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The Journal of Immunology, 2003, 171: 6630-6639.
Copyright © 2003 by The American Association of Immunologists

Neutrophil Gelatinase-Associated Lipocalin Is Up-Regulated in Human Epithelial Cells by IL-1{beta}, but Not by TNF-{alpha} 1

Jack B. Cowland2,*, Ole E. Sørensen*, Maxwell Sehested{dagger} and Niels Borregaard*

Departments of * Hematology and {dagger} Pathology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1{beta} induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-{alpha}, IL-6, and LPS had no effect. Similar IL-1{beta} selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-{kappa}B consensus site was found to control the IL-1{beta}-specific up-regulation. Involvement of the NF-{kappa}B site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-{kappa}B pathway, and by EMSA. TNF-{alpha} activation of NF-{kappa}B, in contrast, did not increase NGAL synthesis, even though induced binding of NF-{kappa}B to the NGAL promoter was observed in vitro. IL-1{beta} specificity was not contained within the NF-{kappa}B site of the NGAL promoter, as determined by exchanging the NGAL promoter's NF-{kappa}B-binding sequence with that of the IL-8 promoter or with the NF-{kappa}B consensus sequence and by testing the NF-{kappa}B-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.




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