Christopher R. Ross

Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity.

Antimicrobial peptides are important host-defense molecules of innate immunity. Cathelicidins are a diverse family of potent, rapidly acting and broadly effective antimicrobial peptides, which are produced by a variety of cells. This review examines the classification, antimicrobial spectrum, mechanism of action, and regulation of cathelicidins.

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 μM, 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. J. Leukoc. Biol. 61: 624–629; 1997.

Molecular cloning and tissue expression of porcine β-defensin-1

Beta‐defensins constitute an emerging family of cysteine‐rich antimicrobial peptides, which are particularly prominent at mucosal epithelial sites in mammals. Here we report the identification of a novel β‐defensin from porcine tissues, porcine β‐defensin‐1 (pBD‐1). The cDNA sequence of pBD‐1 encoded a 64 amino acid prepro‐peptide, which contained the β‐defensin consensus sequence of six invariantly spaced cysteine residues. Northern blot analysis showed that pBD‐1 was expressed abundantly in tongue epithelia and that the expression was regulated developmentally. Using RT‐PCR, pBD‐1 mRNA was detected throughout the respiratory and digestive tracts and also in thymus, spleen, lymph node, brain, liver, kidney, urinary bladder, testis, skin, heart, muscle, bone marrow, peripheral blood neutrophils, alveolar macrophages, and umbilical cord. The wide expression of pBD‐1 suggests that this endogenous peptide antibiotic may contribute to both mucosal and systemic host defenses in pigs, which may have implications for the use of porcine tissues and organs in xenotransplantation.

Myocardial Ischemia/Reperfusion Injury in NADPH Oxidase–Deficient Mice

Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (-/-) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase(-/-) mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55+/-3%) and NADPH oxidase(-/-) hearts (61+/-4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42+/-4%) and NADPH oxidase(-/-) mice (34+/-5%) (P=not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase(-/-) mice (n=8; 27+/-2.5%) and heterozygous control mice (n=10; 23.3+/-3. 3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired (P<0.01) in NADPH oxidase(-/-) mice (n=6) compared with heterozygous mice (n=7) (0.04+/-0.03 versus 2.2+/-0.08 nmol O(2).min(-1).10(6) cells(-1)). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase(-/-) mice (P:=not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.

Identification of a proline-arginine-rich antibacterial peptide from neutrophils that is analogous to PR-39, an antibacterial peptide from the small intestine.

Neutrophil nonoxidative defense mechanisms include several low molecular weight antimicrobial peptides. We have isolated a proline‐arginine‐rich antibacterial peptide from porcine neutrophils. This cationic, low molecular weight peptide has a very high degree of identity (97%) to a bactericidal peptide, PR‐39, that has been found in the porcine small intestine. Isolation of the same antimicrobial peptide from both neutrophils and cells of the small intestine in the same species is unique and suggests an important role for this protein in innate immune defenses. J. Leukoc. Biol. 56: 807–811; 1994.

Innate Immunity and Host Defense Peptides in Veterinary Medicine

Recent years have witnessed a surge in interest directed at innate immune mechanisms. Proper conceptualization of the key elements of innate immunity, however, is still a work in progress, because most research in immunology traditionally has been focused on components of the acquired immune response. The question of why an animal stays healthy in a world filled with many dangers is perhaps as interesting as why it sometimes surrenders to disease. Consequently, studies with an increased focus on inborn mechanisms of animal host defense may help further the development of appropriate preventative and therapeutic measures in veterinary medicine. Host defense peptides (HDPs) are central effector molecules of innate immunity, and are produced by virtually all living species throughout the plant and animal kingdoms. These gene-encoded peptides play a central role in multiple, clinically relevant disease processes. Imbalances in the expression of HDPs can lead to overt pathology in different organ systems and cell types in all species studied. In addition, HDPs are an ancient group of innate chemical protectors, which are now evaluated as model molecules for the development of novel natural antibiotics and immunoregulatory compounds. This review provides an overview of HDPs and is aimed at veterinary practitioners as well as basic researchers with an interest in comparative immunology involving small and large animal species.

Cloning and Characterization of the Gene for a New Epithelial β-Defensin GENOMIC STRUCTURE, CHROMOSOMAL LOCALIZATION, AND EVIDENCE FOR ITS CONSTITUTIVE EXPRESSION

Mammalian β-defensins are endogenous cysteine-rich peptide antibiotics that are produced either by epithelial cells lining the respiratory, digestive, and urogenital tracts or by granulocytes and macrophages. A growing body of evidence has implicated these peptides in host defense, particularly mucosal innate immunity. We previously reported the cloning of the full-length cDNA for a porcine β-defensin (pBD-1), which was found to be expressed throughout the airway and oral mucosa. Here, we provide the structural organization of the pBD-1 gene, showing that the entire gene spans ∼1.9 kilobases with two short exons separated by a 1.5-kilobase intron. Fluorescence in situ hybridization mapped the pBD-1 gene to porcine chromosome 15q14-q15.1 within a region of conserved synteny to the chromosomal locations of human and mouse α- and β-defensins. We also provide several independent lines of evidence showing that thepBD-1 gene is expressed constitutively during inflammation and infection, despite its resemblance to many inducible epithelial β-defensins in amino acid sequence, genomic structure, and sites of expression. First, stimulation of primary porcine tongue epithelial cells with lipopolysaccharide, tumor necrosis factor-α, and interleukin (IL)-1β failed to up-regulate the expression ofpBD-1 mRNA. Second, pBD-1 gene expression was not enhanced in either digestive or respiratory mucosa of pigs following a 2-day infection with Salmonella typhimurium orActinobacillus pleuropneumoniae. Last, direct transfection of the pBD-1 gene promoter into NIH/3T3 cells showed no difference in reporter gene activity in response to stimulation by lipopolysaccharide and IL-1β. The constitutive expression ofpBD-1 in airway and oral mucosa, which is consistent with a lack of consensus binding sites for nuclear factor-κB or NF-IL-6 in its promoter region, suggests that it may play a surveillance role in maintaining the steady state of microflora on mucosal surfaces.

Gene Silencing and Overexpression of Porcine Peptidoglycan Recognition Protein Long Isoforms: Involvement in β-Defensin-1 Expression

ABSTRACT Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide β-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of β-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of β-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.

PR-39, a proline/arginine-rich antimicrobial peptide, exerts cardioprotective effects in myocardial ischemia–reperfusion

OBJECTIVE PR-39, a proline/arginine-rich antimicrobial peptide, has been shown to inhibit the NADPH oxidase activity of polymorphonuclear leukocytes (PMNs) by blocking assembly of this enzyme. We hypothesized that PR-39 could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production. METHODS We examined the effects of PR-39 in isolated ischemic (20 min) and reperfused (45 min) rat hearts administered PMNs at the onset of reperfusion. RESULTS PR-39 (4 or 10 microg/ml) given i.v. 30 min prior to ischemia-reperfusion (I-R) significantly improved left ventricular developed pressure (LVDP, P<0.01) and the maximal rate of development of LVDP (i.e. +dP/dt max, P<0.01) compared to I-R hearts obtained from rats given 0.9% NaCl. PR-39-treated PMNs (10 microg/ml) also significantly attenuated cardiac contractile dysfunction after I-R (P<0.01). Superoxide release was significantly reduced (P<0.01) in N-formylmethionyl-leucylphenylalanine stimulated PMNs pretreated with 4 or 10 microg/ml PR-39. PR-39 also significantly attenuated P-selectin expression on the rat coronary microvascular endothelium and CD18 upregulation in rat PMNs. In addition, PR-39 significantly reduced PMN vascular adherence and infiltration into the post-ischemic myocardium. CONCLUSION These results provide evidence that PR-39 significantly attenuates PMN-induced cardiac contractile dysfunction in the I-R rat heart at least in part via suppression of superoxide release. This cardioprotection occurred both by inhibition of PMN and endothelial NADPH oxidase.

Toll-like Receptor 3 Activation Decreases Porcine Arterivirus Infection

Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus that initiates infection in pulmonary alveolar macrophages (PAMs), elicits weak immune responses, and establishes a persistent infection. To understand the role of dsRNA intermediates in eliciting host immunity, we sought to determine if toll-like receptor-3 (TLR3), a well-known dsRNA sensor, is involved in the regulation of PRRSV infection. TLR3 gene expression was increased in PAMs of congenitally infected 2-wk-old pigs. Stimulation of PAMs with dsRNA increased gene expression for TLR3 and interferon-beta and suppressed PRRSV infectivity. To investigate activation and signaling parameters, expression constructs of wild-type and functional-domain-truncated porcine TLR3 were used in cell transfection studies. When cells that overexpressed porcine TLR3 were stimulated with dsRNA a rapid and robust calcium influx was induced. Moreover, ligand activation of porcine TLR3 expressed in MARC-145 cells elicited an antiviral response to PRRSV. Conversely, transfection of PAMs with small-interfering RNA targeting porcine TLR3 resulted in up to 80% suppression of TLR3 mRNA expression and an increase in PRRSV infectivity. These data provide fundamental genetic and molecular information for porcine TLR3, and implicate its involvement in PRRSV infection, findings that may suggest new strategies to limit this costly pandemic disease.