Cloning And Expression Of HIL-10 And To Study Its Anti-inflammation Property In Mouse Allergy Induced Airway Inflammation

Object: To construct the pBAD/hIL-10 expressing plasmid and induce human IL-10 expression in E.Coli. Purify the rhIL-10 and investigate its anti-inflammation property in a murine model of asthma- like reaction Methods: Peripheral blood mononuclear cells(PBMCs) were isolated from peripheral blood of normal subjects and stimulated with ConA for 24 hours Total RNA were extracted from PBMCs and the intact cDNA of hIL-10 was amplified by RT-PCR. The hTL-IOcDNA was cloned into pBAD/gIIIA vector and transformed into E.coli. The DNA sequence of selected clones were completely consistent with the published hIL- IOcDNA. Induced with arabinose, the pBAD/hIL-10 produced hIL-10 in periplasmid space of E.Coli The expressed hIL-10 was purified and its property of anti-inflammation was investigated in a murine model of asthma-like reaction. Female mice(8 weeks) were sensitized i.p. with 20 μg OVA in 2mg Al(OH)3 adjuvant(alum) at day 1 and 10.Mice were challenged at day 22% 23% 24 via the airway by OVA (1% in PBS)for 20 minutes by ultrasonic nebulization. Experiments were performed in three groups of mice,20 minutes later ,mice in control group were treated with 2ml PBS by ultrasonic nebulization for 20 minutes, mice in treatment group were treated with rhIL-10(10ng) by ultrasonic nebulization for 20 minutes. Mice in protection group were treated with rhIL-10(10ng) by ultrasonic nebulization for 20 minutes at 24hs and 0.5hs before challenge. All mice were killed at day 26,the lung were washed twice with lml PBS .The BAL fluid was centrifuged immediately and cells were resuspended in 0.5ml PBS. Lung tissure were fixed in 10% buffered formalin,embeded in paraffin, sectioned,stained with HE and examined for pathological changes under light microscopy.IL-5 levels of BAL fluid were measured using ELISA. Results: To directly examine the role of IL-10 in allergen-induced pulmonary inflammation, we analyzed cellular components in the bronchoalveolar lavages(BAL) of each group. Although the composition of their inflammation cells was comparable in the three groups, The amounts of infiltrating cells of the treatment group and protection group was less than that of the control group Similar to the difference in BAL fluid, histopathological analysis(HE staining) also showed differential patterns of respiratory tract inflammation Control group showed higher cellular infiltration than treatment group and protection group We also measured the levels of IL-5 of BAL fluid, there was no difference among three groups. Discussion: Cytokine play an important role in modulating inflammatory responses .IL-10 is a regulatory cytokine that has been suggested for treatment of asthma because of its immunosuppressive and anti-inflammatory properties. We constructed a pBAD/hIL-10 vector expressing hIL-10 and found that exogenous recombinant IL-10 can inhibited in vivo IL-5 production. This may be due to the property of IL-10 that it could prevent cellular infiltration in inflammatory response.