Effects of TNF- α on Expression of ICAM-1 in Human Airway Epithelial CellsIn Vitro

Abstract

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.

Acute Hypoxia Induces HIF-Independent Monocyte Adhesion to Endothelial Cells through Increased Intercellular Adhesion Molecule-1 Expression: The Role of Hypoxic Inhibition of Prolyl Hydroxylase Activity for the Induction of NF-κB

“…p65 and p50 then enter the nucleus and induce their target genes. The NF-kB-binding site within the ICAM-1 promoter is activated through binding of p65 in THP-1 cells, as well as in various other cell types (e.g., A549 and HUVECs) (42)(43)(44)(45).…”

Differential regulation of Moraxella catarrhalis-induced interleukin-8 response by protein kinase C isoforms

“…In addition, several studies have demonstrated airway epithelial cell TNF-a responses to be at least partially sensitive to PKC inhibitors. In human bronchial epithelial cells, cigarette smoke-induced, as well as C5a-mediated, IL-8 expression [42] and TNF-ainduced intercellular adhesion molecule-1 expression [43] were blocked by calphostin C, and TNF-a-induced GM-CSF expression was inhibited by the pan-PKC blocker staurosporine [44]. In addition, HEWSON et al [45] reported that PMA-induced expression of the airway mucins MUC5B and MUC5AC by …”

MARCKS Protein Is a Key Molecule Regulating Mucin Secretion by Human Airway Epithelial Cells in Vitro

“…The NHBE cells utilized in the present study were cultured at the air/liquid interface, resulting in fully differentiated primary cell cultures that maintained a well documented structure and function similar to the in vivo one (8,(21)(22)(23). This air/liquid methodology to culture airway epithelial cells was developed several years ago to provide an ideal in vitro model system to study mechanisms involved in various cellular processes in airway epithelium.…”

“…NHBE Cell Culture-Expansion, cryopreservation, and culture of NHBE cells in the air/liquid interface were performed as described previously (8). Briefly, NHBE cells (Clonetics, San Diego, CA) were seeded in vented T75 tissue culture flasks (500 cells/cm 2 ) and cultured until cells reached 75-80% confluence.…”