| Background and ObjectiveAcute lung injury (ALI) commonly caused by infection is one of emergercy and critical diseases of respiratory system. ALI induced by Gram-negative bacteria infection is due to effects of cytokines from inflammatory cells activated by lipopolysaccharide(LPS). Alveolar macrophages (AMs) are the important defence line of the respiratory tract and also the target cells of LPS effection. It is helpful and important to investigate LPS signaling transduction of AMs and the blocking effects to understand the mechanism of ALI and find out a new therapy way.Nucleolin is one protein to shuttle between the nucleus, cytoplasm and cell surface, which has been implicated in controlling regulatory processes and may play a role in virus infection and autoimmunity diseases. Nucleolin can improve the adhesion of pathogens to host cells and assist them to entry into host cells, which will be the new target of anti-microbial treatment. Now many reports have shown that nucleolin was also localized on the cell surface of THP-1 monocytes and involved in the LPS-mediated expression and secretion of inflammatory cytokines such as TNF-αand IL-6. The signaling events that precede activation of these pathways in monocytes, however, have not been thoroughly studied. AMs is drived from THP-1 cells, however, the expression of nucleolin on AMs surface and the role of nucleolin in LPS- induced AMs activation is undetermined. The aim of this study is to examine the expression of nucleolin on AMs, to investigate the relationship between LPS and nucleolin,and to evaluate the potential role of nucleolin in LPS-induced AMs activation and LPS induced ALI with rat.Method1.To detect the presence of nucleolin at the surface of AMsThe primary AMs were obtained from SD rats by bronchoalveolar lavage.The AMs cell-surface localization of nucleolin was investigated by confocal immunofluore -scence microscopy and Fluorescence Activating Cell Sorter.2.To investigate the interaction between cell-surface nucleolin and LPSWe used an LPS affinity column to investigate cell-surface nucleolin assumed to be a ligand for LPS .The relative affinity of nucleolin to LPS was determined by enzyme -linked immunosorbent assay (ELISA). The fate of LPS in AMs and its colocalization with nucleolin was investigated by double immunofluorescence microscopy using LPS-FITC and Cy3-conjugated antibody against nucleolin. Effect of anti-nucleolin antibody on the binding of FITC-conjugated LPS (FITC-LPS) to AMs was determined by flow cytometric analysis (FACS).3.Gene and protein expression of nucleolin induced by LPS in AMs The expression of nucleolin mRNA and cell-surface nucleolin protein in AMs induced by LPS at different time points were detected by reverse transcription -polymerase chain reaction (RT- PCR) and Western blot. The expression of nucleolin mRNA and cell-surface nucleolin protein in AMs induced by different concentration of LPS were detected by RT-PCR and Western blot.4.Experimental studies of RNAi targeting rat AMs nucleolin in vitro before LPS stimulationAMs were isolated and cultured,and transfected with shRNA before LPS stimulation. The internalization of LPS in AM were examined by Immuno- fluorescence and confocal microscop.The expression of cell-surface nucleolin protein in AMs were measured by Western blot.The activity of NF-κB in AMs was tested with EMSA.The concentrations of TNF-αand IL-6 in the supermatant of cultured AMs were also assayed with ELISA.5.Experimental studies of pBS-U6.C23shRNA transfection in vivo before LPS stimulationAfter anesthetized, SD rats were intubated. pBS-U6.C23.shRNA mediated by jetPEI in vivo was administered intratracally.The control group were treated with pBS-U6.control.shRNA.Then,rats were extubated.Forty-eight hours after transfection, rats were anesthetized and treated with LPS intravenously.The lung were excised and lavaged after 4h.Lung tissue samples were stained with Hematoxylin-eosin(HE) staining for histopathologic analysis. The expression of cell-surface nucleolin protein in AMs were detected by Western blot.The activity of NF-κB in AM was tested with EMSA.The concentrations of TNF-αand IL-6 in the bronchoalveolar lavage fluid (BALF) were assayed with ELISA.Results1.By using immunofluorescence microscopy and FACS, we detected the presence of nucleolin on the plasma membrane of AM in addition to nucleolar sites.2.By using LPS affinity columns, we demonstrated that nucleolin may be a new membrance receptor for LPS and can directly binds to LPS. In ELISA experiments,we observed that nucleolin and LPS interacted in a dose–dependent way. In addition,we found that LPS colocalized with nucleolin on both the cell surface(1h) and in the cytoplasm(4h). The FACS results show that anti-nucleolin antibody markedly suppressed the binding of FITC-LPS to AMs.3.The expression of nucleolin mRNA and cell-surface nucleolin protein could be detected in normal AMs. After the stimulations with LPS(1ug/ml), the levels of nucleolin mRNA and cell-surface nucleolin protein was higher then that of the control group (P<0.01).The levels of nucleolin mRNA started to increased at 1h,peaked at 4h,then gradually decreased.The levels of cell-surface nucleolin protein started to increased at 2h,peaked at 8h, then gradually decreased. After the stimulations with different LPS concentration, the levels of nucleolin mRNA and cell-surface nucleolin protein significantly increased (P<0.01).The increases were in a dose-dependent manner. When the concentration of LPS is higher than 100ug/ml,nucleolin mRNA and cell-surface nucleolin protein expression still increase but less significantly.4.Compared with the cells without LPS stimulation,in the AMs before LPS stimulation, without transfection or transfected with control shRNA vector,the expression of cell-surface nucleolin protein, the internalization of LPS ,the NF-κB activity and the levels of TNF-αand IL-6 increased remarkably (P<0.01).The above measurements in AMs transfected with pBS-U6.C23shRNA vector were lower than those in cells without transfection or transfected with control shRNA vector groups respectively (P<0.01).5.Forty-eight hours after trannsfected the pBS-U6.C23shRNA or pBS-U6.Control -shRNA intratracally mediated by jetPEI in vivo,the rats were normal in vital signs. Compared with the control group, after stimulation with LPS,in rat group without transfection or transfected with control shRNA vector,the expression of cell-surface nucleolin protein,the histopathologic,the NF-κB activity and the levels of TNF-αand IL-6 in BALF increased remarkably(P<0.01). After stimulation with LPS,in the rat group transfected with pBS-U6.C23shRNA vector,the expression of cell-surface nucleolin protein,the histopathologic, the NF-κB activity and the levels of TNF-αand IL-6 decreased remarkably in contrast to those in rats group without transfection or transfected with control shRNA vector groups respectively(P<0.01).Conclusion1.We demonstrate that nucleolin is expressed on the surface of AMs and its expression of mRNA and cell-surface nucleolin protein significantly increased after LPS stimulation.2.Our study imply a new functional role of nucleolin may be a new membrane binding receptor for LPS and is essential for LPS cellular entry in AMs.3.The internalization of LPS with cell-surface nucleolin induced the activation of the transcription factors nuclear factor-κB(NF-κB),than increase the protein expression cytokines such as TNF-αand IL-6. It is able to contribute to LPS signaling in AMs.4.pBS-U6.C23.shRNA could be transfected safely and effectively by intratracheal instillation in vivo.It could led to significant reductions in the expression of cell-surface nucleolin protein and NF-κB activity in AMs,and in the protein expression of TNF-αand IL-6 in BALF. It could attenuate lung injury. |
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