| Background Acute respiratory distress syndrome(ARDS)is a common critical illness due to direct injury(contusion,inhalation injury,pneumonia,aspiration,mechanic ventilation)or indirect injury(sepsis,blood transfusion,pancreatitis,traumatic brain injury).It has been well established that ARDS is characterized by endothelial injury and epithelial injury as a consequence of inflammatory reaction.And cytokines and chemokines play an important role in initiating,mediating and maintaining lung inflammation.Macrophage migration inhibitory factor(MIF)is a pleiotropic inflammatory molecule which contributes to the pathogenesis of various diseases such as immune diseases,inflammation,cancers,cardiovascular diseases and endocrine diseases.During early phases of ARDS,MIF can stimulate macrophages to release multiple inflammatory cytokines including IL-1,IL-6,IL-8,IL-10 and TNF-?,as well as chemokines,thus recruiting and activating neutrophils in lung tissues.And oxidants,proteases,leukotriene and other pro-inflammation mediators produced by activated neutrophils can further aggravate the injury of lung tissues.These findings suggest that MIF may be the key initial inflammatory factor participating in cascade reaction.In 2003,cell surface CD74 was proved as a high-affinity membrane receptor for MIF.And mechanisms that MIF mediate pulmonary inflammation include CD74 engagement and phosphorylation of MAPKs.Takahashi et al reported that anti-CD74 antibody could inhibit MIF-induced MAPK phosphorylation and chemokines release.Recently,a soluble form of CD74(sCD74)was identified in serum of patients with autoimmune liver disease and might be released after processing by proteolysis from hepatic stellate cell.In addition,the in vitro experiments using human primary skin fibroblasts showed that circulating sCD74 is bioactive and could neutralize MIF signal transduction activity.Thus,according to previous findings,we hypothesized that sCD74 might exist in circulation or alveolar space under ARDS pathological conditions and sCD74 would be associated with clinical outcomes in patients with ARDS caused by trauma or burn.In the first part of present work,we used a mouse model of lipopolysaccharide(LPS)-instillation induced inflammatory lung injury and a mouse model of cecal ligation and puncture(CLP)induced inflammatory lung injury.First,we detected the expression of CD74 in lung tissues and the level of sCD74 in serum and bronchoalveolar lavage fluids.Then,the association between sCD74 and severity of lung injury as well as the degree of inflammation was analyzed.Finally,we investigated the cell source of sCD74 and its biological function with in vitro experiments.In the second part,we measured sCD74 in serum from patients with ARDS and examined the relationship of serum sCD74 levels to clinical outcomes.Part one.sCD74 levels in murine models of inflammatory lung injury and its biological function(Basic research)Objective(1)To investigate the levels of sCD74 in serum and bronchoalveolar lavage fluids from inflammatory lung injury murine models and test the relationship between sCD74 and severity of lung injury.(2)To investigate the cell source of sCD74 and its biological function.MethodsThe murine models of inflammatory lung injury induced by instillation of lipopolysaccharide(LPS)intratracheally or cecal ligation puncture(CLP)were adopted in this study.The total RNA was extracted from lung tissues and the expression of CD74 mRNA was examined by RT-PCR.And CD74 protein expression in lung tissues was measured by western blot.Immunohistochemistry and immunofluorescence assays were used to test the location of CD74 in lungs.Serum and bronchoalveolar lavage fluids were collected to determine the levels of sCD74 by using dot blotting and ELISA methods.Lung tissues were assayed for histologic and wet/dry ratio to reflect the severity of lung injury.The concentrations of sCD74 in supernants from rmMIF stimulated RAW264.7 cells and MLE-12 cells were detected.In addition,the CD74 expression of stimulated cells were also examined.The cell viability and apoptotic response of RAW264.7 cells relative to rmMIF stimulation were examined by CCK-8 Kit and FITC-Annexin V Kit,respectively.ELISA was used to detect the TNF-? and MIP-2 levels in supernants from RAW264.7 cells under rmMIF stimulation with or without rmsCD74-FC.ResultsIn the two murine models,the expression of CD74 in lungs was increased at certain time.Immunohistochemistry and immunofluorescence assays showed that CD74 was mainly localized on cell surface of pulmonary macrophages.sCD74 in serum and bronchoalveolar lavage fluids was significantly increased compared with controls.BALF sCD74 was observed to be positively correlated with MIF(r= 0.609,p< 0.05),TNF-?(r= 0.511,p< 0.05)and IL-6(r= 0.585,p< 0.05).The sCD74 level was below the detection limit of ELISA in the supernatant of MLE-12 cells under rmMIF stimulation,whereas sCD74 was detected in the supernatant of RAW264.7 cells under rmMIF stimulation.Furthermore,the ability of rmMIF to induce apoptosis was relatively low,which was not signifcant compared to controls.And the cell survival rate was not reduced by diff erent concentrations of rmMIF,either.When the concentration of rmsCD74-FC was at a low level(10–~200 ng/ml),MIF stimulation induced release of TNF-? and MIP-2 decreased in a dose-dependent manner.When the addition of rmsCD74-FC exceeded ~2?g/ml,TNF-? and MIP-2 concentrations in the culture media significantly increased compared to control and MIF groups.ConclusionsThe expression of CD74 and secretion of sCD74 were increased in inflammatory lung injury.And increased BALF sCD74 levels positively correlated with the severity of pulmonary inflammation and lung injury.Macrophage was one of cell sources of sCD74.Notably,sCD74 can stimulate the production of inflammatory cytokines as well as neutralize MIF's pro-inflammatory activity.Part two.Clinical significance of sCD74 in ARDS caused by trauma or burn(Clinical trial)ObjectiveTo observe the changes of serum sCD74 in ARDS patients and to test the hypotheses that higher levels of serum sCD74 would be associated with worse clinical outcomes.MethodsThis retrospective study was registered on ClinicalTrial.gov(NCT02201446)and conducted at the Changhai Hospital,Shanghai.Eighty-one patients were enrolled consecutively and identified as ARDS according to the Berlin definitions of ARDS.Serum sCD74 protein concentrations were investigated in 81 patients and 58 healthy volunteers with ELISA method.Demographic and clinical severity data as well as serum sCD74 were compared between surviving and non-surviving patients with ARDS.Receiver operating characteristic curve analysis was carried out to determine the prediction of serum sCD74 on mortality.Kaplan-Meier curves and log-rank test calculations were performed to display the impact of serum sCD74 levels on survival.Multiple logistic regression analyses were carried out to determine the association between serum sCD74 levels and mortality.ResultsOf the 81 ARDS patients,the average age was 48.02± 16.05 and 54.3% were male.Inhalation injury(56.8%)was the major primary etiology of lung injury,followed by trauma(43.2%).At the time of admission,the average of APACHE II score was 14.35±6.39.And fifty-one patients accepted mechanical ventilation.Median ventilator-free days and length of ICU stay was 22 days(IQR,8.5-28)and 23 days(IQR,15-44),respectively.14 of the 81 patients died(17.3%)during their hospital stay.When compared with survivors,non-survivors were more likely to suff er inhalation injury and receive mechanical ventilation,and had higher AHACHE II score,lower FiO2/PO2 ratio and fewer unassisted ventilation days(p<0.05).The day 1 serum levels of MIF,TNF-?,IL-6 and sCD74 were 73.64±23.27ng/ml,97.09(92.32,100.82)pg/ml,101.63(79.5,138.9)pg/ml and 75.83(65.76,96.36)ng/ml,whereas the day 3 serum levels were 129.27±41.12ng/ml,138.67±64.26pg/ml,305.36±177.14pg/ml and 117.0(105.2,160.1)ng/ml,respectively.The day 1 serum sCD74 levels of non-survivors(77.96(65.82,124.63)ng/ml)were markedly outnumbered those of survivors(75.25(64.99,95.74)ng/ml),but the difference showed no significance(p>0.05).However,the day 3 serum sCD74 levels of non-survivors were significantly higher than those of survivors((160.9(133.7,179.3)vs.115(103.9,147.0),p<0.05).Furthermore,Day 3 serum sCD74 levels significantly correlated with MIF(r=0.333,p<0.01),TNF-?(r=0.409,p<0.01)and IL-6(r=0.303,p<0.05).The areas under the receiver operating characteristic curves(AUROC curves)to predict mortality of ARDS patients were: 0.58 for Day 1 serum MIF(95% CI: 0.38–0.78,p< 0.05),0.52 for Day 3 serum MIF(95%CI: 0.38-0.72,p>0.05),0.47 for Day 1 serum sCD74(95% CI: 0.29-0.64,p>0.05),and 0.75 for Day 3 serum sCD74(95%CI: 0.61-0.89,p<0.05).Survival analysis showed that patients with Day 3 serum sCD74 >151 ng/ml had higher mortality than patients with lower levels(hazard ratio= 5.71;95% CI,1.51 to 21.51;P< 0.01).And the mortality of patients with Day 1 serum sCD74 > 118 ng/m was higher than that of patients with Day 1 serum sCD74 < 118 ng/m,which showed a trend towards statistical significance(p=0.058).Multivariate linear regression analyses showed that there were 0.11 more days on ventilator for each 1 ng/ml increase in Day 3 serum sCD74.In a multivariate logistic regression model,we demonstrated that Day 3 serum sCD74 was an independent predictors of mortality(OR= 6.72,95% CI,1.435–31.4,p = 0.016).Unexpectedly,we did not find Day 1 serum sCD74 to be significantly associated with mortality.But it showed a trend towards statistical significance(OR=13.21,95%CI: 0.83-209.79,p=0.067).ConclusionssCD74 levels were increased in patients with ARDS and elevated serum sCD74 levels were associated with poor clinical outcomes. |
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