Effects Of DOCK2 On The Apoptosis And The TLR4 Expression Of PMNs

Objective:Polymorphonuclear neutrophils (PMNs) are the most important immune cells in human body and play important roles in innate immune response. The toxic substances in cytoplasm of PMNs can not only resist invading pathogenic microorganisms but also cause damage of self-tissue. Studies have shown that the whole progress of adhesion, activation and apoptosis of PMN can be affected by Toll-like receptor (Toll-like receptors, TLRs), which accomplishes the resolution of inflammation and maintains the stability of internal environment. TLR4 is firstly discovered TLRs that can directly mediate pathogen response. Recently, Japanese scientists have discovered a kind of new protein named DOCK2 (Dedicator of cytokinesis 2) which is associated with chemotaxis of PMN. DOCK2 gather toward inflammatory side of PMNs, so that PMNs can change their shapes and move to inflammatory site efficiently. We mainly discuss the relativity between DOCK2 and apoptosis of PMN and the relation between DOCK2 and expression of Toll-like receptor 4, which provides a potential material for the study of the anti-inflammatory ability of neutrophils and a new target for clinical treatment of inflammatory related diseases. METHODS:Heparinized peripheral blood was obtained from healthy volunteers. Neutrophils were isolated by density gradient centrifugation with Ficoll. The purity of neutrophil population was>96% on the Wright-Giemsa stain, as determined by trypan blue dye exclusion. There are four experimental model buildings according to experimental needs:the experimental group DOCK2 (1 mg/mL), positive control group IL-1β(100 ng/mL) and TNF-α(20 ng/mL), negative control group. Observe cell morphology of different cultured conditions by optical microscope. Apoptosis of neutrophil was detected by flow cytometric analysis. Detect TLR4 mRNA expression of different groups by RT-PCR. And detect expression of TLR4 by western blotting. Results:1. Flow cytometry suggested that apoptosis of neutrophil cultured with DOCK2 is down-regulated, compared with the controls.2. RT-PCR implied that DOCK2 maybe up-regulate expression of tlr4 mRNA, compared with the controls (P< 0.01).3. Western blotting showed that TLR4 protein expression is possibly up-regulated by DOCK2, compared with the controls (P<0.01). Conclusion:1. DOCK2 maybe delay neutrophil apoptosis.2. DOCK2 possibly up-regulates expression of TLR4.3. TLR4 maybe delay neutrophil apoptosis.4. DOCK2 maybe delay neutrophil apoptosis and up-regulates expression of TLR4.However, the latter may also be caused by the former or the latter causes the former. Further, the amount of change between the two may exist some relationship.