**Defensins**

186 Antimicrobial Agents

lysozyme and lactoferrin. Dermcidin precursor was also found in human gestational tissue (Lee Motoyama et al., 2007), where it is proposed to play a role in pregnancy by regulating

Cathelicidin is found in eccrine gland cells, but also released into circulation. The CAP18 precursor is produced in skin by keratinocytes and is processed within neutrophils, keratinocytes and mast cells by inflammation or injury. In circulation, the mature form is LL-37, after processing of CAP18 by neutrophil-derived elastase and proteinase-3, but other proteases can also produce KS-30 and RK-21, two peptides active against pathogenic bacteria. Cathelicidin expression is also regulated at the transcriptional level by bacterial LPS, cutaneous injury and pro-inflammatory mediators (IL-6, retinoic acid). The LL-37 is intensively studied and besides its wide antibacterial spectrum, it is considered as a mediator between innate and adaptive immunity (Kai-Larsen & Agerberth, 2008) and cell

Eight known functional RNase A ribonucleases genes are encoding small polypeptides of 15 kDa (Dyer & Rosenberg, 2006). Besides their well-documented ribonuclease activity, some of these proteins display unexpected antimicrobial activities unrelated to their primary function. Eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic peptide (ECP/RNase 3) are proteins secreted by eosinophilic leukocytes and were primarily tested for their toxic role against parasites. *In vitro*, ECP has also an activity against Gram-positive

RNase A7 was identified as a major agent of the innate immune response of the skin acting on Gram-positive and Gram-negative bacteria and also on *C. albicans* (Harder & Schröder, 2002). RNase 7 transcripts were induced in keratinocyte culture by addition of TNF-alpha, interferon-gamma, interleukin-1 beta and in the presence of bacteria (Harder & Schröder, 2002). More recently, RNase 5 was added to the list of antimicrobial molecules present in skin (*stratum corneum*), and the same authors showed that skin proteases are involved in

Psoriasin belongs to the S100 family of calcium-binding proteins. This family is composed of 21 genes and 11 proteins that have been found to be expressed in human epidermis or in cultured keratinocytes. Langerhans cells and melanocytes (Boni et al., 1997; Broome et al., 2003) express S100B and Meissner's corpuscules (sensorial receptors localized in the upper part of dermis) express S100P (Del Valle et al., 1994). These proteins possess two EF hands (helix-loop-helix calcium binding domains) and they act probably as calcium sensors. Several functions have been proposed for S100 proteins in keratinocytes, the main role being an implication in skin inflammatory processes (Jinquan et al., 1996). Another role could be keratinocytes membrane remodeling, that occurs during differentiation: psoriasin and another member of the S100 family, calgranulin-A (S100A9), have been shown to have their expression correlated with the degree of keratinocyte differentiation, suggesting that they are involved in this process (Martinsson et al., 2005]. A third role could be an involvement in the formation of calcium channels, in conjunction with annexins. Other postulated roles

trophoblastic functions.

**RNase A superfamily** 

**Psoriasin (S100A7)** 

**Cathelicidin, LL-37, hCAP18** 

differentiation can also regulate its activity.

and Gram-negative bacteria (Lehrer et al., 1989).

inhibition of RNases 5 and 7 (Abtin et al., 2009).

To date 4 defensins (hBD-1 to -4) in neutrophils and 2 defensins (hBD-5 and hBD-6) produced by Paneth cells were identified. The first inducible human defensin, hBD-2, was identified in psoriatic lesions as the most abundant AMP. It was found to be expressed in terminally differentiated keratinocytes, in a structure located in *stratum corneum*, lamellar bodies that contain lipid-rich secretory granules. It is probably released with lipid-like content of these lamellar bodies (Oren et al., 2003). hBD-2 is also up-regulated locally by infections (Radek & Gallo, 2007) or wounds (Butmarc et al., 2004). It has preferential bactericidal properties against Gram-negative bacteria (Harder et al., 1997) and like LL-37, its effect is sensitive to the concentration of NaCl. hBD-2 derived from neutrophils, promotes prostaglandins production and histamine release from mast cells, playing a role in allergic response (Bals et al., 1998). hBD-2 has also chemotactic properties for immature dendritic cells and memory T cells; it was described to bind to CCR-6, the receptor for macrophage inflammatory protein 3 alpha. In monocytes, hBD-2 expression is stimulated by several cytokines (Ganz, 2003; Kanda & Watanabe, 2008) and Il-1 seems to be the major inducer of hBD-2 production. Bacteria can also stimulate the expression of hBD-2 by epithelial cells, in a cytokine-independent pathway. *P. aeruginosa* is a powerful inducer of hBD-2 by primary keratinocytes (Schroeder & Harder, 2006).

hBD-1 was considered as a constitutively expressed antimicrobial peptide and in particularly not induced by proinflammatory cytokines. However, its production can be induced by peptidoglycan or LPS exposure (Sorensen et al., 2005). It is expressed in malpighian layer and in *stratum corneum* (Ali et al., 2001) and this expression is induced by increasing concentration of calcium (Harder et al., 2004), condition that provokes keratinocyte differentiation *in vitro* (Lichti et al., 2008).

hBD-3 has its expression induced by EGF that provokes keratinocytes proliferation in skin wounds (Sorensen et al., 2006).

It has chemotactic properties for monocytes (Garcia et al., 2001). While its expression is not induced by infection, hBD-3 displays a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria, as well as against fungi (Harder et al., 2001). Regarding the adaptive immune system, hBD-2, 3 and 4 stimulate expression of proinflammatory cytokines, IL-10 and MCP-1 (Niyonsaba et al., 2007). They also stimulate the phosphorylation of STAT-1 and STAT-3 that induce keratinocytes migration and proliferation.

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

Hanssen, 1975).

et al., 2007) (Figure 3).

Lugardon et al., 2000).

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 189

that pancreastatin, a chromogranin A (CGA)-derived peptide inhibits insulin secretion from pancreatic beta-cells, initiated the concept of prohormone (Eiden, 1987; tatemoto et al., 1986). The release of these CGs-derived peptides from chromaffin cells results from the nicotinic cholinergic stimulation and regulates several neuroendocrine functions (Helle & Serck-

Numerous cleavage products of the granins have been characterized, among which some display biological activities (Tatemoto et al., 1986; Aardal et al., 1993; Curry et al., 1992; Fasciotto et al., 1993; Lugardon et al., 2001; Mahata et al., 1997; Strub et al., 1996a,b). Neuroendocrine activities are reported from *in vivo* studies, with modulations of homeostatic processes, such as calcium regulation and glucose metabolism (Helle et al., 2007), cardiovascular functions ( Brekke et al., 2002; Corti et al., 2004), gastrointestinal motility (Amato et al., 2005; Ghia et al., 2004a), nociception ( Ghia et al., 2004b) tissue repair (Gasparri et al., 1997; Ratti et al., 2000), inflammatory responses (Ceconi et al., 2002; Corti et al., 2000) and as host defense agents during infections (Radek et al., 2008). During the past decade, our laboratory has characterized new antimicrobial CGs-derived peptides (Strub et al., 1996a,b; Metz-Boutigue et al., 1998; Lugardon et al., 2000, 2001; Briolat et al., 2005; Helle

Fig. 3. The antimicrobial bovine CGs-derived peptides according to the sequence of CGA (P05059), CGB (P23389) and CGC ((P20616) For each antimicrobial peptide the sequence, the location and net charge are indicated. \*, cysteine residues of the disulfide bridge; phosphorylated residue are underlined and the glycosylated residue is in bold.

They act at micromolar range against bacteria, fungi, yeasts and are non-toxic for mammalian cells. They are recovered in biological fluids involved in defense mechanisms (serum, saliva) and in secretions of stimulated human neutrophils (Briolat et al., 2005;
