Chemoattractant properties of PR-39, a neutrophil antibacterial peptide

The proline-arginine (PR) -rich antibacterial peptide, PR-39, kills bacteria by a non-pore-forming mechanism. Because this neutrophil peptide possesses several distinct functional properties and because other antimicrobial peptides are chemoattractants, we sought to determine whether PR-39 was a chemoattractant for porcine leukocytes. The peptide was synthesized by the solid-phase method using t-Boc chemistry and purified by reversed-phase high-performance liquid chromatography. Leukocyte migration was assessed with the use of a 48-well microchemotaxis chamber. PR-39 induced the directed migration of neutrophils. The peak chemotaxis response occurred at 0.5-2 microM, which was slightly lower than the minimal inhibitory and bactericidal concentrations for PR-39. However, the peptide was not a chemoattractant for mononuclear cells. Truncation of PR-39 suggested that the neutrophil chemoattractant domain may be contained within the first 26 amino acid residues. Intracellular Ca2+ fluxes in response to PR-39 were monitored by flow cytometry and showed a transient increase that peaked at approximately 40 s and approached basal values by 4 min. However, in a Ca2+-free environment, the PR-39-induced Ca2+ increase was abrogated. Furthermore, PR-39 did not induce neutrophil chemotaxis in the absence of extracellular Ca2+, and pertussis toxin inhibited both neutrophil chemotaxis and Ca2+ mobilization. Taken together, these data suggest that PR-39 is a Ca2+-dependent chemoattractant of neutrophils. The finding of a neutrophil antibacterial peptide that is also a neutrophil chemoattractant is intriguing and may indicate an important role for PR-39 in inflammation.