Bactericidal activity of porcine neutrophil secretions

Abstract

Antimicrobial proteins in neutrophil granules exert their bactericidal activity both within the neutrophil phagolysosome and as components of neutrophil extracellular traps. This study evaluated the bactericidal activity of porcine neutrophil secretions against four bacterial pathogens of swine. Porcine neutrophils were treated with or without phorbol myristate acetate (PMA), then the resulting supernatants were incubated with Escherichia coli K-12, Streptococcus suis, Actinobacillus suis, or Pasteurella multocida, and the surviving colony forming units were enumerated. Supernatants of PMA-activated neutrophils killed an average of 95% of E. coli K-12 cells, relative to supernatants from untreated neutrophils. Inhibition of elastase activity using chloromethylketone (CMK) prior to PMA stimulation significantly reduced the bactericidal activity of the neutrophil supernatants; 57% of the PMA-induced bactericidal activity against E. coli K-12 was estimated to be elastase-dependent. The same neutrophil supernatants had lower bactericidal activity against S. suis, A. suis, and P. multocida, with 30%, 36% and 13% reduction in bacterial numbers, respectively. The cathelicidin porcine myeloid antimicrobial peptide (PMAP)-36 and lactotransferrin were among the proteins identified in the supernatants of PMA-stimulated neutrophils by mass spectrometry. These findings imply that elastase-activated proteins, such as cathelicidins, are partially responsible for the bactericidal effect of porcine neutrophil secretions, but non-elastase-dependent proteins such as lactoferrin may also contribute. Further, the secretions of activated neutrophils were effective in killing the avirulent E. coli K-12 but were less effective against the other bacteria tested, suggesting that these pathogens may have evolved mechanisms to resist neutrophil-mediated killing.

Introduction

Neutrophils are important effectors of immunity to extracellular opportunistic bacterial pathogens. Neutrophil-mediated bacterial killing occurs both within the phagolysosome following phagocytosis, and in the extracellular environment following active secretion of chromatin and other intracellular constituents to form neutrophil extracellular traps (Brinkmann et al., 2004, Papayannopoulos and Zychlinsky, 2009). Two-dimensional gel electrophoresis reveals at least 20–40 proteins in the supernatants of phorbol myristate acetate (PMA)-stimulated porcine neutrophils (Brooks et al., 2003), including antimicrobial proteins that are secreted from neutrophil granules and contribute to both intracellular and extracellular mechanisms of bacterial killing (Panyutich et al., 1997).

Of the neutrophil antimicrobial proteins and peptides, defensins are found in the primary granules of neutrophils, whereas cathelicidins and lactotransferrin are stored in the secondary granules (Selsted and Ouellette, 2005, Zanetti, 2005). Cathelicidins are stored as inactive propeptides, and are activated following cleavage of the conserved N-terminal cathelin region by elastase, a serine protease which is sequestered in primary granules (Shinnar et al., 2003, Selsted and Ouellette, 2005, Zanetti, 2005). Since cathelicidins are the only antimicrobial proteins known to be activated by elastase (Cole et al., 2001), and because elastase itself has minimal bactericidal activity (Thorne et al., 1976), elastase inhibition may be used as a method to estimate the contribution of cathelicidins to the bactericidal activity of neutrophils.

Pigs have the largest repertoire of cathelicidins of any species studied, including prophenin (PF)-1, PF-2, proline-arginine-rich 39-amino-acid peptide (PR)-39, protegrins 1 to 5 (PG-1 to PG-5), porcine myeloid antimicrobial peptide (PMAP)-23, PMAP-36, and PMAP-37 (Sang and Blecha, 2009). In contrast, pigs appear to lack α-defensins, which are common constituents of neutrophil granules in many species. Further, in contrast to the rich repertoire of neutrophil β-defensins in ruminants, porcine β-defensin-1 is the only β-defensin gene known to be expressed in porcine neutrophils, and no porcine neutrophil β-defensins have yet been documented at the peptide level (Zhang et al., 1998, Ganz, 2003, Sang and Blecha, 2009). Thus, the molecular basis of neutrophil-mediated bacterial killing is likely to differ between host species, and cathelicidins may be of particular importance in swine.

Previous studies have demonstrated antimicrobial activity of individual porcine cathelicidins, but these studies do not evaluate activity against the important bacterial pathogens of swine (Shi et al., 1996, Zhang et al., 2000, Linde et al., 2001, Scocchi et al., 2005, Sang and Blecha, 2009). Moreover, these studies with purified cathelicidins fail to account for synergism among the contents of neutrophil granules (Rogan et al., 2006). Thus, the objectives of this study were to evaluate the bactericidal activity of unfractionated porcine neutrophil secretions against several important pathogens of swine, and to estimate the proportion of this activity that was elastase-dependent and therefore attributable to cathelicidins.