1.Toll like receptor 2 mediates bleomycin-induced acute lung injury, inflammation and fibrosis in mice.
Han-zhi LIU ; Hong-zhen YANG ; Su MI ; Bing CUI ; Fang HUA ; Zhuo-wei HU
Acta Pharmaceutica Sinica 2010;45(8):976-986
Anti-cancer drug bleomycin (BLM) can cause acute lung injury (ALI) which often results in pulmonary fibrosis due to a failure of resolving acute inflammatory response. The aim of this study is to investigate whether toll-like receptor (TLR) 2 mediates BLM-induced ALI, inflammation and fibrosis. BLM-induced dendritic cells (DCs) maturation was analyzed by flow cytometry and cytokine secretion was detected by the ELISA method. The expression and activity of p38 and ERK MAPK were determined with Western blotting. The roles of TLR2 in ALI, inflammation and fibrosis were investigated in C57BL/6 mice administered intratracheally with BLM. The results demonstrated that BLM-administered mice had higher expression of TLR2 (P<0.001) and its signaling molecules. Blocking TLR2 significantly inhibited the maturation of DCs and reversed BLM-stimulated secretion of cytokines in DCs, such as IL-6 (P<0.001), IL-17 (P<0.05) and IL-23 (P<0.05). TLR2 inhibition attenuated BLM-induced increase of inflammatory cells in bronchoalveolar lavage fluid (BALF), and reversed the immunosuppressive microenvironment by enhancing TH1 response (P<0.05) and inhibiting TH2 (P<0.001), Treg (P<0.01) and TH17 (P<0.01) responses. Importantly, blocking TLR2 in vivo significantly protected BLM-administered mice from pulmonary injury, inflammation and fibrosis and subsequently increased BLM-induced animal survival (from 50% to 92%). Therefore, TLR2 is a novel potential target for ALI and pulmonary fibrosis.
Acute Lung Injury
;
chemically induced
;
metabolism
;
pathology
;
Animals
;
Bleomycin
;
toxicity
;
Bronchoalveolar Lavage Fluid
;
Cells, Cultured
;
Cytokines
;
secretion
;
Dendritic Cells
;
cytology
;
metabolism
;
Inflammation
;
chemically induced
;
metabolism
;
pathology
;
Interleukin-17
;
secretion
;
Interleukin-23
;
secretion
;
Interleukin-6
;
secretion
;
Lung
;
metabolism
;
MAP Kinase Signaling System
;
Male
;
Mice
;
Mice, Inbred C57BL
;
Pulmonary Fibrosis
;
chemically induced
;
metabolism
;
pathology
;
T-Lymphocytes, Regulatory
;
drug effects
;
Th1 Cells
;
drug effects
;
Th2 Cells
;
drug effects
;
Toll-Like Receptor 2
;
metabolism
;
physiology
2.IL-4 and IL-5 Secretions Predominate in the Airways of Wistar Rats Exposed to Toluene Diisocyanate Vapor.
Kouame KOUADIO ; Kui Cheng ZHENG ; Abdoulaye Abba TOURE ; Mireille DOSSO ; Hidemi TODORIKI
Journal of Preventive Medicine and Public Health 2014;47(1):57-63
OBJECTIVES: We established a Wistar rat model of asthma caused by toluene diisocyanate (TDI) exposure, and investigated the relationship between TDI exposure concentrations and respiratory hypersensitivity, airway inflammation, and cytokine secretions in animals, to better understand the mechanism of TDI induced occupational asthma. METHODS: Wistar rats were exposed to two different concentrations of TDI vapor four hours a day for five consecutive days. Bronchoalveolar lavage (BAL) was performed, and differential leucocytes from the BAL fluid were analyzed. Lung histopathological examination was carried out to investigate the inflammatory status in the airways. Production of cytokines interleukin (IL)-4 and IL-5 productions in the BAL fluid in vivo was determined with enzyme-linked immunosorbent assay kits. RESULTS: The TDI-exposed rats exhibited greater airway hypersensitivity symptoms than the control rats. The BAL differential cell count and lung histopathological examination demonstrated that inflammation reactions were present in both the central and peripheral airways, characterized with marked infiltration of eosinophils in the TDI-exposed rats. The cytokine assay showed that IL-4 and IL-5 were predominantly produced in the BAL fluid in vivo. CONCLUSIONS: These findings imply that TDI exposure concentrations may greatly affect the occurrence and extent of inflammatory events and that Th2 type cytokines may play an important role in the immunopathogenesis of TDI-induced occupational respiratory hypersensitivity.
Animals
;
Bronchoalveolar Lavage Fluid/chemistry/cytology
;
Enzyme-Linked Immunosorbent Assay
;
Eosinophils/cytology/immunology
;
Female
;
Gases/chemistry
;
Hypersensitivity/pathology
;
Interleukin-4/*analysis
;
Interleukin-5/*analysis
;
Lung/*drug effects/pathology/secretion
;
Rats
;
Rats, Wistar
;
Toluene 2,4-Diisocyanate/*toxicity
3.Adeno-associated virus-mediated CD40 ligand transfer into human lung cancer cells.
Jian-qing WU ; Wei-hong ZHAO ; Kai-sheng YIN ; Yun-lin CHENG
Chinese Journal of Oncology 2007;29(4):253-257
OBJECTIVETo investigate the transduction efficiency of serotype 1, 2, 5, 6, 7, 8, 9, 10 recombinant adeno-associated viruses (rAAV) in human lung cancer cell line A549 cells and compare the transduction efficiency of conventional AAV vectors with that of self-complementary AAV (scAAV) vectors. Furthermore, the capacity of A549 cells expressing transgenic CD40L to stimulate dendritic cells (DCs) was evaluated.
METHODSLung cancer A549 cells were infected with 1 x 10(4) particules per cell of AAV encoding the green fluorescent protein (GFP) or human CD40L driven by CMV promotor, and transgene expression was analyzed by flow cytometry and fluorescence microscopy. Stimulation of isolated human dendritic cells by CD40L-expressing tumor cells was quantified by measuring secreted interleukin-12 with immunoassay.
RESULTSSerotype AAV2/5 transduced A549 cells much more efficiently than serotypes AAV2/1, AAV2/2, AAV2/6, AAV2/7, AAV2/8, AAV2/9 and AAV2/10. The transduction efficiency of scAAV2/5 was significantly higher than that of conventional AAV2/5. Furthermore, pre-treatment with carboplatin substantially increased AAV-mediated transgene expression. The scAAV2/5 vectors encoding human CD40L was used to generate CD40L. A549 cells transduce by these vectors were co-cultured with immature human DCs. As a consequence, interleukin-12 was released and measured in the culture supernatant. Specificity of immunostimulatory effect of CD40L was confirmed by blocking with a monoclonal antibody binding to human CD40L.
CONCLUSIONscAAV2/5 transduce lung adenocarcinoma A549 cell efficiently, and co-administration of chemotherapeutic agent carboplatin further enhances its transduction efficiency. It is confirmed that lung cancer cells infected with a CD40L-encoding scAAV2/5 construct can activate human DCs to secrete interleukin-12. Our findings provided a basis for future immunotherapeutic approaches including intratumoral transfer of stimulating factors.
Antineoplastic Agents ; pharmacology ; Blotting, Western ; CD40 Ligand ; genetics ; metabolism ; physiology ; Carboplatin ; pharmacology ; Cell Line ; Cell Line, Tumor ; Coculture Techniques ; Dendritic Cells ; cytology ; secretion ; Dependovirus ; classification ; genetics ; Flow Cytometry ; Gene Expression ; drug effects ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Immunoassay ; methods ; Interleukin-12 ; secretion ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Microscopy, Fluorescence ; Recombinant Fusion Proteins ; genetics ; metabolism ; Serotyping ; Transfection
4.Tumor microenvironment elicits primary resistance to afatinib through HGF secretion.
Xiao-hong KANG ; Li-fang WANG ; Fei CAO ; Fang-tian FAN ; Zhen-ye XU
Chinese Journal of Oncology 2013;35(10):732-736
OBJECTIVETo observe the effects of hepatocyte growth factor (HGF) derived from tumor microenvironment and/or afatinib on the growth of human lung adenocarcinoma H1975 cells and explore the potential mechanisms by which HGF induces primary resistance to afatinib.
METHODSThe effects of HGF, TGF-α and afatinib on the growth of H1975 cells were evaluated by MTT assay. The HGF concentrations of normal human fetal lung fibroblasts MRC-5 cells and human lung adenocarcinoma H1975 cells co-cultured or separately cultured were determined by ELISA assay. Western blot was used to detect the expressions of EGFR and Met signal pathway-related proteins and epithelial-mesenchymal transition (EMT) markers in H1975 cells treated with HGF and/or afatinib.
RESULTSThe MTT assay showed that H1975 cells were hyposensitive to afatinib in the presence of HGF. The ELISA assay showed that HGF production by H1975 cells was less than 0.1 ng/2.0×10(6) cells, but HGF production by MRC-5 cells was (151.37 ± 2.07)ng/2.0×10(6) cells incubated for 48 h. When H1975 cells and MRC-5 cells were co-cultured for 72 h, the concentration of HGF in the culture supernatant was (61.13 ± 16.21)ng/ml. In the presence of HGF, the expression of p-Met, p-Akt and p-ERK proteins in the H1975 cells was markedly up-regulated. afatinib inhibited p-EGFR, but did not affect the expression of p-Met, p-Akt and p-ERK proteins. In the presence of afatinib, HGF up-regulated the expression of vimentin and down-regulated the expression of E-cadherin.
CONCLUSIONSHGF secreted by stromal cells in the tumor micro-environment may confer resistance to afatinib in H1975 cells by activation of the Met/PI3K/Akt and Met/MAPK/ERK signaling pathways, and is involved in the epithelial-mesenchymal transition process.
Adenocarcinoma ; metabolism ; pathology ; Antineoplastic Agents ; pharmacology ; Cadherins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Coculture Techniques ; Drug Resistance, Neoplasm ; Epithelial-Mesenchymal Transition ; Fibroblasts ; cytology ; metabolism ; Hepatocyte Growth Factor ; pharmacology ; secretion ; Humans ; Lung ; cytology ; Lung Neoplasms ; metabolism ; pathology ; MAP Kinase Signaling System ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-met ; metabolism ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Signal Transduction ; drug effects ; Transforming Growth Factor alpha ; pharmacology ; Tumor Microenvironment ; Vimentin ; metabolism
5.Inhibitory effect of low molecular weight heparin on the secretion of vascular endothelial growth factor by tumor cells in vitro.
Zhao SUN ; Zong-lan HU ; Xiao-hong NING ; Jian-feng ZHOU ; Ya-juan SHAO ; Jin-hong DUAN ; Xian-da YANG ; Chun-mei BAI
Chinese Journal of Oncology 2009;31(11):826-830
OBJECTIVETo investigate whether low molecular weight heparin (LMWH) may suppress the expression and secretion of vascular endothelial growth factor (VEGF) from tumor cells in vitro and inhibit the VEGF-induced proliferation of human tumor vascular endothelial cells.
METHODSHuman lung cancer cell line A549, human liver cancer cell line HepG2, human colon carcinoma cell lines HCT116 and HCT8 were used in this study. The expression levels of VEGF and TNF-alpha (tumor necrosis factor-alpha) in the tumor cells with or without pretreatment of LMWH/heparin were measured by standard sandwich ELISA technique. The VEGF mRNA level of HepG2 cells cultured with or without LMWH/heparin was determined by RT-PCR and real time PCR. Human umbilical vein endothelial cells (HUVEC) were cultured in tissue culture medium (TCM) with or without LMWH/heparin for 3 days. Then non-radioactive cell proliferation assay (MTS) kit and cell cycle assay by flow cytometry were performed to measure the proliferation of HUVEC.
RESULTSThe VEGF levels in the control, LMWH, and heparin groups of the pulmonary adenocarcinoma cell line A549 were (1045.89 +/- 165.30) pg/ml, (782.45 +/- 67.17) pg/ml and (916.54 +/- 71.25) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups of the colon adenocarcinoma cell line HCT116 were (955.76 +/- 51.14) pg/ml, (822.89 +/- 142.39) pg/ml and (951.77 +/- 188.22) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the colon adenocarcinoma cell line HCT8 were (1290.62 +/- 41.23) pg/ml, (1063.34 +/- 63.82) pg/ml and (1257.14 +/- 11.40) pg/ml, respectively. The VEGF levels in the control, LMWH, and heparin groups in the liver cancer cell line HepG2 were (1083.00 +/- 134.35) pg/ml, (758.00 +/- 84.85) pg/ml and (874.00 +/- 22.62) pg/ml, respectively. The VEGF expression levels in the above mentioned cell lines cultured in TCM were significantly reduced in the LMWH-treated groups compared with that of the control group (P < 0.05). But the level of TNF-alpha in TCM-cultured cells was unaffected by LMWH. The VEGF mRNA was reduced in the LMWH-treated HepG2 cell line. Moreover, TCM exhibited stimulating effect on proliferation of HUVEC and the effect was significantly impaired by LMWH treatment. Flow cytometric analysis revealed that LMWH treatment arrested HUVECs at the G1 phase of cell cycle.
CONCLUSIONLMWH can suppress the expression and secretion of VEGF by tumor cell lines and therefore have a potential inhibiting effect on angiogenesis induced by VEGF.
Adenocarcinoma ; metabolism ; pathology ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media, Conditioned ; Endothelial Cells ; cytology ; HCT116 Cells ; Hep G2 Cells ; Heparin ; pharmacology ; Heparin, Low-Molecular-Weight ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; secretion
6.Shengdi injection on rat model of lung inflammation induced by lipopolysaccharides.
Li LIU ; Lan TANG ; De-Sheng XU ; Hui-Li XIA ; Qiang-Min XIE
China Journal of Chinese Materia Medica 2007;32(6):526-528
OBJECTIVETo investigate the effect of Shengdi injection on rat model of lung inflammation.
METHODThe rat model was established by intratrachea instillation of lipopolysaccharides (LPS). The total and different white blood cell counts in bronchoalvoelar lavage fluid (BALF) were performed and the level of tumor necrosis factor-alpha (TNF-alpha), superoxide anion radical (O2-) and myeloperoxidase (MPO) was measured, as well as pathologic change of pulmonary tissue was tested.
RESULTShengdi injection could depress the increasing of the amount of total white blood cells and neutrophils and inhibit the increasing of TNF-alpha, O2-, MPO caused by LPS, as well as relieve the pathologic change including Neutrophils infiltrating and mucous edema in tracheae after intravenous administration. While it did not show the effect on monocyte, and histological lesion of the lung tissue.
CONCLUSIONShengdi injection shows some anti-inflammatory effect in rat lung induced by LPS and it can be concluded tentatively that anti-inflammatory, inhibiting the release of cytokine and inflammatory medium, and antioxidation are some of the mechanism of its effect on COPD.
Animals ; Anti-Inflammatory Agents, Non-Steroidal ; administration & dosage ; isolation & purification ; pharmacology ; Bronchoalveolar Lavage Fluid ; chemistry ; Cytokines ; secretion ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; pharmacology ; Injections, Intravenous ; Leukocyte Count ; Lipopolysaccharides ; Lung ; drug effects ; metabolism ; pathology ; Male ; Neutrophils ; drug effects ; pathology ; Peroxidase ; metabolism ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Pneumonia ; chemically induced ; metabolism ; prevention & control ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Rehmannia ; chemistry ; Superoxides ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism