The Effects Of CpG-ODN On Airway Remodeling And Expression Of ADAM33 Gene In Chronic Asthmatic Mice

BackgroundAsthma is a common and complex disease, which was estimated to affect about 155 million individuals worldwide. Its prevalence is increasing.It has been recognized that asthma is the type 2 T-helper-cell (Th2) mediated chronic airway inflammation. But airway remodeling is also considered to play an important role in the pathogenesis of asthma. The end result of repeated cycles of inflammation and repair may be imperfect repair resulting in a structurally and functionally abnormal remodeling of the airway. The structural remodeling changes noted in asthmatic airway include epithelial metaplasia, goblet cell hyperplasia, subepithelial fibrosis and smooth muscle hypertrophy and hyperplasia. Airway remodeling may be related to a large component of airway hyperreactivity (AHR), development of fixed airway obstruction and corticosteroid resistance. Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing a unmethylated CpG motifs have many immunostimulatory roles. The effect of CpG-ODN is known to induce antigen-specific Th1 responses and inhibit Th2 responses. Several studies have demonstrated that CpG-ODN has protective effect on inhibiting Th2 responses, eosinophilic airway inflammation and airway reactivity to methacholine in mouse models of acute asthma. It has been reported that CpG-ODN also can reverse established airway inflammation and AHR. Although these studied have demonstrated that CpG-ODN may have protective and therapeutic effects in allergen-induced acute asthma, it is not clear that CpG-ODN can inhibit features of airway remodeling associated with chronic asthma.The multiple genes and environment factors play roles in pathogenesis of asthma. A recent report provided evidence that single nucleotide polymorphisms (SNPs) of a disintegrin and metalloprotease domain 33 (ADAM33) are significantly associated with asthma and AHR. ADAM33 which encodes a metallprotease may link to epithelial repairing after injury. ADAM33 is preferentially expressed in fibroblasts, smooth muscle, myofibroblasts but not in epithelial cells and T cells. The selective expression of ADAM33 strongly suggests that alterations in its activity might be associated to small-airway remodeling. Now it is still not clear whether ADAM33 might express higher or lower in asthmatic models. Objective(1) To investigate that CpG-ODN intervention could have inhibitory effects on the development of airway remodeling and the expression of ADAM33 in an ovalbumin (OVA)-sensitized mouse model of chronic asthma.(2) To study the effect of Th2 cytokines (IL-4 and IL-13) on ADAM33 mRNAexpression in cultured human lung fibroblast. Methods(1) Part One Forty female C57BL/6 mice were randomly divided into four groups (n=10): ?Group A (chronic asthma model): mice were sensitized by means of intraperitoneal injection of OVA (10p,g) precipitated with aluminium hydroxide (lOOug) on days 1 and 14. From day 21, mice were challenged by nebulized 2.5% OVA solution (30min/d, three times a week for 8 weeks). ?Group B (CpG-ODN intervention group): mice were sensitized and challenged as above, also received 60ug CpG-ODN by means of intraperitoneal injection for once two weeks. ?Group C (GpG-ODN control): Mice were received GpC-ODN instead of CpG motifs, otherwise were treated as Group B. ? Group D (saline control): mice were sensitized and challenged by saline.Mice were killed 24h after the final OVA challenge. Blood was obtained for eosinophil counts and measurement of serum IgE, IgGl and IgG2a by enzyme-linked immunoabsorbent assay (ELISA). Bronchoalveolar lavage fluid (BALF) was collected for total and differential counts. The concentration of inteleukin-13 (IL-13) and transforming growth factor-beta 1 (TGF-pl) in BALF was measured by ELISA. Total RNA was isolated from the right lung of each mouse, and mRNA levels for ADAM33, IL-13, interferon-y (IFN-y) and TGF-pi were determined using real-time polymerase chain reaction (real-time PCR). Left lung was isolated for pathological examination. Lung sections were stained with hematoxylin and eosin (HE), Masson's Trichrome, periodic acid Schiff (PAS). Other sections were prepared for immunohistochemistry using two monoclonal antibodies against a-smooth muscle actin (a-SMA) and TGF-pi. Morphometric analysis of the stained sections was performed using computerized image analysis system.(2) Part Two Human fetal lung fibroblasts (MRC-5) were cultured and stimulated with IL-4 or/and IL-13, the expression of ADAM33 mRNA was evaluated by real-time PCR.Results(1) Part One Eosinophil counts and the levels of serum IgE^ IgGl and IgG2a in chronic asthmatic model (Group A) were significantly higher than those of Group D (P<0.01). Compared with Group A and C, the EOS numbers and the levels of IgE, IgGl in Group B significantly decreased, but IgG2a significantly increased (PO.01).The numbers of total cells, eosinophils and the concentration of IL-13 and TGF-pi of BALF in Group A were significantly higher than those of Group D (P<0.05). Compared with Group A and C, those in BALF of Group B all significantly decreased (PO.01). However there was no difference between Group A and Group C (P>0.05).