OBJECTIVE: To investigate the level of mRNA expression of C-reactive proteins (CRP) induced by LPS in human alveolar type epithelial cell line, A549. METHODS: Sputum and plasma specimens were obtained from patients with chronic obstructive pulmonary disease (COPD) diagnosed according to the national criteria. CRP was measured by enzyme-linked immunosorbent assays (ELISA). A549 cells were incubated with lipopolysaccharide (LPS) at different concentrations and for different time. CRP mRNA was extracted from cells and the expression was analyzed by RT-PCR. The CRP in the supernatant was measured by ELISA. RESULTS: The concentration of CRP in the sputum of patients with COPD was significantly higher than that in the plasma (P < 0.05). LPS at different concentrations and for different time induced the expression and secretion of CRP in A549 with a time-dependent increase and the expression of CRP were significantly higher than those of the control (P < 0.05 - 0.01). CONCLUSION Self-secretion of CRP in the respiratory tract may exist. A549 induced by LPS may express CRP mRNA.
C-Reactive Protein ; biosynthesis ; genetics ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Female ; Humans ; Lipopolysaccharides ; Male ; Middle Aged ; Pulmonary Alveoli ; cytology ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Sputum ; metabolism

PURPOSE: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmic reticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluated in the human retinal pigment epithelial cell line, ARPE-19. METHODS: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatment by enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blot analysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levels of CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measured in the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfected ARPE-19 cells. RESULTS: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-κB inhibitor abolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNA expression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. CONCLUSIONS: These results suggest that PTX3 production increased in the presence of tunicamycin-induced ER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinal pigment epithelial cells. Inositol-requiring enzyme 1α and the NF-κB signaling pathway may serve as potential targets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to be further investigated.
Anti-Bacterial Agents/pharmacology ; Apoptosis ; Blotting, Western ; C-Reactive Protein/biosynthesis/*genetics ; Cells, Cultured ; Endoplasmic Reticulum Stress/*drug effects/genetics ; Enzyme-Linked Immunosorbent Assay ; *Gene Expression Regulation ; Humans ; Polymerase Chain Reaction ; RNA, Messenger/*genetics ; Retinal Pigment Epithelium/*metabolism/pathology ; Serum Amyloid P-Component/biosynthesis/*genetics ; Tunicamycin/*pharmacology

The activation of nuclear factor of activated T cells 5 (NFAT5), a well-known osmoprotective factor, can be induced by isotonic stimuli, such as activated Toll-like receptors (TLRs). It is unclear, however, how NFAT5 discriminates between isotonic and hypertonic stimuli. In this study we identified a novel context-dependent suppression of NFAT5 target gene expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) or a high salt (NaCl) concentration. Although LPS and NaCl both used NFAT5 as a core transcription factor, these stimuli mutually inhibited distinct sets of NFAT5 targets within the cells. Although reactive oxygen species (ROS) are essential for this inhibition, the source of ROS differed depending on the context: mitochondria for high salt and xanthine oxidase for TLRs. Specifically, the high salt-induced suppression of interleukin-6 (IL-6) production was mediated through the ROS-induced inhibition of NFAT5 binding to the IL-6 promoter. The context-dependent inhibition of NFAT5 target gene expression was also confirmed in mouse spleen and kidney tissues that were cotreated with LPS and high salt. Taken together, our data suggest that ROS function as molecular sensors to discriminate between TLR ligation and osmotic stimuli in RAW 264.7 macrophages, directing NFAT5 activity toward proinflammatory or hypertonic responses in a context-dependent manner.
Animals ; *Gene Expression Regulation/drug effects ; Interleukin-6/biosynthesis/genetics ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Male ; Mannitol/pharmacology ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Reactive Oxygen Species/*metabolism ; Rotenone/pharmacology ; Sodium Chloride/pharmacology ; Toll-Like Receptors ; Transcription Factors/genetics/*metabolism

We investigated acute effects of intermittent large dose bisphophonate therapy in osteoporotic patients. Peripheral blood mononuclear cells were incubated with alendronate (100 micrometer) for 18 hr, in vitro and cytokine expressions were measured by real-time RT-PCR. Pamidronate 30 mg was administered on 26 osteoporotic patients; and acute phase reactants, inflammatory cytokines and bone biomarkers were measured. The in vitro study showed significant increase in mRNA expression of IL-6, TNF-alpha and IFN-gamma. A notable rise in serum C-reactive protein (CRP) was observed over 3 days after pamidronate infusion (P=0.026). Serum levels of TNF-alpha, IL-6 and IFN-gamma were also significantly increased (P=0.009, 0.014, 0.035, respectively) and the increase in IL-6 levels were strongly correlated with CRP levels (P=0.04). Serum calcium and c-telopeptide levels rapidly decreased after the treatment (P=0.02, <0.001, respectively). This study showed that mRNA expression of inflammatory cytokines at peripheral blood mononuclear cells (PBMC) level were observed within 18 hr and marked elevation of inflammatory cytokines and acute phase reactants were demonstrated after pamidronate infusion at the dose for osteoporosis. Our studies confirmed that intermittent large dose aminobisphosphonate causes acute inflammation.
Acute-Phase Proteins/biosynthesis/genetics ; Adult ; Aged ; Aged, 80 and over ; Alendronate/pharmacology ; Biological Markers/blood ; Blood Cells/drug effects ; Bone Density Conservation Agents/*administration & dosage ; C-Reactive Protein/genetics/metabolism ; Calcium/blood ; Collagen Type I/blood ; Diphosphonates/*administration & dosage ; Female ; Humans ; Injections, Intravenous ; Interferon-gamma/blood/genetics ; Interleukin-6/blood/genetics ; Male ; Middle Aged ; Osteoporosis/*drug therapy ; Peptides/blood ; RNA, Messenger/metabolism ; Tumor Necrosis Factor-alpha/genetics/metabolism