Contained Lipoprotein A-¢ñ In The Body's Acute Phase Response On The Regulation Of Neutrophil Function

This work investigated the alteration of plasma high density lipoprotein (HDL) during the lipopolysaccharide(LPS)-induced acute phase response (APR) and observed the effects of apolipoprotein A-I (apoA-I) on fMLP- and PMA-activated polymorphonuclear leukocyte (PMN) functions.Rabbit was chosen to be the investigation object. Gram-negative bacterial endotoxin LPS was used to induce APR in rabbits to observe the alteration of HDL component during the APR process especially the changes of apoA-I to provide experimental basis for the possibility that apoA-I released from HDL particle during APR to participate the host immuno-modulation. APR was induced by i.v l.Omg/kg LPS and blood was token every 30 minutes. HDL was observed during the period of APR, 1) HDL prepared by ultracentrifugation was undergone sieve chromatography to observe the variation of molecular size. SDS-PAGE and ELISA were applied simultaneously to identify protein component alteration of APR-HDL; 2) HDL Cholesterol and triglyceride was assayed in plasma HDL isolated by PEG-6000 precipitation.HDL prepared by ultracentrifugation was delipidated by regular ethanol-aether method. apoA-1 was isolated from the delipidated HDL apolipoproteins (apoHDL) using anion-exchange column (POROS HQ column) on BIO-CAD workstation. Peaks were collected, desalted by ultrafiltration and identified by SDS-PAGE. Pure apoA-I was collected for the use of PMN functions investigation.PMNs were prepared from stabilizer-free hepann anticoagulated whole blood of healthy New Zealand white rabbit. The anticoagulated whole blood was diluted with an equal volume of PBS (pH7.4) and then carefully layered over lymphocyte separation medium (d. 1.077g/ml) with equal volume. Gradient centrifugation was earned at room temperature at 400 g for 20 mm. The lower layer enriched of granulocytes and erythrocytes was homogenized with equal volume of 2.5% (w/v) Gelatin to allow erythrocyte sedimentation for 30 min on ice. The resulting supernatant was collected to harvest granulocytes and washed twice with PBS. Contaminated erythrocyte wasremoved by Gey's solution treatment. Cell viability was greater than 95% as determined by Trypan Blue exclusion, and Giemsa stains showed greater than 95% PMN purity.fMLP and PMA (with final concentration of 10"7mol/L) were used as receptor-binding and nonreceptor-binding activator to co-incubate separately with PMNs and apoA-I of different concentration, 1) resting PMNs + apo A-I (with final concentration of 0,2.5,5,lOug/mL); 2) f-MLP-activated PMNs + apo A-I (with final concentration of 0,2.5,5, lOug/mL); 3)PMA-activated PMNs + apo A-I (with final concentration of 0,2.5,5, lOug/mL). PMN functions were tested including 1) adhesion, to observe the influence of apoA-I on fMLP- and PMA-activated PMNs adhesion to fibronectin surface; 2) O2" and H2O2 production (oxidative burst), to observe the influence of apoA-I on fMLP- and PMA-activated PMNs oxidative burst; 3)degranulation, to identify activated PMNs released MPO and elastase activity to determine the effect of apoA-I on fMLP- and PMA-activated PMNs degranulation; and 4) the assay of L-929 cells mortality, to observe the L-929 cells mortality rate caused by fMLP- and PMA-activated PMN in the present of apoA-I.Our results showed that, 1) Components of HDL, both apolipoproteins and lipids, altered dramatically during the LPS-induced APR. First, smaller HDL was appeared during the process. Second, apoA-I was displaced by a protein of approximate molecular weight 13000. Third, depressed HDL cholesterol capacity was accompanied by rising triglyceride content. Last, triglyceride become the main lipid component in APR-HDL. 2) apoA-I diminished fMLP- and PMA-activated PMN functions with no influence on resting PMNs at the same time. This kind of depression was much more powerful in the case of fMLP-activated PMNs than that in the case of PMA-activated PMNs. ( i ) Adhesion of fMLP- and PMA-activated PMNs to fibronectin surface determined by crystal violet stain was dramatically diminished in the presence of apoA-I (PO.01) and dose-dependent effect was shown in the case of fMLP-activated PMNs CPO.05, P<0.01, P<0.0\). (ii) Reactive oxygen species (ROS) production of fMLP- and PMA-activated PMNs (oxidative burst) measured by NBT reducing test for O2 and HVA fluorescence spectrophotometry for H2O2 separately was depressed by apoA-I (P<0.0\). Dose-dependent effect was shown in the case of fMLP-activated...