In Vitro Study On Effects Of Lipopolysaccharide On Activation Of Nuclear Factor-κB And Release Of Tumor Necrosis Factor-α By Alveolar Macrophages Harvested From Rats

Background and ObjectiveLipopolysaccharide ( LPS) is a major component of endotoxin which induces acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). LPS binds to the receptors on cell membrane of such cells as macrophages, neutrophils, and endotheJial cells, initiates the inflammatory response, and causes excess production of such cytokines as tumor necrosis factor - a (TNF - a) and interleukin - 1 (IL - 1 ? ?These cytokines further act on other cells and enhance the inflammatory response. Nuclear factor - KB (NF - kB) is required formaximal transcription of many cytokines , including TNF - , IL - 1 ( , and IL - 6 , which are thought to be important in the generation of a-cute inflammatory responses. It is via activation of NF - KB that LPS triggers systemic inflammatory response syndrome , resulting in ALI or ARDS. In quiescent cells , NF - KB is sequestered in the cytoplasm. NF - KB can be activated in cells by a variety of stimuli such as LPS. Following activation , NF - KB migrates to the nucleus , where it binds to specific promoter sites and activates gene transcription. At present , methods for examine NF - KB are complex. To further understand the pathogenesis of a variety of disease states , including ARDS , we investigate the relation between activation of NF - KB detected by confocal laser scanning microscopy ( CLSM) and TNF -a expression. Methodsalveolar macrophages were obtained from Wistar rats, added to 24 - well plates, and incubated with LPS (lOmg/L). Cells in the control group were incubated with saline. NF - KB activation in alveolar macrophages was quantified by CLSM at half an hour after start of stimulation with LPS. Supernatants were collected at 0, 1,2 and 4 h after start of stimulation with LPS for determination of TNF - aby ELISA. ResultsNF - KB/P6 associated green fluorescence can be seen in the nuclei of alveolar macrophages stimulated with LPS. Such fluorescence were not found in the nuclei of cells incubated with saline , but in thecytoplasm. F/F was increased (P < 001) with LPS from a basal level of 1.053 0.138 to 0.412 0. 072. Supernatant TNF - a levels were increased from 133.3 ?. 8, 135. 2 ?. 6, and 138. 5 ?. 3 ng/ L in the control group at 1h, 2h, and 4h after LPS, respectively, to 479.9 ?6. 6, 348.5 ?. 8 ,and 251. 3 ?. 0 ng/L in group incubated with LPS, respectively (all P<0.01). ConcisionsThe activation of NF - KB can be detected by CLSM orientation-ally and quantitively. CLSM can be used to monitor intercellular trans-location of the nuclear factor. LPS induced NF - KB activation in alveolar macrophages, resulting in high production of TNF - a. The concentration of TNF - a in the LPS - stimulated group was significantly enhanced compared with that in the conlrol group after stimulation with LPS and peaked at Ih, demonstrating that LPS activates alveolar macrophages. After activation, these cells release cytokines, resulting in intensifying inflammatory response. Activation of NF - KB by LPS was associated with peak expression of TNI' - , indicating that NF - KB activation caused by LPS regulates the production of TNF - a.