Sputum samples of a well-characterized cohort of stable COPD patients who has not required repeated admissions for exacerbation of their disease has been analyzed in the present study. Relationships between bronchial colonization by H. influenzae, the specific IgA response against this PPM, and metalloproteinase activity in bronchial secretions were examined. Bronchial colonization by H. influenzae was associated with low levels of IgA against this PPM in sputum, a defective specific immunologic response that may difficult the eradication of H. influenzae from the bronchial tree. Levels of proMMP-9 and its active form, additionally, were high in patients colonized by H. influenzae, supporting a significant impact of this PPM on the metalloproteinase activity in bronchial secretions, in the absence of exacerbation symptoms. These findings suggest that bronchial colonization by H. influenzae may be related to a defective immune response against this PPM, and associated with an increase in MMP-9 levels to concentrations that may cause extracellular matrix destruction and airway remodeling.
The low levels of specific IgA against H. influenzae found in the present study in colonized patients may be related to protease production by the PPM or due to a defective response of the bronchial mucosa. The immune response of the bronchial tree is the first defense line for the protection of the respiratory system, with a major role of local immunoglobulins, functionally related to the epithelial barrier and mucociliary clearance
. Secretory IgA originates from the selective transport of polymeric IgA across the epithelial cells, and is the predominant type of antibody in the bronchial mucosa. This immunoglobulin agglutinates colonizing bacteria and participates in the inhibition of bacterial mucosal adherence
. Strains of H. influenzae produce IgA proteases
[39, 40], and levels of sIgA in bronchial secretions are partly influenced by the production of specific enzymes that cleave the subtype 1 of this immunoglobulin (IgA1), separating the antigen recognition fragments from the constant region of IgA, in patients colonized by this PPM
[10, 14, 15, 41–43]. Because the first line of the specific immunologic defense against H. influenzae is provided by sIgA
, determinants of the level of this immunoglobulin in bronchial secretions, related to protease production, local consumption or defective production may play a role in the pathogenesis of bronchial colonization and infection in COPD
, an hypothesis supported by the results of the present study.
We have found high levels of the remodeling protein proMMP-9 and its active form in stable COPD patients colonized by H. influenzae. Previous studies have reported a relationship between bronchial colonization and inflammation in these patients, identifiable through high levels of IL-1β and IL-8 in bronchial secretions
[45–47], with a higher effect when H. influenzae is the colonizer
. Interleukin 1β, among other mediators, stimulate the release of MMP from alveolar macrophages, and upregulate MMP activity in the COPD airway
. Destruction of small bronchi and alveoli, leading to emphysema, involves members of the MMP family
, and there is significant evidence that these proteases play a significant role in COPD pathogenesis. Transgenic mice over-expressing MMP-1 develop emphysema
, whilst MMP-12 knockout mice are protected from emphysema despite prolonged cigarette smoke exposure
. High levels of MMP-8, -9 and TIMP-1 have been found in COPD
[17, 49], and MMP-9 level in bronchial secretions was higher in smokers with COPD than in smokers without functional limitation
[50, 51]. A marked increase in MMP-9 expression and activity in lung parenchyma and increased MMP-9/TIMP-1 ratios in induced sputum have been also reported in patients with COPD, when compared with healthy subjects
. Interaction with TIMPs is the physiological way to control the proteolytic activity of MMPs in normal conditions, and an imbalance between MMPs and TIMPs has been proposed as the cause of the increased levels of MMP-9 that are often detected in COPD patients
[23, 37, 49, 52, 53]. TIMP-1 is the major endogenous inhibitor of MMP-8 and MMP-9, and the levels of this protein are usually elevated in COPD
. As far as we know, the present study is the first to examine the relationship between colonization by H. influenzae, MMP-9 and TIMP-1 in bronchial secretions of stable COPD patients. We have found high levels of both proMMP-9 and its active form in patients colonized by H. influenzae, suggesting that the presence of H. influenzae in the airways of stable COPD patients may have an effect on tissue matrix. The molar ratio between proMMP-9 and TIMP-1 was also found higher in patients colonized by H. influenzae in the present study. ProMMP-9 to TIMP-1 ratios below 1:1 keep the metalloproteinase below its activity level and are the reference in healthy subjects
. Accordingly, values above 1:1 must be considered abnormal and associated with availability of the active form of MMP-9 in bronchial secretions, at levels that may be high enough to be associated with disease
Limitations of the performed study should be considered. Firstly, the present study is cross-sectional, and the correlation between low levels of IgA and persistent or recurrent colonization by H. influenzae cannot be concluded from the available data. Secondly, the PAC-COPD cohort included COPD patients who had mainly moderate disease and has been hospitalized because an exacerbation only once, and this characteristic determines that the obtained results may not be applicable to patients with advanced COPD, who also show a higher prevalence of bronchial colonization due to other PPMs, which are unusual in patients with moderate disease. The proportion of females in the PAC-COPD Study is low, a common finding in epidemiologic studies performed in Spain, attributable to the delayed introduction of smoking habits in women in the region, and determines that the observed results may not be extrapolated to female COPD patients. Finally, we have only analyzed the IgA response against H. Influenzae, because other COPD colonizers had low figures in the PAC-COPD Study. Therefore, further studies will be necessary to analyze the specific immunity against other PPMs in COPD patients.