The Role Of Acidic Mammalian Chitinase In The Pathogenesis Of Bronchial Asthma

Asthma is a chronic inflammatory disorder of the airways in which many cells and cellular elements,such as activated osinophils,activated mast cells,T lymphocytes,neutrophiles and airway epithelial cells,etc., play a role.The number of Th1 and Th2 and the ratio between Th1 and Th2 are associated with allergic diseases including asthma.Recent studies suggest that diverse populations of regulatory T cells(Treg)make a big difference in the regulation of T-helper type 2(Th2)responses to allergy, thus maintaining functional tolerance.Optimization of the Th1/Th2 balance promises to be the focus of future studies on asthma.Chitin microparticles(CMP)are the granules 1～20μm in diameter. Previous studies showed CMP could decrease airway inflammation of asthmatic mice,including the production of the gamma interferon (IFN-gamma),which served as a Th1 adjuvant.Recently,inhibition of acidic mammalian chitinase(AMCase)in a murine asthmatic model was reported to have a profound anti-inflammatory effect.In these experiments, inhibition using anti-AMCase did not alter the expression of IL-4,IL-5, IL-13 or phosphorylated signal transducer and activator of transcription (STAT)-6.Some animal studies found the expression of chitinase could be induced seven days after allergic sensitization.There was evidence to suggest that AMCase might be an important mediator relating to airway remodeling and pathophysiology of asthma. No chitin synthesized enzyme was detected in the mammalian body. With acidic mammalian chitinase identified,our team attempted to explore the association between chitin and AMCase,and their roles in asthma. Although expression of AMCase was demonstrated in the asthmatic lung, more work needs to be done to determine the roles of this enzyme and chitin in human inflammatory pathology and investigate the contribution of AMCase to asthma.Part 1 The effect of chitin microparticles on the expression of Th2 chemokine, transforming growth factor,and acidic mammalian chitinaseObjectives To investigate the effects of chitin microparticles on airway inflammation,the expression of Th2 chemokine,transforming growth factorβ-1,and acidic mammalian chitinase of asthma.Methods Forty female BALB/c mice were randomly divided into the control group,the asthma group,the oral treatment group(CMP mouth group),and the intranasal treatment group(CMP nose group),with 10 BALB/c mice in each group.All the mice except those in the control group were sensitized by ovalbumin(OVA),and only the two CMP groups were treated with CMP(40μg/mouse).The number of total cells and eosinophils in bronchoalveolar lavage fluid(BALF)were counted.Light and electronic microscopes were used to detect the pathologic histology and morphologic change.The levels of TGF-β₁ and IL-10 in BALF were measured by ELISA.Epithelial TGF-β₁ expression was evaluated in immunohistochemistry.In situ hybridization was used to measure AMCase mRNA in the lung.The expressions of TGF-β₁,AMCase,TARC, MDC,NF-κB,STAT1 and C-jun mRNA in the lung were detected by RT-PCR.Flow cytometry was performed for detecting the intracellular IL-4,IFN-γand Foxp3 production in T cells.Results The intranasal and oral application of microgram doses of chitin microparticles are effective treatments to reduce lung inflammation in both allergic models,which results in elevated level of Th1 and lowered TGF-β₁, AMCase,and STAT1 production during the allergen challenge.The expression of TARC mRNA was increased while the production of MDC mRNA was decreased in the lung of the CMP nose group.In contrast, CMP in oral application markedly decreased the expression of MDC mRNA in the lung.Meanwhile,there was increased expression of NF-κB and C-jun mRNA in the nose group.Conclusions Chitin microparticle suspensions have Th1 immunostimulatory properties and are effective when administered intranasally or orally in mice.Chitin microparticles could attenuate OVA-induced airway inflammation and the expression of TGF-β₁,STAT1 and AMCase mRNA,and increase TARC,NF-κB,C-jun mRNA expression in the lung of asthmatic mice. Part 2 The effect of chitinase inhibitor on the expression of Th2 chemokine and transforming growth factorβ-1Objectives To investigate the effects of chitinase inhibitor allosamidin on airway inflammation,the expression of Th2 chemokine,and transforming growth factorβ-1 of asthma.Methods Thirty female BALB/c mice were randomly divided into the control group,the asthma group,the treatment group by chitinase inhibitor,with 10 BALB/c mice in each group.All the mice except those in the control group were sensitized by ovalbumin (OVA),and only the inhibitor group were treated with allosamidin (0.5mg/kg).The total number of cells and eosinophils in bronchoalveolar lavage fluid(BALF)was counted.Light and electronic microscopes were used to detect the pathologic histology and morphologic change.The levels of TGF-β₁ and IL-10 in BALF were measured by ELISA.Epithelial TGF-β₁ expression was evaluated in immunohistochemistry.In situ hybridization was used to measure AMCase mRNA in the lung.The expressions of TGF-β₁,AMCase,TARC,MDC,NF-κB,STAT1 and C-jun mRNA in the lung were detected by RT-PCR.Flow cytometry was performed for detecting the intracellular IL-4,IFN-γand Foxp3 production in T cells.Results The chitinase inhibitor allosamidin is an effective treatment for reducing lung inflammation in allergic model, which results in the elevation of Treg proportion,IL-10 levels in BALF, and c-jun mRNA,and the reduction of TGF-β₁,STAT1,and AMCase during the allergen challenge.Conclusions Chitinase inhibitor allosamidin has Treg immunostimulatory properties and is found to be effective in the decrease of airway inflammation in mice.Chitinase inhibitor could attenuate the expression of TGF-β₁ and STAT1 mRNA,and increase C-jun mRNA expression in the lung of asthmatic mice.Part 3 Expression of acidic mammalian chitinase gene in human bronchial epithelial cellsObjectives To observe the expression and characteristics of acidic mammalian chitinase(AMCase)mRNA in human bronchial epithelial cells and explore the possible effects of chitin microparticles and budisonide on the production of AMCase mRNA by the Human Bronchial Epithelial Cells(HBE).Methods HBE cells were stimulated with cytokines,CMP and budisonide for different times.AMCase mRNA in the lung was detected by Realtime PCR.Results The level of AMCase mRNA was not increased by IL-13 stimulation within 24hrs.The AMCase mRNA expression was up-regulated at 2h,drastically increased at 48h,and declined nearly to base level at 72 h after stimulation with IL-4.The production of AMCase mRNA was increased following the stimulation time with TNF-α,Der P1,IL-13 and Der P1,IL-4 and Der P1 while there was individul peak expression at 4h of Der P1 stimulation and 2h of IL-4 and Der P1 co-stimulation.CMP stimulation resulted in significant increase of AMCase mRNA while HBE cells were cultured with IL-13 and Der P1,IL-4 and Der P1.However,budisonide could down-regulate the expression of CMP stimulation effect.Conclusions IL-4,TNF-α,Der P1 and CMP could induce AMCase mRNA expression of HBE directly. Corticosteroids could downmodulate the expression of AMCase mRNA.Part 4 Role of acidic mammalian chitinase in regulating the expression of TARC,TGF-β₁ and ICAM-1 mRNA in bronchial bronchial cellsObjectives To observe if acidic mammalian chitinase(AMCase)has any effect on the expression of TARC,TGF-β₁ and ICAM-1 mRNA in human bronchial epithelial Cells(HBE).Methods RNA interference(siRNA)and real time quantitative PCR(RQ-PCR)methods were used to identify differentially expressed genes regulated by AMCase.The target genes included TARC,TGF-β₁ and ICAM-1.The expression of IκBαin HBE was analyzed by Western blot.Resultes AMCase RNA interference resulted in up-regulating expression of TGF-β₁ and ICAM-1 mRNA. Conclusions These results suggest that AMCase could play negative role in the expression of TGF-β₁ and ICAM-1 mRNA in HBE.