This is the first study to prospectively study neutrophil adhesion molecules and activation markers in COPD exacerbations. Following rhinovirus infection CD11b, CD162 and CD62L levels were significantly reduced on circulating neutrophils in COPD subjects, and CD11b levels correlated with clinical and inflammatory parameters at exacerbation. The results indicate a role for these adhesion molecules in the recruitment of neutrophils to the lungs in COPD exacerbations and may represent potential therapeutic targets.
COPD exacerbations are associated with neutrophilic inflammation [3
], and neutrophil migration from the blood is mediated by adhesion molecules that are well characterised in vitro
but few studies have examined their role in COPD exacerbations in vivo
. Pharmaceutical agents targeting these molecules are under development as novel anti-inflammatory agents in asthma and COPD [6
], and therefore a better understanding of their role in these diseases is required. We have previously reported that experimental rhinovirus infection induces increases in airway neutrophils in subjects with COPD [4
]. Following on from these observations we examined expression of neutrophil adhesion molecules and activation markers in experimental rhinovirus infection in COPD subjects and a control group of non-obstructed smokers.
We initially examined neutrophil markers at baseline prior to infection and there were no differences in expression in neutrophil markers between the groups. Previous studies have reported increased CD162 [11
], no differences in CD11a and CD62L [12
] and both increased [12
] and unchanged [14
] CD11b on neutrophils in COPD. These discrepant results are likely due to variations between studies in severity of COPD, different control groups and different methods of neutrophil isolation. The COPD subjects in our study had moderate COPD (GOLD stage II) and the comparator group were similarly aged smokers without airway obstruction. Therefore the exclusion of patients with severe COPD and the choice of smokers as the comparator group may account for the lack of differences between the groups. Comparing marker expression on blood and sputum neutrophils we report up-regulation of CD11b, CD63 and CD66 and down-regulation of CD11a, CD31, CD162 and CD62L on sputum neutrophils. Although CD11b is an adhesion molecule it is released from granules in neutrophils on activation and therefore unlike the other adhesion molecules it is upregulated following neutrophil migration. Our results are similar to those reported in allergy [18
], asthma [19
], bronchiolitis [20
] and sarcoidosis [10
]. In COPD only CD11b has been examined previously and increased expression on sputum neutrophils was reported [17
]. CD162 is down-regulated upon neutrophil stimulation in vitro
] but has not been examined in vivo
and therefore this is the first study to report down-regulation of CD162 following neutrophil migration to the lung in vivo
. Following rhinovirus infection there were no changes in neutrophil marker expression on sputum neutrophils. These findings imply that changes in neutrophil surface markers following migration from the blood to the lung are an ‘all or nothing’ phenomenom and not specific to any particular disease process [10
]. Therefore markers on sputum neutrophils are unlikely to be helpful as diagnostic or prognostic markers in airway diseases.
In contrast there were consistent changes in marker expression on blood neutrophils following rhinovirus infection. Only one study has examined neutrophil adhesion molecules in COPD exacerbations and this study reported reduced CD11a and CD11b but not CD62L on blood neutrophils in exacerbated patients [12
]. Studies of neutrophils in virus infections have had conflicting results. Peripheral blood neutrophils collected during RSV infections in infants showed no differences in CD11a, CD11b or CD62L compared to controls [20
]. In children with viral wheeze CD62L was reduced but not CD11b [23
], and in adults with influenza infection CD11b was increased [24
]. These studies were all cross-sectional with different subjects as controls, making interpretation of the dynamics of neutrophil responses difficult. Our study is the first to prospectively study marker expression within the same subjects prior to and during the course of virus infections. We hypothesised that neutrophils expressing higher levels of adhesion molecules would preferentially migrate to the lungs following infection, and therefore the cells remaining would consist of a population with lower surface levels of adhesion molecules. Following rhinovirus infection CD11a on peripheral blood neutrophils was reduced in both COPD and non-COPD subjects, and CD11b, CD62L and CD162 were significantly reduced in COPD subjects only. These data favour our hypothesis and suggest that these adhesion molecules are key to neutrophil recruitment to the lungs following virus infection in COPD. There was no change in CD31, CD66b and CD63 expression on blood neutrophils post-infection, consistent with their role in endothelial transmigration and as neutrophil activation markers respectively. There was a trend towards increased CD54 expression on blood neutrophils post-infection. A role of CD54 in neutrophil migration is unlikely, given our current state of knowledge, as it has no known ligands on endothelial cells, but it has been reported up-regulated on peripheral blood neutrophils in RSV infection [20
], following exposure to endotoxin [25
] and in sarcoidosis [10
], and therefore may be up-regulated in response to systemic inflammation. Further studies are needed to determine whether CD54 is upregulated in more severe exacerbations or bacterial exacerbations and whether it plays a role in neutrophil chemotaxis
There were significant inverse relationships between CD11b expression on blood neutrophils in the COPD subjects and respiratory symptoms and inflammatory markers. Lower levels of blood CD11b post-infection were associated with more symptoms and greater neutrophilic inflammation in both blood and sputum. These data suggest that CD11b is closely linked to virus-induced neutrophilic inflammation in COPD exacerbations and CD11b has potential as both a marker and therapeutic target in COPD exacerbations. In an ozone-induced model of airway inflammation the CXCR2 antagonist SB-656933 inhibited CXCL1-induced CD11b expression on peripheral blood neutrophils and reduced neutrophilic inflammation in sputum [26
]. Our data suggests that this approach may also have potential as a treatment for virus-induced COPD exacerbations. Given our recent observation that secondary bacterial infections occurred in 60% of COPD subjects following experimental rhinovirus infection, and that virus-induced neutrophilic airway inflammation was implicated in precipitating bacterial overgrowth via neutrophil elastase-mediated degradation of anti-microbial peptides, therapeutic approaches that reduce neutrophil migration and activation might have the attractive potential of preventing secondary bacterial infections [7
Our study has a number of limitations that must be considered when interpreting the results. The number of subjects studied was relatively small and experimental rhinovirus infection is limited to patients with moderate COPD, and therefore may not be relevant to more severe disease. Experimental infection studies will always be limited by these factors but we believe that such studies are a useful tool to investigate mechanisms in COPD exacerbations and generate hypotheses that warrant further investigation in naturally-occurring exacerbations.
The surface markers we used to identify sputum neutrophils may not have excluded other cell types particularly eosinophils. However eosinophils represented <2% of sputum cells in these subjects and therefore are unlikely to have had a major impact on the results [4
]. Moreover the results from the blood neutrophils are likely to be valid as purified neutrophils were used.
In conclusion this is the first study to prospectively study neutrophil marker expression in experimental rhinovirus infection. We observed reduced expression of the adhesion molecules CD11a, CD11b, CD62L and CD162 on peripheral blood neutrophils following rhinovirus infection in COPD subjects, and correlations between CD11b and exacerbation severity. Further studies investigating the role of adhesion molecules in other virus-induced disease such as asthma exacerbations, and interventions targeting neutrophil adhesion molecules are warranted.