The Primary Study Of CD14^highCD16⁺Subset Of Monocytes And Its HLA-DR Expression In The Exacerbation Of Chronic Hepatitis B

Background Patients with severe hepatitis suffer from serious hepatocytes necrosis,high incidence and poor prognosis[4,27]. There is a percentage of70%of severe hepatitis inChina, which result from chronic hepatitis B and has a high mortality, though the inspectiontechniques has been improved, and the progress of the antiviral drugs advanced as timegoing. The replication speed of virus, viral gene, immune status, or overlap other viralinfection work solely or together in the development of severe hepatitis B[1,3,27]. Itsimmunological mechanism is particularly complex for immune cells, cytokines,chemokines and adhesion molecules involved. Studies shown that HBV specific CD8T（CTL）[2], non-specific lymphocytes NK[5], Treg[8], and INF-γ，TNF-[30]had a closecontact with destruction of liver cells in the severe exacerbation progression of chronichepatitis B. Because of unclear the mechanism entirely for the progression, deficient forspecificial and effective treatment, mortality of patients is at a high rate of40%-60%. Thus,clinical prevention and treatment work for severe hepatitis B are still the enormouschallenges in the current and future.Peripheral blood monocytes differentiating from bone marrow hematopoietic stem cell,are a group of immune cells, involving in the body’s natural immune response mainly. Monocytes can differentiate into macrophages （M） and dendritic cells（DC） in differenttissue. The existence of different populations of blood monocytes is now well establishedaccording to the expression of CD14and CD16. In addition to the classical monocytes,which are strongly positive expression for the CD14cell surface molecule asCD14^highCD16^-monocytes, and take a percentage of85%-90%, there is a population ofmonocytes coexpressing CD16and low levels of CD14antigens, which are identified asCD14⁺16⁺monocytes and occupy about10%of all monocytes. They are thought to bemore mature than the regular CD14^highCD16^-subset, as they exhibit features of tissuemacrophages and dendritic cells[11,14]. And the latter are further divided into two minorCD14low16⁺and CD14^high16⁺subpopulations[14,16]. Increasing evidence shown that,rised CD14⁺16⁺monocytes collaborating with CD14^high16⁺played an important rolein shaping the inflammatory environment. More recently, elevated CD14^high16⁺monocytes were collaborate with HIV/AIDS[10], sepsis[11,12], epticopyemia[25], systemicinflammatory response syndrome（SIRS）, tuberculosis[13], chronic nephritis[14]and so on.Recently, some studies shown the distinct accumulation of CD14⁺CD16⁺monotytes in theblood during the progression of chronic hepatitis B, liver cirrhosis, and acute-on-Chronicliver failure[15,16,17,31]. Guo et al[25]found that CD14⁺16⁺monocytes activated in theTLR4signal transduction caused by endotoxin, through releasing large amounts of TNF-and other inflammatory cytokines, to promote antibody elevating and CTL activation, thusto trigger specific immune system.Comparing with CD14⁺+CD16^-, CD14⁺CD16⁺subset has a higher expression ofHLA-DR, presents antigens processed to CD4+T lymphocytes similar as DC, further toswitch on the specific immune system[18,21,22]. So, HLA-DR expressed on CD14⁺CD16⁺can be as a marker to reflect the immune function potentially. In SIRS, blood CD14⁺CD16⁺monocytes shown down-regulated HLA-DR sharply[25,26]. Wasmuth et al[20]. found thedecrease of expression of HLA-DR on CD14⁺16⁺monocytes also appears in theprogression of decompensated liver cirrhosis, and the decrease in HLA-DR expression between days0and3was associated with a hazard ratio for later mortality of3.36. In acuteliver failure[30]and acute-on-chronic liver failure[17,19], monocyte HLA-DR expressiondecrease more significant. Yolk et al[26]reported when HLA-DR expression onCD14⁺CD16⁺monocytes declined to30%, human body would occur to immune paralysis,patients with prognosis poor, even to death. These results not only contribute to a betterunderstanding of inflammation, but may also give rise to new therapeutics for temporaryimmunology down-regulated, such as decompensated liver cirrhosis and liver failure.Blood regular report prompted the proportion of monocytes in white blood cellsincreased in patients with chronic hepatitis B, especially in severe hepatitis. In the presentstudy，We focused on the function of CD14^high16⁺monocytes and HLA-DR in severeexacerbation progression of chronic hepatitis B and the temporal relationship to investigate,in order to search for a novel immune mechanism to explain the progression, and provide anew temporal alarm marker for diagnosis in the early period.Objectiveâ‘ To investigate the relationship between the ascendant subsets and theprogression by observing the subsets of blood monocytes in different stages of populationswith chronic hepatitis B;â‘¡To investigate the relationship between HLA-DR and the progression by observingthe expression of HLA-DR on the ascendant monocyte subsets in different stages ofpopulations with chronic hepatitis B;â‘¢Following up patients with CHB in serious phase and severe hepatitis B, toinvestigate the relationship between ascendant subsets of blood monocytes, expression ofHLA-DR and the severe exacerbation of chronic hepatitis B with its prognosis.Methodsâ‘ The peripheral blood was obtained from8healthy control and50patientswith HBV infections,including33CHB in different disease phases and17severe hepatitisB.â‘¡Peripheral blood mononuclear cells （PBMC） were isolated by standard Ficoll-Paque（Pharmacia, Uppsala, Sweden） density gradient centrifugation, and then were stained withMoAb allowing a triple-colour analysis of surface antigens. APC-Cy7-conjugated anti-CD16（DAKO）, FITC-conjugated anti-CD14, APC-A-conjugated anti-HLA-DR.â‘¢The quantities of monocytes, the ascendant subsets and the expression of HLA-DR onmonocytes were analyzed by flow cytometry. And making reflationary analysis betweenand clinical material, such as alanine aminotransferase（ALT）, serum total bilirubin （TBIL）,prothrombin activity（PTA）;Resultsâ‘ Baseline characteristics of different clinical forms of chronic hepatitis Bpatients who were enrolled in the studyâ‘¡Compared with healthy control, the frequencyof monocytes in the patients with CHB and severe hepatitis B exhibited increasedpercentages, especially in severe hepatitis B with a significant elevation.â‘¢monocytesincreased in patients with hepatitis shifted towards the ‘non-classical’ CD14^highCD16＋monocytes subset, and were closely associated with serum （TBIL） and （PTA） levels.â‘£The expression of HLA-DR on CD14^high16⁺monocytes in blood descended in patientswith chronic hepatitis B, especially in severe hepatitis B, and were associated with diseaseprogression. The descent was negatively correlated with TBIL, and positive to PTA.⑤Following up patients with chronic severe hepatitis B （CSHB） and severe hepatitis B （SHB）,patients who ended with recovery or survive exhibited a recovery level in the frequencyCD14^highCD16⁺and expression of HLA-DR on CD14^highCD16⁺, even to the normal, as thenon-survivals had a high frequency of CD14^highCD16⁺monocytes and preserved a lowerlevels of HLA-DR. The experimental data was closely correlated with clinical data.Conclusion It suggested that the increased qualities of CD14^highCD16⁺monocytesand declined expression of HLA-DR on CD14^highCD16＋MO was correlated withimmunology injury of hepatocyte in the exacerbation of chronic hepatitis B;Dynamic monitoring the frequency of CD14^highCD16⁺monocytes and expression ofHLA-DR in the exacerbation of chronic hepatitis B may be a marker to evaluate or predictthe prognosis potentially.