| BackgroundAcute respiratory distress syndrome(ARDS)is a clinical syndrome caused by pulmonary or extrapulmonary factors,which is characterized by intractable hypoxemia.ARDS has a high incidence and mortality in ICU patients such as burn injury,which is difficulty in treatment.ARDS is a common problem in clinic and a hot topic in clinical and basic research.Basic research found that the pathogenesis of ARDS is a systemic imbalance of inflammatory response mediated pulmonary parenchymal injury,in which inflammatory factors,in particular cascade amplification of inflammatory cytokines play an important role.The inflammatory factors produced during the course of ARDS can eventually become independent injury factors and aggravate ARDS.CD74 is a Single transmembrane receptor protein consisting of 232 amino acid(aa).Recently,a study shows that CD74 on the cell surface,can interact with macrophage migration inhibitory factor,contributing the development of ARDS.The membrane CD74 protein is divided into three segments,including cytoplasmic domain(CD741–46),transmembrane segment(CD7447–72)and ectodomain(CD7473–232).Soluble CD74(s CD74)is a membrane truncated protein,ectodomain of CD74,which is approximately 25 KD.Richard Bucala and his team members firstly identify s CD74 in serum of patients with autoimmune liver disease in 2014,and they initially confirmed the important role of s CD74 in immune diseases.Bong-Sung Kim and his team members identify that the serum levels of s CD74 in non-survivors are low in the first 12 hour but gradually increased after 24 hours.Our experimental team has done some research on s CD74 in the early stage.We found that s CD74 was present in both mouse peripheral blood and BALF in ARDS mouse model,and the content of s CD74 was positively correlated with the severity of ARDS.In addition,there was a positive regression relationship between s CD74 content and inflammatory cytokines TNF-α and IL-6 in mouse alveolar lavage fluid.It is found in clinical trial that s CD74 levels were increased in patients with ARDS in the early stage and elevated serum s CD74 levels were associated with poor clinical outcomes.These results above all suggest that s CD74 may be involved in the occurrence and progression of ARDS.What role does s CD74 play in the ARDS process? Is it as an independent injury factor aggravating lung injury as other inflammatory factors? There is no relevant literature report yet.Therefore,this subject has confirmed in vivo and in vitro that s CD74 can promote the inflammatory response of mouse lung and lung-related cells.we preliminarily explored its possible mechanism and confirm that s CD74 promotes inflammatory response by activating the inflammatory classic signaling pathway NF-κB.Part one.Inflammation of mice induced by s CD74 airway instillation(in vivo experiments)Objective:To investigate the phenomenon of s CD74 promoting inflammatory response at the animal levelMethods: The mouse model was established by instillation of s CD74 into the airway.Experiment groups were divided into 2h、6h、8h、12h and 24 h group according to the different stimulation time of s CD74.We set up different concentration groups(0.01、0.1、0.5、1、2mg/kg)according to the stimulus dose of s CD74.Meanwhile,we set up Ig-Fc protein group as a control.Peripheral blood、lung tissue and bronchoalveolar lavage fluids(BALF)were collected in time.The total RNA was extracted from lung tissues and the expression of inflammatory gene was examined by RT-PCR.The content of inflammatory cytokines in BALF was measured by ELISA.The total protein in BALF was detected by BCA protein quantification method.The extent of pulmonary edema was investigated by lung wet / dry ratio.The gross lung injury was detected by HE staining.The content of inflammatory cytokines in peripheral serum was measured by ELISA.Results: Inflammatory genes TNF-α,MIP-2 and IL-6 in lung tissue increased 2.1,3.4 and 2.8 times respectively in 2 hours group after 0.5mg/kg s CD74 airway instillation,after which the inflammatory genes gradually decreased to normal control levels.The variation tendency of inflammatory cytokines increased first and then decreased with time.The concentration of TNF-α and MIP-2 were 560 pg/ml and 107 pg/ml at 8 hours group,which was the peak of inflammatory cytokine secretion.The expression of inflammatory genes and inflammatory cytokines secretion showed a concentration-dependent relationship with the concentration of s CD74 instillation.There was significant difference between sCD74 group and Ig-Fc protein group.There were no significant different between s CD74 group and control in the total protein content in lung lavage fluid、the ratio of dry to wet lung tissue and serum inflammatory factors in peripheral blood after airway instillation of s CD74.Lung HE staining showed no obvious injury.Conclusions:Instillation of s CD74 into the airway could induce the high expression of inflammatory genes and the corresponding secretion of inflammatory cytokines in mouse lungs.But,s CD74 had little effect on the inflammatory injury in the lungs and systemic inflammatory response.Part two.s CD74 induces inflammatory response in lung cells(in vitro experiments)Objective:To investigate the phenomenon of s CD74 promoting inflammatory response at the cells levelMethods: RAW264.7 cells were selected as representative of lung macrophages,A549 cells as epithelial cells and HUVEC as vascular endothelial cells.The biological functions of s CD74 were explored in three cell lines.Experiment groups were divided into 2h,8h,12 h and 24 h groups according to the different incubation time of s CD74.Experiment groups were divided into 10,100 and 1000ng/ml groups according to the concentration of s CD74 incubated with cells.The supernatants of cells and RNA lysate were collected at different time points.The relative expression of inflammatory genes in the cells was detected by RT-PCR,and the secretion of inflammatory cytokines in the supernatant was detected by ELISA.Results: When macrophages were stimulated with 1000ng/ml s CD74,the relative expression levels of inflammatory gene TNF-a,MIP-2,and IL-6 increased 4.0,11.8,and 2.6 times respectively at 2 hours,after which the inflammatory genes gradually decreased to normal control levels.The contents of inflammatory cytokines TNF-a and MIP-2 in macrophage supernatant increased with the stimulation time extension and stimulation concentration increasing.s CD74 could stimulate the expression of inflammatory gene TNF-a and IL-6 in A549 and HUVEC cells at 8h.But,s CD74 had little effect on cytokines secretion of them.Conclusions:s CD74 can stimulate lung-related cellular inflammatory gene expression and stimulate macrophages to secrete inflammatory cytokines.Part three.The initial study is made on the mechanism of s CD74 promoting inflammatory responseObjective:(1)To investigate the signaling pathway of sCD74 to promote inflammatory response.(2)To investigate the cell membrane receptor of s CD74.Methods: The cytoplasm and nuclear proteins were extracted at 0,10,and 30 min after stimulation with 1000ng/ml s CD74.The expression of IκB,phospho-IκB,P65,phosphate-P65 in the cytoplasm and the expression of phospho-P65 in the nucleus were detected by Western Blotting.We detected the above protein expression changes after BAY11-7082 pretreatment cells for 30 min.The changes of inflammatory cytokine secretion in macrophages after pretreatment with BAY11-7082 were detected by ELISA.In cell lines such as A549,HUVEC,HEK293,Jurkat,RBL,and RAW264.7,flow cytometry was used to analyze the binding of hs CD74 to their cell membranes.Results: The protein content of phosphate-IκB,phosphate-P65 in cytoplasm and phosphate-P65 in nucleus was significantly increased after the stimulation of s CD74 in macrophages lysate.After the NF-κB pathway inhibitor treatment,the increased protein in the pathway was significantly decreased,meanwhile the inflammatory cytokines secreted by the cells also decreased.hs CD74 could bind to a variety of human-derived cells(A549、HUVEC、HEK293 、Jurkat)in a dose-dependent manner.hs CD74 cannot bind to rat-derived(RBL)and mouse-derived(RAW264.7)cell membranes in a dose-dependent manner.Conclusions: s CD74 is involved in the inflammatory response of lung through NF-κB pathway.hs CD74 can bind to a variety of human cell membranes. |
PDF Full Text Request