BRP-39 was discovered in mouse breast cancer cells (9
). Subsequently, a variety of homologues were described including human YKL-40, human HcGP-39, GP38K (porcine 38-kD heparin-binding glycoprotein), bovine 39-kD whey protein, and Drosophila melanogaster
imaginal disc growth factors (8
). In the breast, the expression of BRP-39 is increased during the glandular remodeling that is seen after the cessation of lactation (9
). In Drosophila
, BRP-39–like molecules act as growth factors (23
), and in porcine systems, GP38K induces the differentiation of cultured vascular smooth muscle cells (10
). Human YKL-40/HcGP-39 is also produced by chondrocytes and synovial cells where it regulates cell proliferation and has mitogenic effects on fibroblasts and synoviocytes (8
). This limited body of information suggests that BRP-39/YKL-40 plays a role in tissue remodeling. Because the levels of HcGP-39/YKL-40 are elevated in a variety of human diseases, it has also been thought to contribute to pathological remodeling responses (2
). Surprisingly, however, the roles of these BRP-39–like moieties in normal physiology and disease pathogenesis are poorly understood. In fact, our limited understanding of the functions of these strongly conserved (and therefore, presumably, biologically important) moieties in mammals and man is believed to be one of the most pressing issues in chitinase/CLP biology (27
). To address this issue, we generated and characterized BRP-39−/−
mice, YKL-40–overexpressing Tg mice, and mice that were deficient in BRP-39 and expressed YKL-40 selectively in lung epithelial cells. Studies of these mice have added to our understanding of the biology of CLP by demonstrating that BRP-39 plays a critical role in the pathogenesis of Th2 inflammation and IL-13–induced inflammation and remodeling. They also provide insights into the multifaceted mechanisms that underlie these effects by demonstrating that BRP-39 and YKL-40 are important regulators of allergen sensitization and Th2 cytokine effector responses that augment IgE production, DC accumulation and activation, and alternative macrophage activation while inhibiting inflammatory cell apoptosis and CD95 expression and inducing PKB/AKT activation.
We initiated our studies of BRP-39 by comparing the allergen-induced adaptive Th2 responses in WT and BRP-39−/− animals. These studies demonstrated that BRP-39−/− mice do not mount robust antigen-induced Th2 inflammatory reactions. Interestingly, similar defects were seen in experiments with chitin–free and chitin-containing antigens, suggesting that chitin binding does not play an essential role in these reactions. Evaluations of the defective Th2 responses in these mice demonstrated that BRP-39/YKL-40 contributes to Th2 reactions via a variety of mechanisms. First, they demonstrate that BRP-39−/− mice do not sensitize appropriately after antigen exposure when assessed with lymphocyte proliferation assays or assessments of antigen-specific IgE. In accordance with these findings, they demonstrate that BRP-39 is an important stimulator of DC accumulation and activation in this setting. However, our studies also demonstrate that BRP-39–deficient mice also have a significant defect in Th2 cytokine-induced effector responses. This was readily appreciated in the IL-13 Tg mice where, despite producing similar levels of Tg protein, inflammation and fibrosis were diminished in BRP-39–deficient animals. These defective IL-13–induced responses and the diminished antigen-induced responses in BRP-39−/− mice were associated with increased levels of CD4 T cell, macrophage, and eosinophil apoptosis, increased CD95 expression, and diminished levels of CD4 T cell and alternatively activated macrophage accumulation. In vitro studies also demonstrated that rBRP-39 can directly inhibit death receptor–induced T cell and macrophage apoptosis and induce alternative macrophage activation. These observations demonstrate that BRP-39 is an important inhibitor of death receptor–induced inflammatory cell apoptosis and M2 macrophage differentiation. They also suggest that the accelerated cell death responses and diminished M2 differentiation that are seen in BRP-39−/− mice play important roles in the pathogenesis of the defective IL-13 and Th2 responses in these animals. Based on these findings, one can envision how elevated levels of tissue and or circulating BRP-39/YKL-40 can enhance antigen sensitization, increase DC number and activation, inhibit inflammatory cell death, and augment the accumulation of T cells and alternatively activated macrophages, thereby regulating the intensity and natural history of Th2-dominated diseases such as asthma. These findings also provide a potential mechanism, through which YKL-40 can contribute to the severity and activity of diseases in which T cells and macrophages are believed to play important pathogenic roles, such as rheumatoid arthritis, diabetes, inflammatory bowel disease, pulmonary fibrosis, and atherosclerosis.
Many apoptotic signals engage the cell death machinery via the membrane (extrinsic) pathway. This apoptotic mechanism is well documented in T cells, macrophages, and eosinophils (28
) and is triggered when ligands like FasL and TNF-α activate surface death receptors such as Fas (CD95). The present studies demonstrate that BRP-39 and YKL-40 inhibit inflammatory cell apoptosis in vivo and in vitro. They also demonstrate that these protective responses are associated with the activation of the well known apoptosis inhibitor PKB/AKT and the more recently described death receptor apoptosis inhibitor Faim 3 (also called TOSO [reference 30
]). They also demonstrate that the augmented cell death that is seen in the BRP-39–null mice is associated with the exaggerated expression of CD95 and that rBRP-39 ameliorates FasL- and TNF-α–induced spleen cell death. These observations demonstrate that BRP-39/YKL-40 is an important inhibitor of CD95 expression and death receptor–mediated inflammatory cell apoptosis. They also demonstrate that this protection is associated with and potentially mediated by PKB/AKT and/or Faim 3. This speculation is compatible with the known ability of PKB/AKT and Faim 3 to inhibit Fas-FasL–induced cell death responses and the importance of Fas-mediated inflammatory cell apoptosis in the control of Th2 tissue inflammation (31
Excess tissue fibrosis is a well documented consequence of chronic Th2 inflammatory responses, and IL-13 is believed to be a major mediator of these remodeling reactions (22
). The airway fibrosis in the lung-targeted IL-13 Tg mice that were used in these studies is a well appreciated example of this relationship. Previous studies from our laboratory demonstrated that these IL-13–induced fibrotic responses are mediated, in part, by the ability of IL-13 to stimulate TGF-β1 (22
). Thus, studies were undertaken to determine if the diminished levels of fibrosis in IL-13 Tg mice that lack BRP-39 are associated with decrements in TGF-β1 induction. These studies provide insight into the mechanisms that BRP-39 may use in this setting by demonstrating that TGF-β1 induction is mediated by a partially BRP-39–dependent mechanism in IL-13 Tg animals. It is important to point out, however, that these studies do not define a role for BRP-39 in the pathogenesis of TGF-β1–induced responses. This possibility will need to be addressed in separate evaluations.
Our studies demonstrate that BRP-39 is produced by macrophages and epithelial cells at sites of Th2 inflammation. However, the importance of BRP-39 in each of these tissue compartments has not been defined. To begin to address this issue, we bred BRP-39−/− mice with YKL-40 Tg mice to obtain mice that were deficient in BRP-39 and produced YKL-40 only in respiratory epithelial cells. We then compared the antigen-induced responses in WT mice, BRP-39–null mice, and CC10-rtTA-tTS-YKL-40/BRP-39−/− animals. These studies demonstrated that the selective expression of YKL-40 in lung epithelial cells is able to fully rescue the defective Th2 response in BRP-39−/− mice. These observations also demonstrated that YKL-40 can effectively crosse species lines and is able to trigger BRP-39 pathways in mouse modeling systems. Additional investigation will be required to define the roles of macrophage BRP-39 in lung biology.
The 18-glycosyl-hydrolase family contains molecules with true chitinase activity, like AMCase, and molecules like BRP-39/YKL-40 that bind to but do not cleave these polysaccharides (1
). To date, the majority of the investigations of these moieties have focused on AMCase. These studies have revealed a complex effector profile with AMCase contributing to the pathogenesis of adaptive Th2 inflammation in chitin-free experimental systems (4
) and inhibiting type 2 innate immune responses in chitin-driven experimental systems (7
). The present studies demonstrate that BRP-39 and YKL-40 also play key roles in the pathogenesis of adaptive Th2 inflammation. Thus, one could be tempted to speculate that AMCase and BRP-39 play similar roles in this and other settings. However, upon deeper analysis it is becoming clear that this assumption is not correct. It is already well documented that these molecules differ in their ability to cleave chitin. Our data also demonstrates that, in contrast to our present understandings regarding AMCase, BRP-399 has similar effects in chitin-free and chitin-containing antigen-driven systems. This finding suggests that the contributions of AMCase in this setting may also be independent of its chitinase enzyme activity. Our studies also demonstrate that BRP-39 and AMCase are regulated differently, are produced by only partially overlapping populations of cells, and differ in their ability to induce alternative macrophage activation and DC activation. In accord with these findings, we noted that although BRP-39 is need to generate Th2 inflammation and AMCase induction in antigen-driven systems, once IL-13 is elaborated it induces AMCase via a BRP-39–independent mechanism. In addition, in the latter setting (the IL-13 Tg mice), AMCase induction was not able to rescue the defect in IL-13–induced inflammation and fibrosis in BRP-39–deficient animals. The literature is filled with examples of multigene families that were initially defined based on sequence or structural homologies and a small number of shared effector properties. However, in most cases, as more knowledge is obtained, differences are appreciated that clarify the unique roles that each family member plays in biology. Our data suggests that we will eventually obtain a similar level of insight into the 18-glycosyl-hydrolase family members and understand more completely the relationship between AMCase and BRP-39. Our demonstration that BRP-39 is a critical mediator in Th2 inflammation that regulates allergen sensitization, inflammatory cell apoptosis, and M2 macrophage differentiation is in keeping with the important roles that M2 macrophages (35
), DC activation (37
), and inflammatory cell apoptosis (34
) play in asthma and Th2 inflammatory responses. Additional investigation will be required to determine if these effector responses are generalizable to other members of the 18-glycosyl-hydrolase family and have contributed to the retention of this family in species as diverse as plants and man.
In summary, these studies demonstrate that BRP-39/YKL-40 is induced during and plays a critical role in the pathogenesis of aeroallergen-induced Th2 inflammation and IL-13 effector responses. They also highlight the many ways that BRP-39 and YKL-40 contribute to these responses by describing the novel roles of these molecules in allergen sensitization, IgE induction, Th2 cytokine production, DC activation, and alternative macrophage activation. Lastly, they demonstrate that BRP-39 and YKL-40 are potent inhibitors of death receptor–induced inflammatory cell apoptosis and that these protective responses are associated with the induction and activation of PKB/AKT and the enhanced expression of Faim 3. These studies validate BRP-39/YKL-40 as a therapeutic target against which interventions can be directed to control asthma or other Th2- or macrophage-mediated pathologies.