title: Apoptosis induced by Pseudomonas aeruginosa in antigen presenting cells is diminished by genetic modification with CD40 ligand.
abstract: Persistent colonization with Pseudomonas aeruginosa (PA) is a hallmark of the lung disease associated with cystic fibrosis (CF). Based on the concept that PA is not cleared from the lung by the host response in individuals with CF, we analyzed the capacity of PA to induce cell death in human alveolar macrophages (AM) and murine dendritic cells (DC), antigen presenting cells that play a central role in the initiation of pulmonary host defenses against pathogens, and evaluated if genetic modification can lead to protection against PA induced cell death. AM and DC were susceptible to cell death induced by the laboratory PA isolates PAO1, PAK and PA103, as well as a mucoid derivative of PAO1 and PA isolates derived from sputum of individuals with CF. Apoptosis, analyzed by TUNEL assay, was detectable in AM and DC as early as 3 h after infection with PA. In contrast, the same strains and doses of PA had little effect on the lung epithelial cell line A549 and primary cultures of human bronchial epithelial cells in vitro. Pretreatment of DC with the caspase inhibitors VAD-fmk and YVAD-cmk reduced PA induced cell death (p < 0.05). Finally, genetic modification of DC to express CD40L using an adenovirus vector decreased the susceptibility of DC to cell death induced by PAO1 compared with DC infected with a control Ad vector (p < 0.01). The data demonstrate that DC and AM are susceptible to apoptosis induced by PA and that this response can be partially reversed by genetic modification with CD40L, a CD4+ T cell molecule that plays a central role in activating antigen presenting cells. These observations suggest a potential mechanism contributing to the persistence of PA in CF and suggest that genetic manipulation of antigen presenting cells with anti-apoptotic genes may be able to strengthen host defenses in CF.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Department of Pediatrics, Weill Medical College of Cornell University, New York, New York 10128, USA. geneticmedicine@med.cornell.edu
authors.forename: Stefan
authors.initials: S
authors.lastname: Worgall
authors.forename: Katherine
authors.initials: K
authors.lastname: Martushova
authors.forename: Annette
authors.initials: A
authors.lastname: Busch
authors.forename: Leah
authors.initials: L
authors.lastname: Lande
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Crystal
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D019013:CD40 Antigens / Q000502:physiology; D023201:CD40 Ligand / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000150:complications / Q000382:microbiology; D003713:Dendritic Cells / Q000737:chemistry / Q000187:drug effects / Q000276:immunology*; D005822:Genetic Vectors / Q000494:pharmacology; D006801:Humans; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D011552:Pseudomonas Infections / Q000209:etiology / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000302:isolation & purification / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013183:Sputum / Q000382:microbiology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D019013:CD40 Antigens; D015853:Cysteine Proteinase Inhibitors; C098738:N-acetyl-tyrosyl-valyl-alanyl-aspartyl chloromethyl ketone; D011993:Recombinant Fusion Proteins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D023201:CD40 Ligand