Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression.
Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.
Krunkosky
T M
TM
Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA.
Fischer
B M
BM
Martin
L D
LD
Jones
N
N
Akley
N J
NJ
Adler
K B
KB
D000911:Antibodies, Monoclonal; D001980:Bronchi / Q000166:cytology; D002460:Cell Line; D004847:Epithelial Cells / Q000201:enzymology / Q000276:immunology*; D005434:Flow Cytometry; D015870:Gene Expression / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics* / Q000276:immunology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology*; D015398:Signal Transduction / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D016328:NF-kappa B; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D018799:Intercellular Adhesion Molecule-1; D007770:L-Lactate Dehydrogenase