Nomenclature and tissue distribution of AMPs
Antimicrobial peptides (AMPs) are an extremely diverse group of more than 700 small proteins and can be divided into several categories on the basis of their structures. Most AMPs are cationic (polar) molecules. There are two classes of AMPs based on the mechanism of their cellular synthesis: (1) non-ribosomally synthesised (largely produced by bacteria) and (2) ribosomally synthesised, gene- encoded peptides [1]. Non-ribosomally synthesised AMPs are produced by multifunctional peptide synthetases. These substances are already used in clinical applications: polymyxin B, polymyxin E, colimycin, cyclosporin, tripeptide ACV (precursor of penicillin and cephalosporins) [2, 3]. AMPs, in a more narrow sense, refer to gene-encoded peptides. These AMPs can be grouped according to their size, conformational structure or predominant amino acid structure. On the basis of their gross composition and 3D structure, AMPs can be divided in four main groups: I: linear peptides with an α-helical structure; II: β-sheet structures stabilised by disulphide bridges; III: peptides with predominance of one or more amino acids; IV: peptides with loop structures [4]. This review will concentrate on the biological relevance of two mammalian AMPs- families: defensins (from group II) and cathelicidins (from group I).
The defensins families of AMPs
Defensins are typically 28 to 44 amino acids long and contain 6 to 8 cysteine residues that form characteristic intramolecular disulfide bridges. Defensins are found in mammals, and distantly related peptides appear in insects and plants [5]. They are classified into 3 families- the a-defensins, the b-defensins and the q-defensins.
• α-defensins
These defensins are 29 to 35 residues in lenght and contain 3 disulfide bridges. The first human a-defensin was isolated from neutrophils in 1985 [6]. At the present time, six a-defensins have been identified from humans. Human neutrophil peptides 1-4 (HNP1-4) are localised in azurophilic granules [7, 8]. The two other a-defensins, human defensins 5 and 6 (HD5-6) are located in Paneth’s cells of the small intestine [8] and in epithelial cells of the female urogenital tract [9]. Unlike neutrophils, Paneth’s cells do not store defensins as matured peptides; instead, they store them as propeptides [10]. Elevated plasma levels of a-defensins were observed in patients with active pulmonary tuberculosis [11] and children with severe sepsis [12].
• β-defensins
These peptides are 36-42 amino acids in lenght and contain 6 cysteine motifs connected by 3 disulfide bridges. The first β-defensin was isolated from cow airway epithelial cells [13]. In human beings, 4 types of β-defensins have been identified so far. The first human b-defensin, called hBD1 was originally isolated from large volumes of hemofiltrate [14] and is expressed constitutively in epithelial cells of the urinary and respiratory tracts [15, 16] and in human skin epidermis [17, 18]. Human β defensin-2 (hBD2) was isolated from psoriatic skin [19] and in contrast to hBD1 is upregulated in cultured keratinocytes by inflammatory stimuli like IL1 and live Pseudomonas aeruginosa [20] or Staphylococcus aureus, S. epidermidis, E. coli [21]. E. coli LPS is a very weak direct inducer of hBD2 mRNA and peptide, but the induction is amplified by IL1-mediated signaling [22]. Inducible human β defensin-3 was isolated from lesional psoriatic scales and investigations of different tissues revealed skin and tonsil to be major hBD3 mRNA- expressing tissues [23]. Human b defensin-4 was identified solely by searches of genomic database [24]. Recently, 28 new genes for human β defensins were identified by screening of human genome database [25]. Peripheral blood monocytes, monocyte-derived macrophages and monocyte-derived dendritic cells all express hBD1mRNA and hBD2mRNA. HBD2 peptide was demonstrated in blood monocytes and alveolar macrophages [26].
• θ-defensins
This family of defensins has been isolated from rhesus monkey neutrophils [27] and no data about the presence of these molecules in tissues are aviable at this time.
Cathelicidins
The term „cathelicidins” was coined as an attempt to unify a variety of peptides 12 to 80 or more amino acids in length, related by similar „cathelin” precursor domain (cathepsin L inhibitor) [28]. To date, cathelicidins have been found only in mammals [29]: mouse (CRAMP), rat (rCRAMP), pig (protegrin, PMAP-23, PR-39), monkey (rhLL37, RL37), rabbit (CAP-18), sheep (SMAP-29, SMAP-34) and human (hCAP-18/ LL37). Human LL37 was isolated from human bone marrow and is present in neutrophils [30], γδT cells, NK cells, B cells, monocytes/ macrophages from peripheral blood [31]. LL37 is also present in epithelial cells in the respiratory tract, urogenital tract, gastrointestinal tract [32, 33] and skin epidermis, mainly in response to infammatory stimuli [34-36] and cutaneous injury [37]. Human CAP-18/LL37 is also induced in keratinocytes by growth factors like IGF1 and TNFα, both important in wound healing process [38]. In addition, plasma has been reported to contain LL37 [39]. The mouse cathelin related antibicrobial peptide (CRAMP) expression resembles that of LL37 in human [40].
The role of AMPs in immune response
In higher vertebrates such as mammals, there are two types of immunity: innate (or natural) and adaptive (or acquired). The effector branch of innate immunity consists of two major aspects. One is the generation of humoral antibacterial mediators such as complement and AMPs (including defensins and cathelicidins), as well as large antimicrobial substances (lysozyme, cathepsin G, lactoferrin). The other is the recruitment and activation of phagocytic granulocytes, monocytes/ macrophages, and NK cells to sites of microbial (bacteria, fungi, enveloped viruses) invasion. HBD1 and LL37 are produced constitutively by keratinocytes and other epithelial cells and contribute to the barrier functions in the first line of antimicrobial defence. In mice, a deficiency in CRAMP production leads to the increased susceptibility to necrotic skin infections caused by Group A Streptococcus [41]. Neonatal skin in mice and humans expresses increased levels of AMPs compared to adult [42]. Furthermore, hBD1 expression was described in milk and breast tissue during lactation [43]. Recombinant hBD2 produced by baculovirus had a direct antimicrobial activity in vitro [20]. Human defensins, hBD2 and LL37 induce the activation and degranulation of mast cells resulting in the release of histamine and PDG2 [44, 45]. Because mast cell granule products increase neutrophil influx, AMPs can indirectly promote accumulation of these cells at inflammatory sites. Degranulation of neutrophils releases more defensins and upregulates innate host innflammatory defenses against microbial invasion. HNP1 and HNP2 are chemotactic for human monocytes [46] dendritic cells and T cells [47, 48]. Murine β defensin 2 acts directly on immature dendritic cells as an endogenous ligand for Toll-like receptor 4 (TLR4), inducing up-regulation of costimulatory molecules and, subsequently, dendritic cells maturation, acting as the natural adjuvant [49].
In addition to their role in host defense, defensins may also contribute to some pathophysiologic mechanisms. Neutrophil defensins can inhibit fibrinolysis and modulate tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells [50]. Cathelicidin LL37 can also play a part in wound closure and its reduction in chronic wounds can impair re-epithelialization [51].
AMPs as therapeutic agents
The broad spectrum of activity and the low incidence of bacterial resistance are attractive features of AMPs. Animal studies provided first proof that AMPs can be used to modify the course of infection and inflammatory diseases. Small biotech companies in association with larger pharmaceutical companies carried out human studies using AMPs of animal origin. AMPs from the skin of frogs, called magainins, have been one of the first substances to go through a drug development process. Its derivative, pexiganan was investigated in phase III trials of 926 patients and topical use has been found to show equivalence to oral ofloxacin against polymicrobic diabetic foot ulcer [52]. The porcine neutrophil-derived protegrins are the first of the mammalian cathelicidin peptides used in clinical trials [53]. A phase II study showed that this peptide used orally by patients undergoing bone marrow transplantation significantly reduced mucositis and number of febrile days. A phase III study of protegrin is now underway [54]. In future, animal studies may resolve the role of AMPs in tissue protection, wound healing and interaction with the aquired immune system. Better understanding of AMPs relationship between the innate and the aquired immune systems is the critical step to give us insite into how we naturally prevent infections and how to design new drugs to fight diseases.
Concluding remarks
AMPs have emerged as effector substances of the innate immune system involving not only activities as endogenous antibiotics but also as mediators of inflammation. Several important topics will have to be addressed in the future: (I) AMPs might contribute to the development of diseases as pro- or anti- inflammatory substances; (II) clinical useful of AMPs as novel therapeutic agents promises to revolutionize treatment of many inflammatory and infectious diseases.
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Correspondence: Hanna Gałkowska, PhD, Department of Surgery and Transplantation, Medical Research Center, Polish Academy of Sciences, 5 Pawinskiego, 02-106 Warsaw, Poland. Phone: +48 22 608 64 05, fax: +48 22 668 53 34, e-mail: hgalk@cmdik.pan.pl
