Polymorphonuclear (PMN) cells play a pivotal role in protecting higher organisms against microbial invasion. They destroy the invading microbes by two principal mechanisms. One is an oxygen dependent mechanism in which H₂O₂, O⁻₂ and HOCl are involved (Lehrer and Ganz, 1990). The other non-oxidative, microbicidal activities are mediated by the concerted action of various cationic proteins such as the bactericidal permeability increasing protein (BPI), lactoferrin, CAP-57, and several small molecular weight peptides. In addition, enzymes such as lysozyme, collagenase and several neutral serine proteases (azurocidin, cathepsin G, elastase etc.) are also associated with oxygenindependent killing of the microbes (Lehrer and Ganz, 1990). These proteins are located in the granular fractions of the neutrophils.

Major species differences exist between these oxygenindependent antimicrobial substances, e.g., cattle lack lysozyme and horses are devoid of defensins (Pellegrini et al., 1991). Gennaro et al. (1983a) reported that, in contrast to neutrophils of other species, bovine neutrophils contained, in addition to azurophilic and specific granules, a third larger and much denser type of granule, which contained cationic proteins that are unique to these granules, except for lactoferrin.

As there are no similar reports on the PMN cells of goats, the antibacterial property of caprine PMN granular proteins and peptides was studied.

AU-PAGE and SDS-PAGE of goat PMN granular acetate extracted cationic proteins and peptides are given in Fig. 1 and 2. AU-PAGE revealed presence of peptides with maximum cationicity and nearer to lysozyme, while SDS-PAGE separated proteins based on their mass, which in the presence study showed 6 bands in the range of 4 to 1.5kD.

Presence of goat PMN granular cationic peptides was confirmed by both AU-PAGE as well as SDS-PAGE. The results are similar to the reports published in buffalo (Roy et al., 1997).

Antibiotic peptides are key components of innate immunity. Their distribution throughout the animal kingdom is ubiquitous reflecting the importance of these molecules in host defense (Zasloff, 1992). Among the best studied of these peptides are mammalian defensins. Although defensins were discovered initially in neutrophils and monocytes granules, they have more recently been identified as a part of antimicrobial barrier of the mucosal surface (Jones and Bevins, 1992). ß defensins are cationic, cysteine rich molecules and most have broad-spectrum antimicrobial activity against bacteria, fungi, viruses (Zasloff, 2002). β-defensins contribute to a protective barrier on mucosal surfaces including tongue, nasal, intrapulmonary airways and small intestine (Mathews et al., 1999). Cattle express at least 15 different β-defensin genes including two found in epithelial tissues, tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) (Diamond et al., 1991; Schonwetter et al., 1995). Several diseases in humans and laboratory animals are characterized by impairment in the function of antimicrobial peptides (Zasloff, 2002). There is also considerable data indicating epithelial defensins are upregulated in response to infections (Diamond et al., 1996). In cultured epithelial cells, TAP message is induced in response to lipopolysaccharide exposure.

Stolzenberg et al. (1997) investigated the distribution and expression of ß defensin class in normal cattle and in animals in varying states of disease and proposed that expression of antimicrobial peptide is an integral component of the inflammatory response. The observation that last peak which contained the smallest proteins such as peptides was associated with the highest bactericidal potency against E. coli and S. aureus is compatible with the reports of Selsted et al. (1984), Ganz et al. (1985) and Marzari et al. (1988) in rabbits, humans and cattle, respectively. As has been shown by other authors, the antimicrobial effects of these goat PMN peptides were strongly pH dependent, the pH optimum being 7.2. Gennaro et al. (1983b) recorded that the bovine neutrophil granular proteins were very active in vitro in media of physiological pH and containing mono and divalent cations. Selsted et al. (1984) observed that purified antibacterial peptides from rabbit granulocytes NP-1, NP-2, NP-3a, NP-3b, NP-4 and NP-5 were best expressed at around neutral pH under conditions of low ionic strength. However, purified azurocidin showed its maximum antibacterial activity at pH 5.5 (Campanelli et al., 1990). Obviously there is variation in the pH optima for the antimicrobial activity of different PMN cationic proteins or peptides in different animals. As far as in vivo relevance of pH is concerned, surprisingly human PMN phagocyte vacuoles were not found to be strongly acidic, and studies with fluorescent pH probes showed that the newly formed phagosomes were neutral to mildly alkaline for the first 15 min, with subsequent acidification to pH 6.0 (Segal et al., 1981; Mayer et al., 1989). Accordingly the present observation that a neutral pH allowed the peptides to display optimal antibacterial activity may be extrapolated to in vivo conditions. Similar observations regarding the requirement for nutrients were recorded by Ganz et al. (1985). They reported that human defensins killed S. aureus, Pseudomonas aeruginosa and E. coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but had little or no bactericidal activity in nutrient free buffer. Lehrer et al. (1989) reported that when stationary phase E. coli ML-35 were exposed to human PMN peptides no β–galactosidase was released for >90 mn. In contrast, when these organisms were exposed to defensins in buffer containing nutrient (Sodium phosphate buffer-tryptic soy broth), hydrolysis of ONPG occurred, signifying that the inner membrane of the bacteria had become permeable. We also observed that the gram positive S. aureus required a higher concentration of the factor IV to check its growth, compared to E. coli. Weiss et al. (1980) observed that rough strains of S. typhimurium were more sensitive to the bactericidal, membrane-active cationic proteins from human and rabbit polymorpho-nuclear leukocytes, than smooth strains. Marzari et al. (1988) reported that the more cationic bovine PMN peptides of 4-8 kDa inhibited the growth of 5 × 10⁴ CFU of E. coli at a MIC of 12-50 µg/ml. In addition a 1.6 kDa, highly cationic peptide also inhibited the growth of S. aureus and S. epidermis at MICs of 3-8 µg/ml.

Our attempts to find out the minimum incubation period for utmost bactericidal effect of the peptides revealed that the bacterial viability decreased sharply after 10 min incubation in case of E. coli whereas viability declined sharply after 30 min in case of S. aureus. Our finding of increased β-galactosidase in the incubation medium after incubation of the E. coli cells with fraction IV suggested increased permeability in the inner and outer membranes. Lehrer et al. (1989) reported similar findings of inner and outer membrane permeabilization when E. coli bacteria were treated with human defensins. Because of their cationic nature these peptides adhere to the negatively charged surfaces of their targets and act as antibiotics by disturbing the membrane permeability in the microorganisms by opening voltage gated channels (Martin et al., 1995).

It is concluded that caprine PMN cells also contain antimicrobial agents, which probably play a significant role in the immunodefence system.

References