Though the induction of BRP-39 is observed in a wide variety of inflammatory conditions and has been debated as a biomarker of certain disease states, relatively little investigation into its relevance in inflammatory responses has been made; necessitating additional study with in vivo
models (reviewed in [33
]). Thus, the objective of this study was to determine the expression and relevance of the chitinases BRP-39 and AMCase in cigarette smoke-induced airway inflammation and contrast this to HDM-induced allergic inflammation because of previously established chitinase expression in allergic airways disease.
To pursue this study, we utilized a murine whole body cigarette smoke exposure system. Mice were exposed to cigarette smoke for 4 consecutive days. This time point was chosen based on previous time course experiments to determine when a robust inflammatory response could first be reliably detected (data not shown). Though this time point is ideal for assessing cellular inflammation, the smoke exposure period is not long enough to measure lung destruction characteristic of emphysema. The inflammation induced is largely neutrophilic in nature, an observation similar to that described in COPD patients [34
]. As further validation of this model, we previously reported levels of carboxyhemoglobin (a measurement of the saturation of hemoglobin with carbon monoxide) and cotinine (a metabolic product of nicotine) similar to the human reference [19
]. Similarly, the HDM model utilized a 2 week time point as this has been previously established as the earliest time point to observe robust eosinophilic inflammation [36
], while prolonged exposure is required to induce airway remodeling. Thus, the focus of both models is the inflammatory response, which is believed to drive, at least in part, the pathogenesis of COPD and asthma.
The increase in BRP-39 expression after smoke exposure is a robust event observed across inbred strains and outbred stock. This induction is in agreement with clinical observations of increased YKL-40 expression levels in smokers and COPD patients. Unlike models of allergic airway inflammation where both AMCase and BRP-39 have been shown to be elevated [15
], increased expression levels of AMCase were not observed following smoke exposure, thus distinguishing the chitinase expression profile elicited by cigarette smoke from the one elicited by allergens.
The induction of BRP-39 and the infiltration of cells into the lungs were concurrent phenomena after 4 days of cigarette smoke exposure. IHC on lung sections implicated epithelial cells and macrophages as the primary producers of BRP-39 in this model, which is in agreement with the YKL40 expression pattern in humans and other smoke exposure models [17
]. Others have found that neutrophils are capable of producing YKL-40 in humans [37
]; however, no evidence in our model suggests that this prominent inflammatory cell type is contributing to BRP-39 production. Regardless of the relevance of BRP-39 in disease pathology, its expression is closely associated with the inflammatory response and BRP-39 remains a biomarker of inflammatory disease.
Following the initial observation of BRP-39 induction in allergic disease, Th2 mechanisms were postulated as being responsible for driving this process [15
]. Th2 responses are believed to be crucial for parasitic defense and the induction of enzymes with the potential to break down the protective sheaths of parasitic nematodes would be of great efficacy to such responses. The finding that enzymatically active AMCase is induced in an IL-13 dependent manner in Th2 driven inflammation reinforced this hypothesis [16
]. Though Th2 cytokines, including IL-13, have been detected in the smoke exposure model utilized in this study [19
], IL-13 KO mice revealed that BRP-39 induction by cigarette smoke is IL-13 independent. This is not entirely surprising as IL-13 does not appear to be a critical mediator of inflammation in the smoke exposure system for its deficiency also has no effect on cellular inflammation. Conversely, it was rather unexpected that in HDM-induced allergic inflammation; which is Th2-driven, IL-13 was unnecessary for the induction of BRP-39; in other words BRP-39 induction was unaltered and yet eosinophilic inflammation was markedly attenuated. These results are at variance with previous work that implicated BRP-39 as a crucial inflammatory component in similar HDM models [15
]. This represents a significant finding and expands on previous work by Lee et al
in which IL-13 dependence for BRP-39 induction in allergic airway inflammation was strongly implied by experiments where transgenic amounts of IL-13 had been over-expressed in the lungs [15
]. The experiments by Lee et al, however, did not utilize an IL-13 KO strain and as such these data only demonstrate that IL-13 is able to induce BRP-39 and not whether IL-13 is essential for BRP-39 induction. Our data show that although IL-13 is capable of inducing BRP-39 expression, it is redundant in models of cigarette smoke- and allergen-induced airway inflammation in the induction of BRP-39.
IL-1 has been implicated in vitro
in BRP-39 induction [38
]. The IL-1R1 KO mice were chosen for this reason and because IL-1R1 deficiency was sufficient to attenuate smoke-induced neutrophilic inflammation. The observation that smoke-exposed IL-1R1 KO mice did not up-regulate expression of BRP-39 suggests a crucial role of IL-1 in this phenomenon. This provides further evidence that the induction of BRP-39 is closely tied to inflammatory pathways. Further investigation of the importance of IL-1 in the induction of BRP-39 in allergic inflammation revealed that IL-1R1 was not crucial in the HDM model, highlighting the different inflammatory pathways engaged by these two models. Our data which confirms the importance of BRP-39 in HDM-induced inflammation imply that BRP-39, in the context of allergy, is part of an immune inflammatory pathway crucial to mononuclear cell and eosinophil recruitment that is not dependent on IL-1 or IL-13.
Recently Matsuura et al
have implicated IL-18 as a mechanistic component of BRP-39 induction in a murine model of smoke exposure [18
]. These data complement previous experiments that implicate IL-18 as a crucial component of cigarette smoke-induced inflammation [10
]. Our data generated in IL-18 KO mice suggest that IL-18 is redundant in the inflammatory response and in the induction of BRP-39 which was confirmed by experiments with IL-18 receptor KO mice (data not shown). This discrepancy could be the result of different smoke exposure conditions as Matsuura et al
utilized a nose only smoke exposure apparatus characterized by Shapiro et al
], as opposed to a whole body smoke exposure system. A more likely explanation of the discrepancy is the length of smoke-exposure, as our study exposed mice to smoke for four days while Matsuura et al
exposed mice to smoke for a month to determine the mechanistic relevance of IL-18. The four day time point was chosen for this study because experiments showed a greater induction of BRP-39 at subacute time points when compared to the chronic setting (data not shown). These findings taken in context with the data from IL-1R1 KO mice imply a timeline for cigarette smoke induced inflammation where IL-1 inflammatory pathways are more important early on in disease progression with IL-18 mediated pathways engaged after sustain cigarette smoke stimuli.
Evidence such as the stimulation of cells with YKL-40 inducing inflammatory chemokines has implied a role for this YKL-40 and BRP-39 in cellular inflammation [17
], yet BRP-39 deficiency did not lead to significantly attenuated lung-infiltrating cell types after smoke exposure. The redundant nature of BRP-39 in this inflammatory response represents the most striking finding of this study and again contrasts the work by Matsuura et al
]. As stated before, this is likely the result of the different durations of smoke exposure as Matsuura et al
did not witness reduced inflammation in smoke-exposed BRP-39 KO mice until at least 3 months of smoke-exposure. This implicates BRP-39 in the survival of inflammatory cells in a chronic inflammatory setting and not in the initial recruitment of cells to the lungs. The lack of significant difference in tissue neutrophils, DCs, and CD4 T cell activation more specifically reinforces the redundant nature of BRP-39 in the early stages of cigarette smoke-induced inflammation.
Another striking conclusion of these experiments was that although BRP-39 has been shown to be crucial for allergic sensitization, it is redundant in the adjuvant properties of cigarette smoke. This implies a different mechanism of sensitization when cigarette smoke is utilized as an adjuvant. This is not an unprecedented assertion as HDM models of allergic sensitization and models of cigarette smoke induced OVA sensitization have been shown to utilize different inflammatory pathways [40
]. Lee et al
postulated that the attenuation of allergic responses in BRP-39 deficient mice was due to an increase in apoptosis of a key mediating cell type [15
]. Apoptosis was not assessed in this study but if there was increased apoptosis in BRP-39 deficient animals it was not sufficient to impede sensitization or decrease the amount of activated DCs, implying that an increase in apoptosis may not be sufficient to interrupt sensitization when alternate pathways are driving sensitization. This is likely the case when cigarette smoke is utilized as an adjuvant.