The central hypothesis of the research proposal is that airway epithelial and vascular endothelial cells modulate inflammation through the coordinated expression of cell surface adhesion receptors. Preliminary studies demonstrate that intercellular adhesion molecule-1 (ICAM-1) is selectively expressed on airway epithelial cells after interferon-gamma (IFN-gamma) stimulation and that ICAM-1 interaction with corresponding beta2-integrins on neutrophils and T-lymphocytes is a critical mechanism for epithelial-leukocyte adhesion. The epithelial cell profile for cytokine responsiveness is distinct from the one for vascular endothelial cells which express functional ICAM-1 in response to other cytokines but not after IFN-gamma treatment. The complimentary profile of cytokine responsiveness is proposed to allow for efficient localization of leukocytes under different inflammatory conditions. The level of ICAM-1 expression appears to be regulated at the level of mRNA transcription in both airway epithelial and vascular endothelial cells, but the mechanism for the differences in gene activation between cell types is uncertain We propose to study the transcriptional control of ICAM-1 gene in airway epithelial cells with initial emphasis on the mechanism(s) that control IFN-gamma selectivity. The principal cell system to be employed for this work is primary cultures of human airway epithelial cells. A model cell system will also be developed using transformed bronchial epithelial cells which also express functional ICAM-1 and exhibit selective responsiveness to IFN-gamma. Distinct features of the epithelial ICAM-I gene will be defined by comparison to (i) ICAM-I gene regulation in endothelial cells and (ii) HLA-A gene regulation in epithelial and endothelial cells.
Specific aims are to: 1) Identify cis-acting regulatory elements in the ICAM-1 gene promoter that mediate activation by IFN-gamma in airway epithelial cells.
This aim will be accomplished using a transient DNA transfection system with cultured airway epithelial cells and plasmid constructs composed of the 5'-flanking region of the ICAM-1 gene linked to a reporter gene. 2) Identify and characterize the specific trans-acting proteins which bind to IFN-gamma response elements in the ICAM-1 gene.
This aim will be accomplished using nuclear protein extracts from IFN-gamma stimulated cells in DNA-protein binding assays such as the electrophoretic mobility shift assay.
| Holtzman, M J; Sampath, D; Castro, M et al. (1996) The one-two of T helper cells: does interferon-gamma knock out the Th2 hypothesis for asthma? Am J Respir Cell Mol Biol 14:316-8 |
| Holtzman, M J; Brody, S L; Look, D C (1995) Does gene therapy call for ""STAT"" immunity and inflammation at the epithelial barrier? Am J Respir Cell Mol Biol 12:127-9 |
| Nakajima, S; Look, D C; Roswit, W T et al. (1994) Selective differences in vascular endothelial- vs. airway epithelial-T cell adhesion mechanisms. Am J Physiol 267:L422-32 |
| Look, D C; Pelletier, M R; Holtzman, M J (1994) Selective interaction of a subset of interferon-gamma response element-binding proteins with the intercellular adhesion molecule-1 (ICAM-1) gene promoter controls the pattern of expression on epithelial cells. J Biol Chem 269:8952-8 |
