Increased intracellular Cl- concentration promotes ongoing inflammation in airway epithelium.

Airway epithelial cells harbor the capacity of active Cl- transepithelial transport and play critical roles in modulating innate immunity. However, whether intracellular Cl- accumulation contributes to relentless airway inflammation remains largely unclear. This study showed that, in airway epithelial cells, intracellular Cl- concentration ([Cl-]i) was increased after Pseudomonas aeruginosa lipopolysaccharide (LPS) stimulation via nuclear factor-κB (NF-κB)-phosphodiesterase 4D (PDE4D)-cAMP signaling pathways. Clamping [Cl-]i at high levels or prolonged treatment with LPS augmented serum- and glucocorticoid-inducible protein kinase 1 (SGK1) phosphorylation and subsequently triggered NF-κB activation in airway epithelial cells, whereas inhibition of SGK1 abrogated airway inflammation in vitro and in vivo. Furthermore, Cl--SGK1 signaling pathway was pronouncedly activated in patients with bronchiectasis, a chronic airway inflammatory disease. Conversely, hydrogen sulfide (H2S), a sulfhydryl-containing gasotransmitter, confers anti-inflammatory effects through decreasing [Cl-]i via activation of cystic fibrosis transmembrane conductance regulator (CFTR). Our study confirms that intracellular Cl- is a crucial mediator of sustained airway inflammation. Medications that abrogate excessively increased intracellular Cl- may offer novel targets for the management of airway inflammatory diseases.

Relaxant Effect of Sodium Tanshinone IIA Sulphonate on Mouse Tracheal Smooth Muscle.

Sodium tanshinone IIA sulphonate, a water-soluble derivative of tanshinone IIA, has been proven to possess versatile biological properties, but its pharmacological effect on tracheal smooth muscle remains elusive. This paper presents a study on the relaxant effect and underlying mechanisms of sodium tanshinone IIA sulphonate on mouse tracheal smooth muscle. The relaxant effect of sodium tanshinone IIA sulphonate was evaluated in mouse tracheal rings using a mechanical recording system. Intracellular Ca2+ concentration was measured in primary cultured tracheal smooth muscle cells using confocal imaging system. The results showed that sodium tanshinone IIA sulphonate induced dose-dependent relaxation of mouse tracheal rings in a ß-adrenoceptor- and epithelium-independent manner. Pretreatment with the ATP-sensitive K+ channel blocker glibenclamide partly attenuated the relaxation response. Administration of sodium tanshinone IIA sulphonate notably inhibited the extracellular Ca2+-induced contraction. High KCl or carbachol-evoked elevation in the intracellular Ca2+ concentration was also abrogated by sodium tanshinone IIA sulphonate in tracheal smooth muscle cells. In conclusion, the tracheal relaxant effect of sodium tanshinone IIA sulphonate was independent of ß-adrenoceptor and airway epithelium, mediated primarily by inhibition of extracellular Ca2+ influx via L-type voltage-dependent Ca2+ channels and partially by activation of the ATP-sensitive K+ channel. These results indicate the potential therapeutic value of sodium tanshinone IIA sulphonate for asthma treatment.

[Outcome and significance of pulmonary pathological changes induced by a single intramuscular injection of chemokine-like factor 1 in mice].

OBJECTIVE: To investigate the significance of pathological changes in murine lung by a single intramuscular injection of chemokine-like factor 1 (CKLF1). METHODS: A total of 120 gender-matched BALB/c mice were randomly and evenly divided into treatment group and control group (60 in each). One hundred nanomilligram of pcDNA3.1-CKLF1-Myc-His, CKLF1-expressing plasmid, in 100 microl of pyrogen-free saline was injected into the anterior tibial muscle of mice followed by the delivery of electric pulses. Mice in the control group received 100 microg of pcDNA3.1-Myc-His in 100 microl of pyrogen-free saline. At the end of week 1, 4 and 8 respectively after injection of CKLF1, 20 mice were sacrificed in every group and the cellular profiles in bronchoalveolar lavage fluid (BALF) and the pulmonary pathological changes were observed. RESULTS: At the end of week 1 and 4 respectively after CKLF1 injection, the neutrophils [(35.0 +/- 5.2)% and (22.9 +/- 2.2)% respectively] and lymphocytes [(34.5 +/- 2.8)% and (22.0 +/- 2.0)% respectively] in BALF of the treatment group were higher than those of the control group [neutrophils: (6.7 +/- 2.2)% and (7.0 +/- 2.4)% respectively, lymphocytes: (5.9 +/- 1.6)% and (6.1 +/- 2.7)% respectively, all P < 0.01]. Pathological studies demonstrated shedding of bronchiolar epithelium, congestion and edema in interstitial tissue and inflammatory cell infiltration in mice at 1 week after CKLF1 injection. Week 4 after CKLF1 administration, the alveolar wall was shown significantly thickened with proliferation of neutrophils, macrophages and fibroblasts as well as remarked collagen deposition in the interstitium. At the end of week 8 after CKLF1 administration, the remarkable morphological changes of the lung gradually subsided and the structure of the lung returned to normal. CONCLUSIONS: CKLF1 causes injury of inflammation and remodeling in airway in mice. The pulmonary pathological changes induced by a single intramuscular injection of CKLF are reversible.

[Expression and significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein in lungs of SARS patients].

OBJECTIVE: To investigate the significance of severe acute respiratory syndrome associated coronavirus (SARS-CoV)-X4 protein expression in lungs of patients with SARS. METHODS: Pathological features of the lungs from 4 SARS patients were examined and the expression of SARS-CoV-X4 protein in the lungs was evaluated with immunohistochemical staining using specific antibodies against protein X4. RESULTS: Microscopically, all lungs from 4 cases showed edema, erythrocyte and fibrin exudates in the alveoli, hyperplasia of alveolar epithelium, necrosis, hyaline membrane formation and fibroblast foci. Immunohistochemical stains showed a strong positivity of X4 protein in denudation cells, vascular endothelial cells and also erythrocytes and neutrophils in the alveoli of the lung tissues from the 4 cases. CONCLUSIONS: Expression of SARS-CoV-X4 protein in the lungs may be involved in the pathogenesis and progression of SARS.

The protein X4 of severe acute respiratory syndrome-associated coronavirus is expressed on both virus-infected cells and lung tissue of severe acute respiratory syndrome patients and inhibits growth of Balb/c 3T3 cell line.

BACKGROUND: The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) includes sequences encoding the putative protein X4 (ORF8, ORF7a), consisting of 122 amino acids. The deduced sequence contains a probable cleaved signal peptide sequence and a C-terminal transmembrane helix, indicating that protein X4 is likely to be a type I membrane protein. This study was conducted to demonstrate whether the protein X4 was expressed and its essential function in the process of SARS-CoV infection. METHODS: The prokaryotic and eukaryotic protein X4-expressing plasmids were constructed. Recombinant soluble protein X4 was purified from E. coli using ion exchange chromatography, and the preparation was injected into chicken for rising specific polyclonal antibodies. The expression of protein X4 in SARS-CoV-infected Vero E6 cells and lung tissues from patients with SARS was performed using immunofluorescence assay and immunohistochemistry technique. The preliminary function of protein X4 was evaluated by treatment with and over-expression of protein X4 in cell lines. Western blot was employed to evaluate the expression of protein X4 in SARS-CoV particles. RESULTS: We expressed and purified soluble recombinant protein X4 from E.coli, and generated specific antibodies against protein X4. Western blot proved that the protein X4 was not assembled in the SARS-CoV particles. Indirect immunofluorescence assays revealed that the expression of protein X4 was detected at 8 hours after infection in SARS-CoV-infected Vero E6 cells. It was also detected in the lung tissues from patients with SARS. Treatment with and overexpression of protein X4 inhibited the growth of Balb/c 3T3 cells as determined by cell counting and MTT assays. CONCLUSION: The results provide the evidence of protein X4 expression following SARS-CoV infection, and may facilitate further investigation of the immunopathological mechanism of SARS.

Evaluation by indirect immunofluorescent assay and enzyme linked immunosorbent assay of the dynamic changes of serum antibody responses against severe acute respiratory syndrome coronavirus.

BACKGROUND: Severe acute respiratory syndrome coronavirus (SARS-CoV) is a newly emerging virus that gives rise to SARS patients with high rates of infectivity and fatality. To study the humoral immune responses to SARS-CoV, the authors evaluated IgG and IgM specific antibodies in patients' sera. METHODS: Two methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent assay (IFA), were used to detect specific serum IgG and IgM against SARS-CoV in 98 SARS patients and 250 controls consisting of patients with pneumonia, health-care professionals and healthy subjects. The serum antibody profiles were investigated at different times over one and a half years in 18 of the SARS patients. RESULTS: The sensitivity and specificity of ELISA for detecting IgG against SARS-CoV were 100.0% and 97.2% and for IgM 89.8% and 97.6% respectively; the figures using IFA for IgG were 100.0% and 100.0% and for IgM 81.8% and 100.0% respectively. During the first seven days of the antibodies trace test, no IgG and IgM were detected, but on day 15, IgG response increased dramatically, reaching a peak on day 60, remaining high up to day 180 and decreasing gradually until day 540. On day 15, IgM was detected, rapidly reached a peak, then declined gradually until day 180 when IgM was undetectable. CONCLUSION: The detection of antibodies against SARS virus is helpful in the clinical diagnosis of SARS.

Chemokine-like factor 1, a novel cytokine, contributes to airway damage, remodeling and pulmonary fibrosis.

BACKGROUND: Chemokine-like factor 1 (CKLF1) was recently identified as a novel cytokine. The full-length CKLF1 cDNA contains 530 bp encoding 99 amino acid residues with a CC motif similar to that of other CC family chemokines. Recombinant CKLF1 exhibits chemotactic activity on leucocytes and stimulates proliferation of murine skeletal muscle cells. We questioned whether CKLF1 could be involved in the pathogenesis of inflammation and proliferation in the lung. Therefore we used efficient in vivo gene delivery method to investigate the biological effect of CKLF1 in the murine lung. METHODS: CKLF1-expressing plasmid, pCDI-CKLF1, was constructed and injected into the skeletal muscles followed by electroporation. Lung tissues were obtained at the end of week 1, 2, 3 and 4 respectively after injection. The pathological changes in the lungs were observed by light microscope. RESULTS: A single intramuscular injection of CKLF1 plasmid DNA into BALB/c mice caused dramatic pathological changes in the lungs of treated mice. These changes included peribronchial leukocyte infiltration, epithelial shedding, collagen deposition, proliferation of bronchial smooth muscle cells and fibrosis of the lung. CONCLUSIONS: The sustained morphological abnormalities of the bronchial and bronchiolar wall, the acute pneumonitis and interstitial pulmonary fibrosis induced by CKLF1 were similar to phenomena observed in chronic persistent asthma, acute respiratory distress syndrome and severe acute respiratory syndrome. These data suggest that CKLF1 may play an important role in the pathogenesis of these important diseases and the study also implies that gene electro-transfer in vivo could serve as a valuable approach for evaluating the function of a novel gene in animals.

[Protective effect of specific antibody in serum of convalescent patient with SARS].

OBJECTIVE: To investigate inhibitory effect of serum severe acute respiratory syndrome (SARS) -specific antibodies from convalescent patients after half an year of onset on SARS-CoV-mediated cytopathic response. METHODS: SARS-CoV immunoglobulin G (IgG) antibody was determined by enzyme linked immunoadsorbent assay (ELISA). Twelve serum samples from convalescent patients, diluted by 1:8 with maintenance medium, were mixed with the three dilution supernatants of SARS-CoV. SARS-CoV were isolated, cultured and identified by the Guangzhou Institute of Respiratory Disease, and cultured with Vero E6 cell suspension. The extent of cytopathic response was observed. RESULTS: The absorbance (A) value of SARS-CoV IgG antibody ranged from 0.81 to 2.06 in patients after half an year of SARS onset, and form 0.79 to 2.01 in patients before half an year of SARS onset. The extent of cytopathic response was decreased by more than 25% in all 12 convalescent patients, as compared with control serum. CONCLUSION: The A value of SARS-CoV IgG antibody in serum of convalescent patients tended to elevate in half an year after SARS onset. SARS-CoV IgG antibody could inhibit SARS-CoV-mediated cytopathic response, indicating it might be one of protective antibodies.