The surface antigen CD14 plays an important role in innate immunity, serving as a pattern recognition receptor for lipopolysaccharides (LPS). The aim of this study was to investigate the proliferation, NFκB activation, and chemokine secretion of BEAS-2B cells, a human bronchial epithelial cell line, after LPS stimulation, and some details of inVolved signaling. The presence of CD14 was investigated by flow cytometry. Cell proliferation was measured with a [ 3 H]-thymidine incorporation assay. sCD14, RANTES, and IL-8 concentrations in cell supernatants were measured by ELISA. BEAS-2B cells express CD14 on their surface and secrete soluble CD14 into the supernatant. Cells react on LPS with increased proliferation, activation of NFκB, and the secretion of the pro-inflammatory chemotactic cytokines IL-8 and RANTES, which proves the functionality of the CD14 receptor. Neither CD14 nor sCD14 are regulated by LPS. Specific inhibitors of various intracellular signaling pathways diminish the LPS-induced proliferation and IL-8 secretion: Thus MAP-Kinases p38 and JNK, tyrosine kinases, and PI3-kinase are involved in the signaling cascade from the LPS-CD14-complex on the cell surface to the increased cell proliferation and expression of IL-8; furthermore, ERK 1/2, IRAK 1/4, and the NFκB pathway are inVolved in the latter. The data show the existence and functionality of CD14 receptors on BEAS-2B cells and elucidate the signaling pathways inVolved. LPS is able to increase cell prolife-ration, various cytokines which are dependent on endogenous CD14. Three MAPK pathways, PI3 kinase and tyrosine kinase may be involved. Also CD14 is present/involved which was controversial.
KeywordsLPSChemokinesBEAS-2B Cells
M. Guha, “LPS Induction of Gene Expression in Human Monocytes,” Cell, Vol. 13, No. 2, 2001, pp. 85-94.
L. Guillot, “Response of Human Pulmonary Epithelial Cells to Lipopolysaccharide InVolves Toll-Like Receptor 4 (TLR4)-Dependent Signal Pathways,” Journal of Biological Chemistry, Vol. 279, No. 4, 2003, pp. 2712-2718. doi:10.1074/jbc.M305790200
J. Pugin, “CD14 Is a Pattern Recognition Receptor,” Immunity, Vol. 1, No. 6, 1994, pp. 509-516. doi:10.1016/1074-7613(94)90093-0
B. Schmeck, “Pneumococci induced TLR- and Rac1-Dependent NFkB-Recruitement to the IL-8 Promoter in Lung Epi-thelial Cells,” American Journal of Physiology, Vol. 290, 2006, pp. 730-737.
A. Haziot, “The Monocyte Differentiation Antigen, CD14, Is Anchored to the Cell Membrane by a Phosphatidylinositol Linkage,” Journal of Immunology, Vol. 141, No. 2, 1988, pp. 547-552.
V. Bazil, “Biochemical Characterization of a Soluble form of the 53-kDa Monocyte Surface Antigen,” European Journal of Immunology, Vol. 16, No. 12, 1986, pp. 1583-1589. doi:10.1002/eji.1830161218
V. Bazil, “Structural Relationship between the Soluble and Membrane-Bound Forms of Human Monocyte Surface Glycoprotein CD14,” Molecular Immunology, Vol. 26, No. 7, 1989, pp. 657-662. doi:10.1016/0161-5890(89)90048-5
J. Pugin, “Li-popolysaccharide Activation of Human Endothelial and Epithelial Cells Is Mediated by Lipopolysaccharide-Binding Protein and Soluble CD14,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 90, 1993, pp. 2744-2748. doi:10.1073/pnas.90.7.2744
S. D. Wright, “CD14, a Receptor for Complexes of Lipopolysaccharide (LPS) and LPS Binding Protein,” Science, Vol. 249, No. 4975, 1990, pp. 1431-1433. doi:10.1126/science.1698311
D. P. Funda, “CD14 Is Expressed and Released as Soluble CD14 by Human Intestinal Epithelial Cells in Vitro: Lipopolysaccharide Activation of Epithelial Cells Revisited,” Infection and Immunity, Vol. 69, No. 6, 2001, pp. 3772-3781. doi:10.1128/IAI.69.6.3772-3781.2001
H. P. Jersmann, “Time to Abandon Dogma: CD14 Is Expressed by Non-Myeloid Lineage Cells,” Immunology and Cell Biology, Vol. 83, No. 5, 2005, pp. 462-467. doi:10.1111/j.1440-1711.2005.01370.x
H. P. Jers-mann, “Synthesis and Surface Expression of CD14 by Human Endothelial Cells,” Infection and Immunity, Vol. 69, No. 1, 2001, pp. 479-485. doi:10.1128/IAI.69.1.479-485.2001
J. Nakano, “Endotoxin and Pro-Inflammatory Cytokines Stimulate Endothelin-1 Expression and Release by Airway Epithelial Cells,” Clinical & Experimental Allergy, Vol. 24, No. 6, 1994, pp. 330-336. doi:10.1111/j.1365-2222.1994.tb00243.x
I. Striz, “The CD14 Molecule Participates in Regulation of IL-8 and IL-6 Release by Bronchial Epithelial Cells,” Immunology Letters, Vol. 62, No. 3, 1998, pp. 177-181. doi:10.1016/S0165-2478(98)00046-7
M. N. Becker, “CD14-Dependent Lipopolysaccharide-Induced Beta-Defensin-2 Expression in Human Tracheobronchial Epithelium,” Journal of Biological Chemistry, Vol. 275, 2000, pp. 29731-29736. doi:10.1074/jbc.M000184200
A. K. Mayer, “Differential Recognition of TLR-Dependent Microbial Ligands in Human Bronchial Epithelial Cells,” Journal of Immunology, Vol. 178, No. 5, 2007, pp. 3134-3142.
R. R. Reddel, “Transformation of Human Bronchial Epithelial Cells by Infection with SV40 or Adenovirus-12 SV40 Hybrid Virus, or Transfection via Strontium Phosphate Coprecipitation with a Plasmid Containing SV40 Early Region Genes,” Cancer Research, Vol. 48, No. 7, 1988, pp. 1904-1909.
V. L. Kinnula, “Primary and Immortalized (BEAS 2B) Human Bronchial Epithelial Cells Have Significant Antioxidative Capacity in Vitro”, Cell and Molecular Biology, Vol. 11, No. 5, 1994, pp. 568-576.
C. Schulz, “Differences in LPS-Induced Activation of Bronchial Epithelial Cells (BEAS-2B) and Type II-Like Pneumocytes (A-549),” Scandinavian Journal of Immunology, Vol. 56, No. 3, 2002, pp. 294-302. doi:10.1046/j.1365-3083.2002.01137.x
C. Fearns, “Murine CD14 Gene Expression in Vivo: Extramyeloid Synthesis and Regulation by Lipopolysaccharide,” Journal of Experimental Medicine, Vol. 181, No. 3, 1995, pp. 857-866. doi:10.1084/jem.181.3.857
M. Marini, “Expression of the Potent Inflammatory Cytokines, Granulocyte-Macrophage-Colony-Stimulating Factor and Interleukin-6 and Interleukin-8, in Bronchial Epithelial Cells of Patients with Asthma,” Journal of Allergy and Clinical Immunology, Vol. 89, No. 5, 1992, pp. 1001-1009. doi:10.1016/0091-6749(92)90223-O
T. L. Wu, “A Panel of Multiple Markers Associated with Chronic Systemic Inflammation and the Risk of Atherogenesis Is Detectable in Asthma and Chronic Obstructive Pulmonary Disease,” Journal of Clinical Laboratory Analysis, Vol. 21, No. 6, 2007, pp. 367-371. doi:10.1002/jcla.20197
G. Liu, “TNF-Alpha and IL-8 of the Patients with Allergic Asthma,” Journal of Huazhong University of Science and Technology—Medical Sciences, Vol. 25, No. 4, 2005, pp. 274-275, 309.
T. Akinyama, “Use and Specificity of Genistein as Inhibitor of Protein-Tyrosine Kinases,” Methods in Enzymology, Vol. 201, 1991, pp. 362-370. doi:10.1016/0076-6879(91)01032-W
T. Pera, “Cigarette Smoke and Lipopolysaccharide Induce a Proliferative Airway Smooth Muscle Phenotype,” Respiratory Research, Vol. 11, 2010, p. 48. doi:10.1186/1465-9921-11-48
J. Park, “Lipopoly-saccharide Induces Cholangiocyte Proliferation via an Interleukin-6-Mediated Activation of p44/p42 Mitogen-Activated Protein Kinase,” Hepatology, Vol. 29, No. 4, 1999, pp. 1037-1043. doi:10.1002/hep.510290423
K. Takami, “Interferon-Gamma Inhibits Hepatocyte Growth Factor-Stimulated Cell Proliferation of Human Bronchial Epithelial Cells: Upregulation of p27(kip1) Cyclin-Dependent Kinase Inhibitor,” American Journal of Respiratory Cell and Molecular Biology, Vol. 26, No. 2, 2002, pp. 231-238.
S. Bhattacharyya, “Lipopolysaccharide Activates NF-kappa B by TLR4-Bcl10-Dependent and Independent Pathways in Colonic Epithelial Cells,” American Journal of Physiology—Gastrointestinal and Liver, Vol. 295, No. 4, 2008, pp. G784-G790.
A. Freitag, “Effects of Bacterial Lipopolysaccharides (LPS) and Tumour Necrosis Factor-Alpha (TNF Alpha) on Rat Tracheal Epithelial Cells in Culture: Morphology, Proliferation and Induction of Nitric Oxide (NO) Synthase,” Pulmonary Pharmacology, Vol. 9, 1996, pp. 149-156. doi:10.1006/pulp.1996.0017
D. Preciado, “Pseudomonas Aeruginosa Lipopolysaccharide Induction of Keratinocyte Proliferation, NF-Kappa B, and Cyclin D1 Is Inhibited by Indomethacin,” Journal of Immunology, Vol. 174, No. 5, 2005, pp. 2964-2973.
M. E. Dawes, “In Vitro Effects of Lactoferrin on Lipopolysaccharide-Induced Proliferation, Gene Expression, and Prostanoid Production by Bovine Peripheral Blood Mononuclear Cells,” American Journal of Veterinary Research, Vol. 69, No. 9, 2008, pp. 1164-1170. doi:10.2460/ajvr.69.9.1164
LPS-Induced Proliferation and Chemokine Secretion from BEAS-2B Cells — Oak Academic Publishing