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Inhibition of Tumor Necrosis Factor-–Induced RANTES Secretion by Alkaline Protease in A549 Cells

Department of Anatomy and Radiology, College of Veterinary Medicine, and Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia; and Department of Microbiology, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland

Correspondence and requests for reprints should be addressed to Thomas M. Krunkosky, MS, DVM, Ph.D., Department of Anatomy and Radiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30606. E-mail: tmkrunko{at}vet.uga.edu

Pseudomonas aeruginosa is a gram-negative bacterium that is an opportunistic pathogen in patients with cystic fibrosis and in immunocompromised hosts. This bacterium produces a variety of proteolytic enzymes, including alkaline protease (AP), which has multiple biological effects. This study investigated the effects of AP on the A549 pulmonary epithelial cell line. Results demonstrate that AP inhibited tumor necrosis factor (TNF)-–induced RANTES gene expression and secretion in a concentration-dependent manner. The TNF-–induced RANTES gene expression and secretion was attenuated with a neutralizing monoclonal antibody directed against the TNF receptor type 1 (TNFR1). Conversely, a neutralizing monoclonal antibody directed against TNF receptor type II had no effect, suggesting that these events were regulated through the TNFR1 receptor. In addition, we observed that soluble TNF receptor type 1 (sTNFR1) levels were significantly increased in culture supernatants of AP-treated cells in a concentration-dependent manner. Finally, membrane-associated TNFR1 was decreased after AP exposures. In these studies, the enzymatically inactive form of AP had no effect on TNF-–induced RANTES secretion, shedding of sTNFR1, or membrane-associated TNFR1. These results demonstrate that AP stimulates shedding of cell-surface TNFR1, resulting in an increase in sTNFR1. Consequently, these events decrease the cells' ability to stimulate RANTES gene expression and secretion through TNFR1.

Key Words: Pseudomonas aeruginosa • tumor necrosis factor alpha • cytokine • airway epithelium