We characterized the relative contribution of both active human chitinases, CHIT1 and AMCase, to chitinase activity in the lung from healthy subjects, asthmatics, and habitual smokers with mild COPD. We determined chitotriosidase to be the primary active chitinase in the lung, and we found that it’s expression is strongly dependent on genetics and on smoking habit.
We used a multi-faceted approach to establish that CHIT1, not AMCase, is the principal active chitinase in the human lung, including determination of enzymatic activity, gene expression, and protein expression. We found that the pH profile for lung chitinase activity was consistent with the activity of CHIT1, not AMCase, showing high activity at near neutral pHs values and a complete absence of activity at low pH values 9, 24
. In addition, we found a lack of chitinase activity in BAL from all six subjects genetically deficient in CHIT1 activity. Indeed, there appeared to be an additive effect of the CHIT1 duplication on lung chitinase activity, with carriers of the duplication variant having reduced chitinase activity compared to non-carriers. In addition, CHIT1 gene expression in both macrophages and epithelial cells was strongly correlated with chitinase activity in lung secretions, further solidifying the primacy of CHIT1 in contribution to lung chitinase activity.
AMCase transcript numbers have been reported to be increased in asthmatic airways 13
, but our data do not confirm this. Rather, we find that inactive AMCase variants are expressed in the lung and that these variants are not differentially expressed in asthmatics or in smokers. Specifically, our data show that the numbers of “total” AMCase trancripts are higher than the numbers of active AMCase transcripts in epithelial cells and macrophages in all subject groups. This finding agrees with a previous report showing expression of splice variants of AMCase in the lung, variants which lack regions of exon-six containing the conserved active site residues required for enzymatic activity 10
. Consistent with this is our finding that BAL samples which had no chitinase activity still had detectable AMCase protein, indicating that the protein lacks chitinase activity.
Our results have important implications for the role of chitinases in asthma. Paradoxically, mouse studies have implicated lung chitinase activity in both the promotion of allergen-induced airway inflammation and in reduction of chitin-induced airway eosinophilia 13, 14
. Our results indicate that the upregulation in lung chitinase activity in mouse models of asthma does not extend to human asthmatics. In fact, our results do not support a pro-inflammatory role for lung chitinase activity in asthma pathology; rather, we find that chitinase activity tends to be lower than normal in asthmatics. Furthermore, we find that AMCase protein levels in BAL are lower than normal in asthma. These results are more supportive of the proposed protective role of chitinase activity and chitin-binding proteins in asthma and allergy, as evidenced by the inhibition of polymeric chitin’s Th2 inflammatory effects after digestion with AMCase 14
. It is important to note several limitations with regard to interperation of this data. First, our observations were restricted to stable mild-moderate adult asthmatics. It will be important to determine whether our observations extend to subjects with more severe disease or in response to acute allergen challenge. Second, although the lung expressed AMCase is inactive, it does retain an intact chitin-binding domain, and as such it could bind chitin-containing environmental allergens and affect airway inflammation. The function of chi-lectins is unknown but these molecules have been implicated in mechanisms of tissue remodeling and inflammation 25–28
Chitinase activity was markedly higher than normal in bronchoalveolar lavage from the subgroup of habitual smokers with mild COPD. To our knowledge, this is the first report of increased chitinase activity in airway secretions from smokers. CHIT1 gene expression was much higher in macrophages than in epithelial brushings, and we found a positive correlation between lavage chitinase activity and CHIT1 gene expression in macrophages, findings which point to macrophages as the main cellular source of the chitinase activity in these subjects. The mechanism by which cigarette smoke induces CHIT1 expression remains to be determined. Plant chitinases have well defined roles in pathogen response 29
and the role of human chitinases has generally been assumed to be in defense against chitin containing pathogens 30, 31
. One possibility is that there are chitin particles in inhaled tobacco smoke, which occur as a consequence of fungal infection of tobacco leaf. It is estimated that as many as 270 fungal spores may be present in a single cigarette, and fungal spores from A. alternata
have been detected in cigarette ash 32–34
. Polymeric chitin is known to be pro-inflammatory 14
, and if present in cigarette smoke, may activate macrophages and airway epithelial cells to increase chitinase expression and/or secretion. Other fungal-derived compounds such as beta glucans can also induce chitinase production from plants and are potent macrophage stimulators 35
In conclusion, we have used multiple lines of evidence including biochemical, genetic, and gene and protein expression data in determining that chitotriosidase, not acidic mammalian chitinase, is the primary chitinase responsible for chitinase activity in the lung. We found that chitinase activity tended to be lower than normal in asthma, a finding which supports a protective role for chitinolytic activity in allergic inflammation. In contrast, chitinase activity and CHIT1 gene expression are increased in habitual smokers, probably because cigarette smoke induces activation of pulmonary macrophages. Taken together, our findings show that chitotriosidase activity in lung disease is modulated in ways which reflect underlying disease susceptibilities and specific environmental exposures.