levels of this chemokine are significantly increased in the lungs in studies of a rat model of radiation pneumonitis-related pulmonary fibrosis [41].

Recent reports using experimental animal models have suggested that CCL4 may play an important role in the induction of lung inflammation following exposure to tobacco smoke. Ma et al. report in increase in production of CCL4 in the lungs of mice subjected to daily administration of cigarette smoke [42]. The receptor for this chemokine is CCR5, and the administration of tobacco smoke to CCR5-deficient mice shows substantially attenuated lung inflammation when compared to mice which express normal levels of CCR5 [42, 43]. These results suggest that the CCL4-CCR5 response loop may make a substantial contribution to the development of lung inflammation associated with smoke exposure. However, it should be pointed out that Meuronen et al. have reported that CCL4 levels are significantly decreased in the BAL of asymptomatic long-term smokers [44]. This report is in contrast to the results reported herein, or with the results of studies with chronic bronchitis patients in which the levels of CCL4 were found to be increased in lavage fluid [45]. An explanation for the disagreement in the results from these studies is not clear at this time. Less is known about CCL7 in COPD, but it has been described in other inflammatory diseases including asthma, multiple sclerosis, and rheumatoid arthritis [46]. We found that CCL7 plasma levels were greatest in the COPD group. Further study in a larger cohort is needed to validate these findings.

In the present study, some cytokines, and GCH, are downregulated in COPD patients compared to healthy smokers. Other studies have found that proinflammatory cytokines and GCH are upregulated in COPD [4, 5, 14]. Corticosteroids were withheld for four weeks prior to phlebotomy and bronchoscopy, so it is doubtful that the prior use of inhaled corticosteroids is responsible. We suggest that current smoking has a more powerful influence on circulating cytokine levels in this cohort.

This study has several limitations. Firstly, the sample size is small, meaning studies of greater magnitude are needed in order to confirm these findings. The biopsies are of the large airways, which may not represent disease of the smaller airways where airflow obstruction is thought to occur. Sampling error of the mucosa is a potential issue, but the biopsies were performed in systematic fashion and therefore less likely to be the cause of the findings. There were significant differences in racial distribution between the 3 groups, which may have had bearing on the results. By design, the study recruited those with moderate to severe COPD, so little can be said about those with milder disease. Finally, our data suggest that plasma levels of four chemokines statistically correlate with COPD, and three of these (CXCL8, CCL4 and CCL22) also correlate with mucous volume density. Nevertheless, at this point little can be said about immune trafficking of inflammatory cells into the lung, as plasma cytokine levels and GCH were measured separately and are purely associations at this point. Additional studies will be necessary to more fully assess the contribution of chemokine-driven inflammatory cell recruitment to the degree of lung disease, particularly mucous production, in COPD.