Veterinary Immunology and Immunopathology 149 (2012) 58–65 Contents lists available at SciVerse ScienceDirect Veterinary Immunology and Immunopathology j o ur nal ho me p age: w ww.elsevier.com/locate/vetimm Research paper Adenosine-5 ′ -triphosphate release by Mannheimia haemolytica, lipopolysaccharide, and interleukin-1 stimulated bovine pulmonary epithelial cells Michael Craddick a , Rakhi Patel a , Amanda Lower a , Sarah Highlander b , Mark Ackermann c , David McClenahan a,∗ a University of Northern Iowa, Department of Biology, Cedar Falls, IA 50614, United States b Baylor College of Medicine, Department of Molecular Virology and Microbiology, Houston, TX 77030, United States c Iowa State University, Department of Veterinary Pathology, Ames, IA 50011, United States a r t i c l e i n f o Article history: Received 23 June 2011 Received in revised form 1 June 2012 Accepted 6 June 2012 Keywords: Bovine respiratory disease complex Lung inflammation Extracellular adenosine-5 ′ -triphosphate Shipping fever a b s t r a c t Mannheimia haemolytica, one of the agents associated with bovine respiratory disease com- plex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5 ′ -triphosphate (ATP). Therefore, we wanted to determine if ATP levels were ele- vated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non- treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addi- tion, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Bovine respiratory disease complex (BRDC) is a costly disease to both beef and dairy producers in North Amer- ica. Even though the organisms that cause the disease have been identified, molecular features that underlie the pathology associated with these infections are not ∗ Corresponding author. Tel.: +1 319 273 2218; fax: +1 319 273 2893. E-mail address: david.mcclenahan@uni.edu (D. McClenahan). fully understood. One organism associated with BRDC, Mannheimia haemolytica, causes extensive leakage of blood components into the alveoli and airways of the lung within the first couple of hours of infection (McClenahan et al., 2000). The cause of this leakage could be due to products produced by the bacteria itself, or due to the host’s response to the bacteria in the lung, or a combination of both. Because the bacteria associated with BRDC are all Gram-negative organisms, one common virulence prod- uct produced by the group is lipopolysaccharide (LPS). In addition, M. haemolytica produces a unique virulence 0165-2427/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetimm.2012.06.011