| Objective:To study the proinflammatory role of interleukin-33(IL-33)in pulmonary acute respiratory distress syndrome(ARDS)and extrapulmonary ARDS and discuss the clinical significance of IL-33 in ARDS.Methods:1.Plasma samples of eight pulmonary ARDS patients,six extrapulmonary ARDS patients and twenty-four healthy controls were randomly selected from all collected samples,the level of plasma IL-33,TNF-α,IL-1β,IL-2,IL-6,IFN-γ,CXCL1,CXCL8 and CXCL10 were detected by ELISA or cytokine/chemokine magnetic bead panel kit.2.120 male C57BL/6 wild-type mice were randomly divided into six groups: extrapulmonary ARDS group(CLP group),sham group,pulmonary ARDS group(LPS group),Control group,IL-33 antibody group(LPS+anti-IL-33 group),LPS+Ig G group(n=20/group).To induce extrapulmonary ARDS model,cecal ligation and puncture(CLP)was performed in CLP group,and sham group didn’t perform cecal ligation and puncture.LPS group mice were administrated 50 μg of LPS intratracheally in 50 μl phosphate buffered saline(PBS)to induce pulmonary ARDS,50 μl sterile PBS was intratracheally given to control mice.Mice were injected intraperitoneally with 100 mg IL-33 antibody or IgG 30 minutes prior to LPS administration in LPS+anti-IL-33 group or LPS+IgG group.All mice were evaluated at 6,12,24 hour after treatment;the lung wet-to-dry weight ratios were calculated to evaluate lung edema;histopathological examination was observed by HE stain;immunohistochemistry and qRT-PCR were used to detect the expression of IL-33 protein and IL-33 mRNA in lung tissue;the level of serum IL-33,TNF-α,IL-6,TGF-β1,IFN-γ,CXCL1,CXCL8 and CXCL10 were detected by ELISA.Results:1.The level of plasma IL-33 in human and the correlation between IL-33 and other cytokine The concentration of plasma IL-33 was significantly higher in patients with ARDS than healthy controls(P<0.001),and IL-33 concentrations were clearly higher in the plasma from patients with pulmonary ARDS than patients with extrapulmonary ARDS(P<0.01).There was no significant difference between patients with extrapulmonary ARDS and healthy controls(P>0.05).ROC curve analysis was carried out on the concentration of plasma IL-33 in all ARDS patients,the resulted showed that a cut-off level of plasma IL-33 for the diagnosis of ARDS was set at 3.24 pg/ml.The specificity,sensitivity,positive predictive value,and negative predictive value were 79%,86%,71%,and 90%,respectively.ROC curve analysis was carried out on the concentration of plasma IL-33 in patients with pulmonary ARDS,the resulted showed that a cut-off level of plasma IL-33 for the diagnosis of ARDS was set at 5.29 pg/ml.The specificity,sensitivity,positive predictive value,and negative predictive value were 100%,87.5%,96%,and 100%,respectively.Based on correlation analyses between IL-33 and cytokines in patients with ARDS,the concentration of IFN-γ(r=0.715,P<0.01)and IL-2(r=0.680,P<0.05)were significantly correlated with plasma IL-33.2.The general situation in miceCLP group and LPS group began to exhibit a series of character at 6 h after treatment campared to sham group and Control group,including reduced activity,shortness of breath,depressed,hypothermia,anorexia,increased eye purulent secretions,erected and dull hair,diarrhea and so on,and this character gradually worse over time.The survival rate of CLP group at 24 h was 50%(5/10 mouse),and it was 100% in LPS group.The symptoms of LPS+anti-IL-33 group were better than LPS group.There was no significant difference between LPS+IgG group and LPS group.3.Pathological changes in the lung tissue of miceIn the CLP group and LPS group,the lungs stained with hematoxylin–eosin indicated widespread alveolar wall thickness caused by edema,alveolar collapse,severe hemorrhage in the alveoli and a prominent inflammatory cell infiltration.LPS+anti-IL-33 group showed that the above changes were obviously lighter.There was no significant difference between LPS+IgG group and LPS group.In sham group and Control group,no obvious lung injury and inflammation were observed.4.The lung wet-to-dry weight ratios of miceThe lung wet-to-dry weight ratios of CLP group and LPS group mice were significantly increased after treatment campared to sham group and Control group(P<0.001).Pretreated with IL-33 antibody showed decreased the lung wet-to-dry weight ratios compared to LPS group(P<0.001).There was no significant difference between LPS+Ig G group and LPS group(P>0.05).5.Changes of serum IL-33 and inflammatory cytokines in miceIn CLP group,the level of serum IL-33 was not significantly different compared with the sham group at different time points(P>0.05),and the level of serum IL-6,TNF-α and TGF-β1 were significantly increased compared with the sham group(P<0.001).In LPS group,the level of IL-33 in serum was significantly increased after LPS administration(P<0.001),and the level of serum TNF-α,IL-6,IFN-γ,CXCL1,CXCL8 and CXCL10 were all increased compared with the Control group(P<0.05).The LPS induced increase in TNF-α,IL-6,IFN-γ,CXCL1,CXCL8 and CXCL10 levels were significantly reduced by administration of IL-33 antibody(P<0.05).There was no significant difference between LPS+IgG group and LPS group(P>0.05).6.IL-33 mRNA expression in the lung tissue of miceThe expression of IL-33 mRNA in CLP group and sham group weren’t elevated at different time points(P>0.05).The expression of IL-33 mRNA in LPS group was substantially increased 6 h after LPS administration(P<0.05).IL-33 mRNA levels in the lung wasn’t increased in Control group(P>0.05).7.IL-33 protein expression in the lung tissue of miceImmunohistochemistry staining showed that the expression of IL-33 in CLP group wasn’t increased compared with sham group,the expression of IL-33 in LPS group was significantly increased compared with Control group.LPS+anti-IL-33 group showed decreased IL-33 expression compared to LPS group.There was no significant difference between LPS+IgG group and LPS group.Conclusion:IL-33 plays a proinflammatory role in pulmonary ARDS,but we haven’t find the proinflammatory role of IL-33 in extrapulmonary ARDS.Inhibition the expression of IL-33 could effectively reduce the lung inflammatory response and injury of pulmonary ARDS mice,suggesting that IL-33 might be a potential therapeutic target in pulmonary ARDS. |
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