Acidic Mammalian Chitinase in Asthmatic Th2 Inflammation and IL-13 Pathway Activation

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Science  11 Jun 2004:
Vol. 304, Issue 5677, pp. 1678-1682
DOI: 10.1126/science.1095336


Chitin is a surface component of parasites and insects, and chitinases are induced in lower life forms during infections with these agents. Although chitin itself does not exist in humans, chitinases are present in the human genome. We show here that acidic mammalian chitinase (AMCase) is induced via a T helper-2 (Th2)–specific, interleukin-13 (IL-13)–mediated pathway in epithelial cells and macrophages in an aeroallergen asthma model and expressed in exaggerated quantities in human asthma. AMCase neutralization ameliorated Th2 inflammation and airway hyperresponsiveness, in part by inhibiting IL-13 pathway activation and chemokine induction. AMCase may thus be an important mediator of IL-13–induced responses in Th2-dominated disorders such as asthma.

Asthma is a chronic inflammatory disease that affects millions of people worldwide. Current concepts of pathogenesis center on the notion that asthma is the result of exaggerated Th2 airway inflammation (1). Striking similarities also exist between the Th2 responses observed in asthma and the hallmark Th2 responses of antiparasite immunity (2, 3). As a result, it is believed that Th2 inflammation originally evolved to deal with parasites and that allergy and atopic asthma arise as a consequence of poorly controlled Th2 responses elicited independently of parasitic infection (2, 3).

Chitin, the second most abundant polysaccharide in nature, is found in fungal cell walls, in the exoskeletons of crustaceans and insects, and in the microfilarial sheaths of parasitic nematodes (47). These chitin coats provide protection for pathogens from harsh conditions inside the host (6). Chitin accumulation is regulated by the balance of chitin synthase-mediated biosynthesis and degradation by chitinases. Thus the viability of chitin-containing pathogens is decreased by host pathways that diminish chitin accumulation (58). Chitinase production is also a common feature of antiparasite responses of lower life forms against chitin-containing organisms (7, 9, 10). Paradoxically, while chitin and chitin synthase do not exist in mammals, human chitinase family members such as acidic mammalian chitinase (AMCase) have recently been described (4). Little is known, however, about the regulation and functions of AMCase or other mammalian chitinase moieties, or their potential roles in antiparasite or Th2 responses.

On the basis of the importance of chitinases in antiparasite responses in lower life forms, we hypothesized that mammalian chitinases, such as AMCase, might also contribute to the pathogenesis of Th2 immune responses. To test this, we evaluated the expression of AMCase during pulmonary Th2 inflammation, elicited by sensitization and aerosol challenge with the aeroallergen ovalbumin (OVA) (11) in mice. Significant increases in AMCase mRNA and protein (Fig. 1A) and bronchoalveolar lavage (BAL) fluid chitinase activity (Fig. 1B) were detected in lungs of antigen-sensitized/challenged animals, but not controls. Immunohistochemistry of lungs from sensitized and challenged mice revealed AMCase protein in both epithelial cells and macrophages (Fig. 1C). To define the potential T helper specificity of this response, we compared the AMCase expression in lungs from mice that had been reconstituted with either polarized Th2 or Th1 cells and subsequently challenged with antigen (11). In contrast to Th1 cells, which did not induce AMCase, Th2 cells were potent stimulators of AMCase mRNA and protein (Fig. 1D). To define the Th2 cytokines participating in AMCase induction, Th2 cells from wild type were compared with cells from IL-13–deficient or IL-4–deficient mice (11). AMCase up-regulation did not occur in the specific absence of IL-13, but was only modestly prevented by the absence of IL-4 (Fig. 1D). Similarly, in the absence of IL-13, only modest induction of AMCase mRNA and protein were apparent in the OVA mouse model (Fig. 1E). Specific chitinase bioactivity was increased in lungs from mice in which transgenic IL-13 was constitutively (CC10-IL-13) or inducibly (CC10-rtTA-IL-13) overexpressed in a lung-specific manner (Fig. 1F) (11). In contrast, transgenic expression of IL-11 and IL-10 did not result in an increase in AMCase expression (11). These results demonstrate that AMCase is prominently and selectively induced during Th2 inflammation and that this induction is mediated, to a large extent, by IL-13.

Fig. 1.

AMCase in the OVA asthma model and in IL-13 overexpressing transgenic mice. (A) Reverse transcription polymerase chain reaction (RT-PCR) (top) and immunoblot (bottom) analyses of AMCase and chitotriosidase mRNA and protein 24 hours (h) to 7 days (d) after OVA sensitization/challenge. (B) BAL chitinase activity after OVA challenge (*P < 0.01 versus 0 h). (C) Immunohistochemical detection of AMCase in lungs from sensitized and challenged mice and from phosphate-buffered saline (PBS)–challenged control mice. Evaluations were undertaken in the presence and absence of excess AMCase peptide. (D) RT-PCR comparison of the levels of OVA-stimulated mRNA encoding AMCase, eotaxin, and IP-10 in mice that did not receive passively transferred cells (none) and mice that received in vitro polarized Th1 or Th2 cells. Cells from wild-type (+/+) mice were used in the top panel; cells from (+/+), IL-13 null (–/–), or IL-4 null (–/–) mice were used in the bottom panels. (E) Western blot (top) and RT-PCR (bottom) comparison of the induction of AMCase in mice with normal (+/+) or null (–/–) IL-13 loci after OVA sensitization/challenge. (F) RT-PCR (top left), immunoblot (bottom left), and bioassay (right) analyses of AMCase in 1- to 2-month (M)–old CC10-IL-13 mice (*P < 0.01).

Administration of anti-AMCase sera in the aeroallergen challenge model caused a marked decrease in Th2 inflammation with dose-dependent decreases in BAL and tissue eosinophilia and lymphocyte accumulation at all time points (1 to 7 days after antigen challenge) (Fig. 2, A and B) (11). Similar decreases in BAL and parenchymal inflammation were also noted with the chitinase inhibitor allosamidin (10, 12, 13), even at doses as low as 1 mg/kg (Fig. 2C) (11). In vivo, the anti-AMCase and allosamidin treatments caused a substantial decrease in BAL chitinase activity (Fig. 2, D and E). Anti-AMCase also markedly diminished the airway hyperresponsiveness seen in the OVA sensitization/challenge protocol (Fig. 2F). Anti-AMCase did not alter the inflammation in mice in which IL-11 was targeted to the lung in which AMCase was not induced (11).

Fig. 2.

Role of AMCase in OVA-induced responses. Comparison of the BAL total cell, eosinophil, and lymphocyte recovery (A) and morphometric tissue inflammation (B) in OVA-sensitized mice that were challenged with OVA (+ OVA) or vehicle (–OVA) and treated with control serum or anti-AMCase starting 1 day before OVA challenge (*P < 0.05). (C) Inhibitory effects of intraperitoneal allosamidin on BAL cellularity after OVA sensitization and challenge (*P < 0.01 versus vehicle control). BAL chitinase activity 48 hours after OVA or PBS challenge of OVA-sensitized mice treated with allosamidin (D) or anti-AMCase/preimmune (control) serum (P < 0.01) (E) starting the day before the aerosol challenge. (F) Methacholine sensitivity of OVA-sensitized mice was assessed 1 day after antigen challenge, using noninvasive barometric whole-body plethysmography (*P < 0.05 versus the other three groups) (11).

Although OVA sensitization and challenge caused a significant increase in the levels of mRNA encoding IL-4, IL-5, and IL-13 (Fig. 3A), anti-AMCase and allosamidin had no measurable effect on these inductive responses and did not induce gamma interferon (Fig. 3A) (11). In contrast, anti-AMCase markedly decreased the ability of constitutive and inducible transgenic IL-13 to induce BAL as well as tissue eosinophilia, lymphocyte accumulation, and inflammation (Fig. 3, B and C). Thus, chitinase inhibition is effective, even in the setting of established IL-13–induced pathology (11). In these experiments, inhibition using anti-AMCase did not alter the ability of IL-13 to stimulate the expression of IL-13 receptor (R)α1, IL-13Rα2, IL-4Rα, or phosphorylated signal transducer and activator of transcription (STAT)-6 (Fig. 3D) (11). However, the ability of IL-13 to stimulate the expression of eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)–1, MCP-2, macrophage inflammatory protein (MIP)–1β, C10, and ENA-78 was markedly diminished (Fig. 3E) (11). Interestingly, the levels of expression of MIP-2, Gro-α, RANTES, and Gro-β were not altered, whereas interferon-inducible protein-10 (IP-10) and interferon-inducible T cell alpha chemoattractant (ITAC) were induced after this intervention (Fig. 3E). The likelihood that this was, at least partially, due to direct effects of AMCase was suggested by the fact that epithelial cells produced exaggerated levels of MCP-1 and eotaxin after transfection with an AMCase-expressing construct or exposure to recombinant AMCase protein (Fig. 3F) (11). These studies demonstrate that AMCase does not play a role in Th2 cytokine induction but rather mediates pathogenesis by contributing to the effector responses of IL-13, including the chemokine response that is central to tissue inflammation. They also demonstrate that this is due, at least in part, to a STAT-6–independent pathway contributing to optimal IL-13–induced chemokine expression.

Fig. 3.

Effect of anti-AMCase on Th2 and Th1 cytokines and IL-13 effector pathway activation. (A) RT-PCR analysis of the mRNA encoding IL-4, IL-5, IL-13, interferon-γ, and β-actin in lungs from OVA-sensitized mice that were challenged with OVA (+ OVA) or vehicle (–OVA) and treated with preimmune serum or anti-AMCase. (B) CC10-rtTA-IL-13 transgenic (+) and littermate control (–) mice were treated with preimmune serum or anti-AMCase starting 1 day before doxycycline administration, and BAL cellularity (top) and morphometric tissue inflammation (bottom) were evaluated 12 days later. (C) Five-week-old CC10-IL-13 transgenic (+) and littermate control (–) mice were treated with preimmune serum or anti-AMCase, and BAL cellularity was evaluated 14 days later. (D) RT-PCR was used to characterize the levels of expression of IL-13 receptor components (top), and immunoblotting with antiphospho STAT-6 (bottom) was used to detect STAT-6 activation in CC10-rtTA-IL-13 transgenic (+) and negative (–) mice on doxycycline water. (E) The levels of chemokine mRNA and protein in lungs from CC10-rtTA-IL-13 transgenic (+) and negative (–) mice on doxycycline for 2 weeks were evaluated by RT-PCR (*P < 0.05 for all comparisons) (11). (F) Effects of AMCase on mouse lung epithelial cell (MLE) chemokine production. In the top panel, MLE cells were transfected with expression constructs containing AMCase or vector alone (11). Forty-eight hours later, the induction of eotaxin and MCP-1 was compared with the effects of in vitro culture with rIL-13 (10 ng/ml). In the bottom panel, MLE cells were incubated for 48 hours with rAMCase or rIL-13 (10 ng/ml), and MCP-1 protein production was assessed by enzyme-linked immunosorbent assay (11).

Although expression of AMCase was not readily apparent in human lung samples derived from control patients with nonpulmonary disease, expression was readily detected in epithelial cells and macrophages in lung tissue samples taken from patients with asthma (Fig. 4, A and B). In these studies, the percentage of the epithelium with AMCase mRNA expression and the number of AMCase-positive subepithelial cells in samples from asthmatics were significantly greater than seen in controls (Fig. 4B). Thus, in keeping with observations made in the murine asthma model, AMCase expression is also markedly increased in human asthma.

Fig. 4.

AMCase mRNA in human asthma. (A) In situ hybridization was undertaken with human AM-Case probes by using lung tissues from autopsies from asthmatic patients and patients who died from nonpulmonary diseases (panels a to d) and a lung biopsy from an asthmatic patient (panel e). (B) Quantitation of AMCase expression in biopsies from asthmatic subjects (n = 9) and non-asthmatic subjects (n = 7). Positive staining of epithelial cells is expressed as a cellular percentage, and positive subepithelial cell staining is expressed as cells per microscopic field.

The present study demonstrates that AMCase is potently and selectively induced during Th2-mediated, IL-13–induced inflammation. The fact that AMCase-based interventions diminish Th2 inflammation and that this response is the result of an IL-13 receptor and STAT-6–independent pathway demonstrate that AMCase contributes downstream during IL-13–induced pathology. The relevance of these findings to disease is also highlighted by the exaggerated expression of AMCase in human asthma.

These findings add to our understanding of the pathogenesis of Th2 inflammation and asthma in a number of ways. First, because chitin is a potent T and B cell adjuvant (14), which can shift developing immune responses in a Th1 direction (7, 15), it is reasonable to speculate that chitinases might contribute to the eradication of chitin-containing pathogens and shift of tissue inflammation in a Th2 direction. The ability of a host to produce chitinases could thus be an important determinant in an individual's propensity toward atopy and/or asthma. Second, the “hygiene hypothesis” suggests that the increase in the prevalence of asthma seen in recent decades is the result of improved hygiene and health care within industrial societies. It is believed that this decreased the incidence of childhood infections that would normally stimulate the immune system in a direction that would mitigate against asthma (3, 16). Because infections with chitinase-containing pathogens can induce Th1 responses, one can speculate that a decrease in infections with these pathogens, or an enhanced ability to metabolize chitin, could contribute to these asthmatic Th2 responses. Last, AMCase has a pH optimum of 2.3, and airway acidification that resolves with steroid therapy is well described in acute asthma (17). These findings suggest that airway pH may be an important controller of AMCase activity and that the beneficial effects of steroids in asthma may be mediated, in part, by their ability to increase pH and decrease AM-Case bioactivity. In combination with studies highlighting the expression of the chitinase-like proteins, YM-1 and YM-2 (18) and AMCase (19) in animal models, these studies emphasize the potential importance of chitinases as mediators of Th2 responses. The present studies also suggest that AMCase is a potential therapeutic target that can be manipulated to control asthma and other forms of IL-13– or Th2-mediated pathology.

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Materials and Methods

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Table S1

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