Effect Of TLR Ligand On Intracellular Bax Expression Of Neutrophils

Objective: Polymorphonuclear neutrophils (PMNs) are the most important effector cells in earlier inflammation and they play a core role in host infection against microbial infection. Neutrophils are terminally differentiated cells which come from marrow hemopoietic stem cell. Senescent neutrophils are recognized and phagocytized from nomal and inflammatory sites. Neutrophils undergo spontaneous apoptosis in vito and in vivo, and the progress has been recognized as a crucial mechanism for accomplishing resolution of inflammation and maintaining the stability of internal environment. During the study of apoptosis, an increasing number of reports have suggested that apoptosis is not exclusive form of programmed cell death (PCD). The cause and the pass of programmed cell death are very complicated. Bcl-2 family also plays a key role in regulating the cell apoptosis of neutrophils and Bax is one of the important regulated genes of apoptosis. Bax may regulate cell apoptosis by the pathway of the mitochondrion. TLRs, Toll-like receptors, are pattern recognition acceptors which recognize pathogen associated molecular pattern (PAMP). There are 11 members of TLRs, named TLR1-TLR11. Generally, TLRs are expressed in various kinds of rhagiocrine cell, and TLRs except TLR3 can be expressed in the human's PMNs. TLRs ligand are PAMPs, but different TLRs recognize different PAMPs. A lot of studies about TLRs in PMNs are concentrated. But the research about TLRs in PMNs is at primary stage, specially, no study that the Bcl-2 family is activated by TLRs is reported. In order to explore new progress about neutrophil survival, the effects of TLR ligands on intracellular Bax (Bcl-2 homologous antagonist / killer) protein expression were studied. Various TLR ligands were used, such as LPS, PGN, Loxoribine, Flaglline, Pam3CSK4, R-848 and MALP-4. Methods: 1. Heparinized peripheral blood was obtained from healthy volunteers. Neutrophils were isolated using 3% dextran sedimentation followed by density gradient centrifugation with Ficoll. The purity of neutrophils population was > 96% with the Wright- Giemsa stain, and neutrophil viability was > 96%, as determined by trypan blue dye exclusion. 2. The neutrophils were cultured at the presence or absence of TLR ligands for the indicated time periods. The TLR ligands were LPS (TLR4), PGN (TLR2), loxoribine (TLR7), flagellin (TLR5), Pam3CSK4 (TLR1/2), R-848 (TLR7/8), MALP-2(TLR2/6). 3. The neutrophils were cultured in the presence or absence of TLR ligands for the indicated time periods. The whole cell lysates were subjected to SDS-PAGE. 4. Western blotting analysis was performed with specific Bax monoclonal antibody and a horseradish peroxidase (HRP)-conjugated secondary antibody. To confirm equal amounts of loaded proteins, the membranes were reprobed with HRP-conjugated GAPDH monoclonal antibody as an internal control. Results: 1. Flow cytometry results suggested that the purity of neutrophil population was > 96%. 2. After 12 hours, the nature and quanlity of the PMNs cell protein was were changed. 3. Western blotting showed that the expression of Bax protein was down-regulated by TLR ligand that delayed apoptosis. The influence that different TLR ligands down-regulated the expression of Bax may have no relation with the type of TLR receptor. Conclusions: 1. The seven TLR agonists, including LPS, PGN, loxoribine, flagellin, Pam3CSK4, R-848 and MALP-2, can reduce the expression of Bax protein in neutrophils, which may be involved in TLR agonist-induced signal transduction pathway of neutrophil death. 2. The seven TLR agonists, including LPS, PGN, loxoribine, flagellin, Pam3CSK4, R-848 and MALP-2, were not statistically significant on the expression of Bax protein, indicating that the expression of Bax protein may be unrelated to different TLR agonists.3. The seven TLR agonists, including LPS, PGN, loxoribine, flagellin, Pam3CSK4, R-848 and MALP-2, may be effective at delying spontaneous apoptosis of PMN.