BRP-39/YKL-40 is a member of the CLPs, and belongs to glycohydrolase family 18. BRP-39/YKL-40 is a phylogentically highly conserved chitin-binding, heparin-binding, and collagen-binding protein, with homologues in vertebrates and invertebrates (1
). Elevated concentrations of YKL-40 were demonstrated in a variety of diseases that are pathologically characterized by tissue inflammation and remodeling. Because of its strong association with disease, YKL-40 is regarded as a useful prognostic or diagnostic marker and potential therapeutic target (9
). However, the molecular processes governing the induction of YKL-40 and the roles of YKL-40 in normal physiology and in the pathogenesis of diseases remain poorly understood. This is partly attributable to the lack of appropriate animal models, such as specific gene-targeted null or overexpressing transgenic mice. In this study, we demonstrated that BRP-39, the mouse homologue of human YKL-40, plays an important role in the development of CS-induced inflammation and emphysema, by using BRP-39 null mutant mice recently generated in our laboratory (23
The mRNA and protein expression of BRP-39 was significantly induced in CS-exposed mice compared with RA-exposed controls. We further demonstrated that macrophages, airways, and alveolar epithelial cells are the major BRP-39–expressing cells in the lungs of CS-exposed mice. Previous in vitro
studies showed that proinflammatory cytokines IL-1β and TNF-α efficiently stimulated articular chondrocytes to produce YKL-40 (34
). Because these proinflammatory cytokines are elevated in the lungs of patients with COPD (27
) and in CS-exposed animal models of COPD (18
), CS-induced proinflammatory cytokines could play a role in the induction of BRP-39 in the lungs of CS-exposed mice. Although we could not detect a significant induction of IL-1β and TNF-α, the expression of proinflammatory cytokine IL-18 was significantly induced in mice exposed to CS, which is consistent with previous findings from our laboratory (27
). Interestingly, the CS-stimulated induction of BRP-39 was dependent on the IL-18–signaling pathway, at least in part, because the expression of BRP-39 was substantially decreased in mice with the IL-18 receptor null mutation. On the other hand, the induction of IL-18 was not significantly modulated in the absence of BRP-39, suggesting that BRP-39 is a downstream mediator of IL-18. The presence of the IL-18 receptor in bronchoalveolar epithelial cells and macrophages (37
) further supports the potential regulatory role of IL-18 in the induction of BRP-39 in these cells after exposure to CS. However, we cannot exclude the possibility of BRP-39 induction through CS-induced proinflammatory cytokines other than IL-18, such as TNF-α or IL-1β, because previous studies demonstrated that these cytokines regulate the expression of YKL-40 in differentiated macrophages or articular chondrocytes (38
). Interestingly, we did not observe increased expression of TNF-α, IL-6, or IL-1β in the lungs of IL-18R−/−
after exposure to CS (data not shown). On the other hand, when we overexpressed IL-18 in the lung, we noted significant increases in the expression of TNF-α, IL-6, and IL-1β, together with BRP-39 (unpublished data). These studies suggest that IL-18 is an upstream mediator that regulates the expression of multiple proinflammatory cytokines together with BRP-39. The specific contributions of these proinflammatory cytokines in the induction by CS of BRP-39 remain to be determined. Recent studies from our laboratory and others suggest a potential regulatory role of BRP-39/YKL-40 in inflammation. The administration of recombinant YKL-40 can directly stimulate alveolar macrophages and fibroblasts to release proinflammatory and profibrogenic mediators such as IL-8, MCP-1, and MIP-1α to levels comparable with those obtained upon cell activation by TNF-α (8
). Those studies raised the possibility that BRP-39/YKL-40 could mediate or synergize the effects of IL-18 or other proinflammatory cytokines for the generation of optimal inflammatory responses by exposure to CS. In addition, our laboratory demonstrated the direct role of BRP-39/YKL-40 in the regulation of lung inflammation in an animal model of allergen-induced Th2 inflammation (23
). BRP-39, as a downstream mediator of IL-13, directly regulates allergen-induced BAL and tissue inflammation through the inhibition of inflammatory cell apoptosis (23
). Similar to the induction of BRP-39 and the regulatory mechanism in Th2 inflammatory responses, the expression of BRP-39 was also induced by a proinflammatory cytokine (IL-18), and endogenous BRP-39 was required for CS-induced inflammation. However, BRP-39/YKL-40 itself seems insufficient to induce inflammation, because the lung-specific overexpression of YKL-40 only enhanced the allergen-induced inflammatory response, but did not induce an inflammatory response without additional challenge (23
). In combination, these studies demonstrate that BRP-39 is induced in macrophages or epithelial cells after exposure to CS, and plays a proinflammatory role through regulating cellular apoptosis.
Several mechanistic pathways were suggested for the pathogenesis of COPD (17
). They include protease/antiprotease imbalance, oxidative stress, and deregulated inflammatory and immune responses. Recent data from both animal models of COPD and human studies strongly suggest another mechanism of COPD pathogenesis, that is, the disruption of the balance between apoptosis and the replenishment of structural cells in the lung (22
). Specifically, the critical role of epithelial-cell apoptosis in the development of emphysema is supported by a number of studies. The treatment of vascular endothelial cell growth factor (VEGF) receptor blockers was shown to induce alveolar septal-cell apoptosis, resulting in emphysematous lung destruction (21
). Studies from our laboratory demonstrated that epithelial-cell apoptosis was significantly augmented in animal models of emphysema (28
). Intervention with apoptotic tissue responses by nonspecific pan-caspase inhibitors, such as Z-VAD-fmk, significantly reduced IFN-γ–stimulated or TGF-β–stimulated airway and alveolar remodeling, further supporting the critical role of apoptosis in alveolar and airway remodeling processes (29
). In the present study, we also observed significantly increased TUNEL-positive and annexin V-positive apoptotic macrophages and alveolar and airway epithelial cells after chronic exposure to CS. However, the CS-induced structural cell death response and emphysematous destruction were significantly enhanced in the absence of BRP-39. These results suggest a protective role of endogenous BRP-39 from CS-induced apoptosis or cell-death response. In previous studies, the proactive role of BRP-39/YKL-40 in cellular proliferation and tissue remodeling was demonstrated. YKL-40 was shown to act as a growth factor for fibroblasts and chondrocytes in synergy with insulin-like growth factor–1 (13
), and to limit the catabolic effects of TNF-α and IL-1β (14
). YKL-40 is also synthesized by vascular smooth muscle cells, and it promotes their migration and attachment (46
). In addition, YKL-40 was shown to stimulate endothelial cells directly, and to increase angiogenesis in vivo
and in vitro
). Interestingly, the absence of BRP-39 did not significantly alter epithelial-cell proliferation in the lungs of mice after exposure to RA or CS ( in the online supplement). This result suggests that the deregulation of BRP-39 contributes to CS-induced emphysema, mainly via the regulation of the CS-induced cell death response rather than through epithelial-cell proliferation. The present study further reveals that BRP-39 regulates the CS-induced cell death response via the modulation of Fas expression in inflammatory and epithelial cells. When these findings are viewed in combination, we can envision that BRP-39/YKL-40 plays an important role in structural cell survival, and that the endogenous expression of BRP-39/YKL-40 is essential for the maintenance of normal alveolar structures in the lung.
In the present study, we noted that a deficiency of BRP-39 diminishes CS-induced inflammation while augmenting tissue destruction. On superficial analysis, this may appear confusing. However, these and other studies make it very clear that BRP-39/YKL-40 exerts important effects on both inflammatory and structural cell death (23
). Specifically, superphysiologic levels of BRP-39 and YKL-40 appear to inhibit inflammatory cell death (23
). In contrast, physiologic concentrations of BRP-39 appear to inhibit oxidant injury and maintain the survival of structural cells such as epithelial cells and endothelial cells (50
). Thus, in the absence of BRP-39, we can envision heightened levels of CS-induced injury and heightened levels of structural cell apoptosis. Simultaneously, we would also see heightened levels of inflammatory cell apoptosis, and thus a diminished inflammatory response. Thus, not only are these results consistent with each other, but they represent a heightened level of understanding of the biologic roles of BRP-39 in the setting of CS-induced injury. This study also highlights the importance of maintaining physiologic levels of BRP-39 or YKL-40 in the lungs, to prevent both excessive inflammatory responses and excessive alveolar destruction. In this regard, the physiologic balance of YKL-40 should be considered in future therapeutic applications of YKL-40 in diseases associated with an elevated concentration of YKL-40, such as asthma or COPD.
To evaluate the applicability of our murine findings to human diseases, studies were undertaken to investigate the expression of YKL-40 in human lung tissues. Corresponding to the murine data, the tissue expression of YKL-40 was highest in smokers, followed by former smokers and never-smokers, representing a significant impact of CS on the expression of YKL-40 in the lung. In addition, the major YKL-40–expressing cells in the lungs of smokers were also mostly macrophages, airway cells, and alveolar epithelial cells. Letuve and colleagues reported that YKL-40–expressing macrophages and interstitial cells were significantly increased in bronchial biopsies from smokers with COPD compared with never-smokers (8
). In contrast to our findings, they did not directly correlate YKL-40 expression in the lungs with a history of CS. However, a higher percentage of cells expressing YKL-40 in alveolar macrophages was isolated from smokers than from never-smokers (8
), indicating substantial effects of CS on the expression of YKL-40 in the lung.
To see whether correlations exist between the expression of YKL-40 and the development of COPD in humans, serum concentrations of YKL-40 in nonsmoking control subjects, smokers, and smokers with COPD were evaluated. These studies demonstrated that only smokers with COPD had significantly higher serum YKL-40 concentrations compared with smokers without COPD or never-smokers. These findings are consistent with those reported by Levute and colleagues (8
). Interestingly, only a borderline correlation existed between serum concentrations of YKL-40 and smoking history (P
= 0.049). Although the levels of CS exposure (pack-years) were higher in COPD patients with higher YKL-40 concentrations than with lower YKL-40 concentrations (58.43 ± 12.35 versus 34.82 ± 7.09 pack-years [mean ± SEM] for patients with > 100 ng/ml versus < 100 ng/ml, respectively), these reults did not reach statistical significance. On the other hand, serum YKL-40 concentrations were significantly correlated with the age of individuals, irrespective of smoking history, as demonstrated in a previous study (51
). These results suggest that concentrations of circulatory YKL-40 in patients with COPD are profoundly affected by other local or systemic factors in addition to CS exposure. Because activated inflammatory cells express YKL-40, concentrations of serum YKL-40 could be influenced by the presence of low-grade systemic inflammation in patients with COPD (52
). In the present study, the severity of COPD according to GOLD stages did not indicate a significant difference in serum concentrations of YKL-40. In contrast to our studies and others, Agapov and colleagues found no significant differences in serum concentrations of YKL-40 between simple smokers and smokers with COPD, or between stratified groups based on GOLD stages (54
). As suggested Agapov and colleagues (54
), the differences between studies may be attributed to the heterogeneity of a complex disease. Additional investigations on the potential source or other modulating factors of serum YKL-40 concentrations and their clinical implications in a larger cohort of patients with COPD are warranted.
In conclusion, these studies demonstrate that BRP-39/YKL-40 is accumulated during exposure to CS, and plays a significant role in the pathogenesis of CS-induced inflammation and emphysema. These studies also underscore that maintaining the balance of physiologic levels of YKL-40 will be therapeutically important in preventing excessive inflammatory responses and emphysematous alveolar destruction.