Introduction
Chicken heterophils are polymorphonuclear leukocytes that constitute the first line of defence against invading microorganisms. They are the most abundant leukocytes that play key roles in the innate immunity which refers to the first-line host defence that serves to limit infection in the early hours after exposure to microorganisms (Hoffmann et al., 1999). These cells are the equivalent of neutrophils in other species; they are the predominant leukocyte in the acute inflammatory response in birds. Heterophils are highly phagocytic cells with broad spectrum antimicrobial activity. They accumulate in inflamed tissue, causing tissue damage and forming heterophil granulomas (Harmon, Reference HARMON1998). The initial response of the body to microbial invasion and the events in the first few hours are crucial in determining the outcome of infection (Namavar et al., Reference NAMAVAR, VERWEIJ-VAN VUGHT, VEL, BAL and MACLAREN1984) and bacterial infections induce recruitment of large numbers of heterophils to the site of infection with marked heterophilia (Kogut et al., Reference KOGUT, MCGRUDER, HARGIS, CORRIER and DELOACH1994; Kogut et al., Reference KOGUT, MCGRUDER, HARGIS, CORRIER and DELOACH1995).
Antimicrobial activity
In contrast to the bactericidal capacities of mammalian neutrophils with their different chemical properties involving proteins, peptides and oxygen-dependent radicals (Hoffmann et al., 1999) generated during the respiratory burst including active oxygen derivatives (superoxides, hydrogenperoxide, hydroxyl radical, singlet oxygen) are essential for intracellular killing activity (Nagahata et al., Reference NAGAHATA, YATSU and NODA1986; Hirabayashi et al., Reference HIRABAYASHI, TANIUCHI and KOBAYASHI1985). The release of large amounts of hydrogen peroxide was observed in stimulated neutrophils (Biggar and Sturgess, Reference BIGGAR and STURGESS1978). However, avian heterophils lack myeloperoxidase, an essential enzyme of the respiratory burst (Brune et al., Reference BRUNE, LEFFELL and SPITZNAGEL1972; Brune and Spitznagel, Reference BRUNE and SPITZNAGEL1973; Penniall and Spitznagel, Reference PENNIALL and SPITZNAGEL1975; Styrt, Reference STYRT1989; Harmon, Reference HARMON1998) and produce small amounts of oxygen radicals (Conlon et al., Reference CONLON, SMITH and GOWLETT1991). The avian antimicrobial activity of heterophils depends mainly on oxygen-independent mechanisms like cationic antimicrobial peptides and lysozyme (Harmon et al., Reference HARMON, GLISSON and NUNALLY1992). Many researchers in the field of natural cationic antimicrobial peptides have been attracted by avian heterophils due to their antimicrobial activity which depends primarily on oxygen-independent mechanisms (Harmon, Reference HARMON1998), and heterophils are useful in studying oxygen independent killing activity.
Heterophils and the cationic antimicrobial peptides
The killing activity of polymorphonuclear leukocytes against bacteria and fungi is conserved in anaerobic conditions and disease where their ability to generate reactive oxygen intermediates is completely abolished (Vel et al., Reference VEL, NAMAVAR, WERWEIJ, PUBBEN and MACLAREN1984; Ingham et al., Reference INGHAM, SISSON, MIDDLETON, NARANG, CODD and SELKON1981; Mandell, Reference MANDELL1974). In addition, there appears to be antibacterial activity of the crude extract of the polymorphonuclear leukocytes as well as some granule fractions from the cell homogenates of the polymorphonuclear leukocytes (Zeya and Spitznagel, Reference ZEYA and SPITZNAGEL1966a; Zeya and Spitznagel, Reference ZEYA and SPITZNAGEL1966b; Odeberg and Olssen, 1975; Weiss et al., Reference WEISS, ELSBACH, OLSSON and ODELBERG1978 and Hodinka et al., 1983). These findings suggest that mechanisms other than those dependent on oxygen may have a key role in the killing of some bacteria and fungi, and many investigative studies have been conducted to purify the molecules which contribute to the oxygen-independent antimicrobial activity.
The heterophils’ cationic arginine-rich proteins were first demonstrated by Brune and Spitznagel (Reference BRUNE and SPITZNAGEL1973) and the existence of such cationic proteins was proved using the ammoniacal silver reaction (Macrae and Spitznagel, Reference MACRAE and SPITZNAGEL1975). The cationic antimicrobial peptides generally have two to nine excess, positively charged amino-acids, either arginine or lysine (Hancock and Chapple, Reference HANCOCK and CHAPPLE1999). Three cationic antimicrobial peptides have been isolated from chicken leukocytes. They are cysteine-rich molecules with 36 -39 amino acid residues and with sequence homology to the human β-defensins and nine invariant residues (six cysteines, two glycines and one proline) common to bovine β-defensins, named ‘gallinacins’: Gal-1α, Gal-1, Gal-2, (Harwig et al., Reference HARWIG, SWIDEREK, KOKRYAKOV, TAN, LEE, PANYUTICHA, ALESHINA, SHAMOVA and LEHRER1994). In the same year, two antimicrobial peptides were isolated from the granule extract of the chicken heterophils and named chicken heterophil peptides (CHP1 and CHP2). They were cationic and rich in cysteine, arginine and lysine; CHP1 was characterized and consists of 39 amino acid residues with a molecular weight of 4481 (Evans et al., Reference EVANS, BEACH, WUNDERLICH and HARMON1994). The gallinacins, like the cationic antimicrobial peptides of mammalian species, are stored in the granules of chicken heterophils (Evans et al., Reference EVANS, BEACH, WUNDERLICH and HARMON1994). The gallinacin-3 expression was found to be strongly prominent in the chicken tongue, bursa of Fabricius and trachea and was also moderately expressed in the skin, oesophagus and air sacs; weaker expression of GaL-3 was seen in the large intestine, kidney and ovary. In contrast to GaL-3, the expression of GaL-1 (and –1α) and GaL-2 was limited to bone marrow cells (Zhao et al., Reference ZHAO, NGUYEN, LIU, SACCO, BROGDEN and LEHRER2001).
Antibacterial activity of chicken cationic peptides
The cationic antimicrobial peptides have a wide killing activity; they are active against enveloped viruses, gram-negative and/or gram-positive bacteria, fungi, parasites and even cancer cells (Hancock and Diamond, Reference HANCOCK and DIAMOND2000). Bacterial killing occurs in minutes, and, in most cases, requires bacterial cell growth (Huttner and Bevins, Reference HUTTNER and BEVINS1999). The avian cationic peptides isolated from heterophils, like those of mammals, were found to have antibacterial activity against either gram-positive or gram-negative bacteria.
Quantitative assays reveal that CHP-1 kills 80% of E. coli at 2.7 µg/ml, while 80% of the S. aureus was killed at only 0.7 µg/ml. The antibacterial activity of CHP-2 tested by the radial diffusion method indicated that it was bactericidal against both S. aureus and E. coli. The turkey heterophils peptide-1 (THP-1) is less potent than chicken heterophil peptides, and kills 80% of S. aureus at 2.6 µg/ml. Killing of E. coli required higher concentrations and 80% of the E. coli was killed at 5.3 µg/ml. The other two turkey heterophil peptides (THP-2 and THP-3) failed to kill E. coli in vitro at a concentration of 21 µg/ml and killed 80% of the S. aureus at 10.5 µg/ml (Evans et al., Reference EVANS, BEACH, WUNDERLICH and HARMON1994).
The antimicrobial activity of CHP-1 and CHP-2 was tested against a variety of microorganisms including avian pathogens and human pathogens which are harboured by birds. Both peptides were effective against all the bacteria tested at a concentration of 16 µg/ml, except for Pasteurella multocida serotype A:3, strain 1059, also the THP-3 has no effect on Bordetella avium, E. Coli, and Salmonella typhimurium at the maximum concentration of 16 µg/ml and the peptides were unable to neutralize infectious bronchitis virus (IBV) in experimental conditions (Evans et al., Reference EVANS, BEACH, MOORE, JACKWOOD, GLISSON and HARMON1995).
Tested against E. coli ML-35P, Listeria monocytogenes EGD and C. albicans by the ultrasensitive radial diffusion method, Gal-1 and Gal-1α were powerful against E. coli and were 10 fold more potent than HNP-1. Gal-2 (CHP-2) expressed less efficiency against E. coli but far exceeded that of HNP-1. All three gallinacins have almost equal antibacterial activity against L. monocytogenes. Gal-1 and Gal-1α killed C. albicans, whereas Gal-2 was ineffective even at a concentration of 400 µg/ml (Harwig et al., Reference HARWIG, SWIDEREK, KOKRYAKOV, TAN, LEE, PANYUTICHA, ALESHINA, SHAMOVA and LEHRER1994). It has been found that components of lysed heterophils were sufficient to kill E. coli and S. aureus (Harmon et al., Reference HARMON, GLISSON and NUNALLY1992). Gallinacins 4, 7 and 9 have antibacterial activity against salmonella serovars (Milona et al., Reference MILONA, TOWNES, BEVAN and HALL2007).
Mode of action
The mode of action of cationic antimicrobial peptides has been extensively studied in human neutrophil peptides. Usually the cationic antimicrobial peptides interact directly by attraction between the positively charged peptides and the negatively charged bacterial surfaces; causing the loss of membrane integrity and increased membrane damage leading to lysis of bacteria (Gudmundsson and Agerberth, Reference GUDMUNDSSON and AGERBERTH1999).
The bactericidal activity of human neutrophil peptide against E. coli ML-35 is due to sequential permeabilisation of the outer membrane and inner membrane, with the latter appearing to be the lethal event (Lehrer et al., Reference LEHRER, BARTON, DAHER, K, HARWIG, GANZ and SELSTED1989). For Gram-positive bacteria, only a single membrane is involved (Hancock, Reference HANCOCK1997). The cationic antimicrobial peptides disrupt the normal barrier property of the outer membrane which permits the passage of a variety of molecules, including the uptake of the peptide itself (Hancock, Reference HANCOCK1997; Hancock and Chapple, Reference HANCOCK and CHAPPLE1999).
Antiendotoxinic activity of cationic antimicrobial peptides
Septicemia is usually caused by the release of lipopolysaccharides from the outer membrane of Gram-negative bacteria and the toxicity of this endotoxin (LPS) is contained within its lipid A portion. Gram-positive septicemia is also presumed to be caused by the release of bacterial cell wall components (Hancock and Scott, Reference HANCOCK and SCOTT2000). In contrast to most antibiotics which induce endotoxinaemia by releasing LPS, some cationic peptides have the ability to neutralize LPS (Hancock, Reference HANCOCK1997). Most antimicrobial peptides have the ability to block the activation of macrophages by LPS and this reduces or prevents the LPS-induced inflammatory responses (Scott et al., Reference SCOTT, VREUGDENHIL, BUURMAN, HANCOCK and GOLD2000). Also the cationic antimicrobial peptides inhibit the production of cytokines such as tumour necrosis factor TNF-α and IL-6 by macrophages stimulated with LPS (Hancock and Scott, Reference HANCOCK and SCOTT2000). Other small α-helical cationic peptides, MBI-27 and MBI-28 derived from cecropin and bee melittin have the ability to overcome lethal endotoxemia, and they bind to LPS and prevent its ability to induce TNF response, this latter is a major mediator in endotoxinic shock (Gough et al., Reference GOUGH, HANCOCK and KELLY1996).
Action on mammalian cells
Beside their role as natural antibiotics, the cationic antimicrobial peptides have cytotoxic effects on the host cells. In contrast to bacteria, the outer leaflet of the cell membrane is less negatively charged in eukaryotic cells and this may partially explain the different effects of the peptides on prokaryotic cells compared to eukaryotic cells, whose membrane contains a mixture of negatively charged and zwitterionic phospholipid (Gudmundsson and Agerberth, Reference GUDMUNDSSON and AGERBERTH1999).
The cytotoxic activity was demonstrated in assays of cytotoxicity against various cell types especially erythrocytes. The chicken peptides gallinacins 4, 7 and 9 showed less than 10% haemolysis with their microbial killing concentrations (Milona et al., Reference MILONA, TOWNES, BEVAN and HALL2007).
The haemolytic activity is strongly dependent on the conditions under which the haemolytic assay is performed. Histatin and magainins were strongly active against C. albicans and human erythrocytes only in low ionic strength buffer (Helmerhorst et al., Reference HELMERHORST, REIJNDERS, HOF, VEERMAN and AMERONGEN1999).
The cytotoxic peptides found in antimicrobial secretions function primarily by providing a first line of defence against invading microorganisms, whereas some cytotoxic peptides present in venoms (e.g. bee, scorpion or spider) function as toxins and are used for defence against predators (Kourie and Shorthouse, Reference KOURIE and SHORTHOUSE2000).
Other roles of cationic antimicrobial peptides
It was suggested that higher concentrations of defensins in the airways of mice may play an important role in the pathogenesis of inflammatory lung diseases (Zhang et al., Reference ZHANG, PORRO, ORZECH, MULLEN, LIU and SLUTSKY2001) and defensins from human neutrophils and other species have histamine releasing activity and degranulate rat peritoneal mast cells (Befus et al., Reference BEFUS, MOWAT, GILCHRIST, HU, SOLOMON and BATEMAN1999).
The leukocyte defensin has a role in lipid metabolism and causes increased binding of Lp(a) to endothelial cells and to smooth muscle cells. Defensin caused also a comparable increase in the internalization of Lp(a) by each cell type without concomitant increase in degradation and it was suggested that defensin may contribute to the development of atherosclerosis (Higazi et al., Reference HIGAZI, LAVI, BDEIR, ULRICH, JAMIESON, RADER, D, USHER, KANE, GANZ and CINES D.B.1997).
Cationic peptides in pathologic states
The epithelial Gal-3 was found in the trachea and its tracheal expression was increased in response to experimental infection with Haemophilus paragallinarum whereas, its expression in the other organs was unaffected (Zhao et al., Reference ZHAO, NGUYEN, LIU, SACCO, BROGDEN and LEHRER2001). Cationic peptides provide protection against colibacillosis in chickens (Jiang et al., Reference JIANG, SIMS and CONWAY2005) and give higher protection against bacterial pathogens with immunostimulation of chicken heterophils when used as feed additives mainly during the first week post-hatch where the innate immune response is inefficient (Kogut et al., Reference KOGUT, GENOVESE, HE, LI and JIANG2007). Heterophils participate in chicken protection against salmonella infection by releasing another potent cationic antimicrobial peptide, cathelicidin-2 (Van Dijk et al., Reference VAN DIJK, TERSTEEG-ZIJDERVELD, TJEERDSMA-VAN BOKHOVEN, JANSMAN, VELDHUIZEN and HAAGSMAN2009). Thus heterophils form a major barrier against invading microorganisms either by phagocytosis or by their effective non-oxidative antimicrobial activity depending on cationic antimicrobial peptides within cytoplasmic granules and they constitute the best model for studying the oxygen independent mechanisms.
