Early Recognition Of Immune Dysfunction In Burned Patients With Delayed Fluid Resuscitation And The Intervention Study

Object:1.To investigate changes in the expression of HLA-DR antigen on CD14⁺ monocytes and lymphocyte apoptosis rate in peripheral blood of burned patients with delayed fluid resuscitation and its relationship with the development of sepsis.2.To investigate the regulating effect of cholinergic drug carbachol and thymosinα₁ on immune function of monocyte and lymphocyte in human peripheral blood stimulated by LPS.3.To investigate the effect of carbachol on production of cytokines from human monocytes stimulated by lipopolysaccharide and its receptor mechanism.Methods:1.50 patients with a total burn surface area of at least 30 percent were divided into two groups:delayed resuscitation group and undelayed resuscitation group.Peripheral blood was collected on day1,3,7,14 and 28 after burn injury.After the occurrence of sepsis,peripheral blood was-drawn consecutively for two days.20 healthy people were taken as controls.Expression of HLA-DR antigen on CD14⁺ monocyte and lymphocyte apoptosis rate was determined by direct immunofluorescence on a flow cytometer,and TNF-αand IL-10 were measured by ELISA.2.Monocyte and lymphocyte were separated from peripheral blood of healthy adults,and were divided into four groups: control,LPS stimulation alone,LPS stimulation + carbachol and LPS stimulation + thymosinα₁ groups.Monocytes and lymphocytes in 1×10⁶ cells ml^-1were added with different doses of carbachol(100,10,1,0.1,0.01μmol/L)or thymosinα₁(100, 10,1,0.1,0.01μg/mL)followed by stimulation of LPS(100ng/mL).After 12 hours of incubation,expression of HLA-DR antigen on CD14⁺ monocyte and lymphocyte apoptosis rate were determined with direct immunofluorescence on a flow cytometer.3.Primary blood mononuclear cells from healthy donors were isolated by density-gradient centrifugation with Ficol/Hypaque,suspended in RPMI 1640 medium with 10%heat inactivated fetal calf serum,and seeded in 6-well plates.After incubation for 2 h at 37℃,adherent cells were detached with 0.25%Trypsin-EDTA,resuspended(2×10⁵ cells ml^-1)in RPMI 1640 medium with 10%heat inactivated fetal calf serum,and seeded in 96-well plate coated with capture antibody.The cells for test were divided into 6 groups:control,LPS stimulation alone,LPS stimulation + carbachol,LPS stimulation + carbachol + atropin(an antagon of cholinergic M receptor),LPS stimulation + carbachol +α-Bungarotoxin(an antagon ofα₇ subunit of cholinergic N receptor),and nicotine groups.After the manipulation,cells were incubated at 37℃for an appropriate length of time.Remove cells,and add biotinylated detection antibody, streptavidin-alkaline phosphatase conjugated,and substrate BCIP/NBT in order. Content of TNF-α,IL-6 and IL-10 were determined by Automated Elisa-Spot Assay Video Analysis Instrument.Results:1.Expression of HLA-DR antigen on CD14⁺ monocyte in peripherial blood of burned patients was significantly lower than that of controls(P＜0.01),and delayed resuscitation group were lower than that of undelayed resuscitation group,which reached statistical difference at every time point(P＜0.01 or P＜0.05),and was much lower when complicated by sepsis. Lymphocyte apoptosis rate of bum patients was higher than that of controls(p＜0.05).Lymphocyte apoptosis rate of delayed resuscitation group was higher than that of undelayed resuscitation group,and the difference reached statistical significance on day 3 and 28(P＜0.05).When complicated by sepsis, lymphocyte apoptosis rate was much higher.The detection rate of TNF-αin delayed resuscitation group were higher than that of controls and undelayed resuscitation group(P＜0.05),but the concentrations of TNF-αshowed no difference between the delayed and undelayed resuscitation group.When sepsis occurred,the detective rate and concentrations of TNF-αwere both higher than that of controls and patients without sepsis,which showed statistical differences. Expression of HLA-DR on CD14⁺ monocytes was negatively correlated with IL-10 levels,the association reaching statistical significance on dayl,7,and 28 after burns(P＜0.01).2.HLA-DR expression was significantly lower in monocytes with LPS stimulatin than those without.When pretreated with carbachol or thymosinα₁,HLA-DR expression significantly increased in a concentration-dependent manner.Apoptosis rate in lymphocytes stimulated by LPS alone,was significantly higher than those in controls,and much decreased, when pretreated with carbachol or thymosinα₁,which also showed a concentration-dependent manner.3.When monocytes were stimulated by LPS alone,concentrations of TNF-α,IL-6 and IL-10 were obviously higher than that of controls(P＜0.01).After being pretreated by carbachol,concentrations of TNF-αand IL-6 obviously decreased compared with that of LPS stimulation alone group (P＜0.01).But there's no significant change in IL-10 concentration.Decrease of TNF-αand IL-6 showed concentration-dependent manner when stimulated with carbachol fluctuated within 0.01-100μmol/L.When pretreated by atropine before addition of carbachol,there's no significant changes in concentrations of TNF-αand IL-6.When pretreated withα-Bungarotoxin before carbachol administration, inhibitive effect of carbachol on production of TNF-αand IL-6 were blocked.Conclusions:1.Immune function was suppressed in severely burned patients with delayed resuscitation,especially when sepsis was complicated.Expression of HLA-DR antigen on CD14⁺ monocyte and lymphocyte apoptosis rate appears to be an useful parameter for monitoring the immune function of burned patients. Expression of HLA-DR antigen on CD14⁺ monocyte of lower than 10%and lymphocyte apoptosis rate of higher than 15%were considered to be the signal of the happeness of sepsis.2.Inflammatory mediators were markedly produced in severely burned patients with delayed resuscitation.IL-10 had an inhibitive effect on the expression of HLA-DR antigen on CD14⁺ monocyte 3.carbachol and thymosinα₁ had a significant inhibitory effect on LPS induced immunosuppression in human monocyte and lymphocyte.4.Carbachol exerts strong anti-inflammatory effect on monocytes by activatingα₇ subunit of cholinergic N receptor.