Pulmonary Pharmacology & Therapeutics 18 (2005) 321–327 www.elsevier.com/locate/ypupt The human cathelicidin LL-37: a multifunctional peptide involved in infection and inflammation in the lung G. Sandra Tjabringa, Klaus F. Rabe, Pieter S. Hiemstra* Department of Pulmonology, Leiden University Medical Center, Building 1, C3-P, P.O. Box 9600, NL-2300 RC Leiden, The Netherlands Received 8 June 2004; revised 16 December 2004; accepted 6 January 2005 Abstract Antimicrobial peptides play an important role in innate immunity of the lung by acting as effector molecules in host defence against inhaled pathogens. Various families of antimicrobial peptides have been identified, including the cathelicidins. Cathelicidins are characterized by a conserved N-terminal cathelin domain and a variable C-terminal antimicrobial domain that can be released from the precursor protein after cleavage by proteinases. LL-37 is the C-terminal part of the only human cathelicidin identified to date called human cationic antimicrobial protein (hCAP-18), which is mainly expressed by neutrophils and epithelial cells. In addition to killing a broad spectrum of microorganisms, LL-37 was demonstrated to display various cellular activities related to inflammation including cytotoxicity to host cells, chemotaxis, epithelial cell activation, angiogenesis and epithelial wound repair. Focussing on this recent information, this review discusses the role of LL-37 in infection and inflammation in the lung. In addition, the importance of the fact that antimicrobial peptides such as LL-37 display a range of activities for the design and development of antimicrobial peptides for therapeutic use is discussed. q 2005 Elsevier Ltd. All rights reserved. Keywords: Infection; Inflammation; Antimicrobial peptides; Innate immunity 1. Introduction human cationic antimicrobial protein (hCAP-18) is the only member of the cathelicidin family that is expressed in The innate immune system forms an essential defence humans. General information on cathelicidin peptides can system that helps to protect the lung against infection caused be found in recent reviews [1–4]. This review is focussed on by inhaled pathogens. Recent studies have highlighted the the role of the human cathelicidin hCAP-18 and its major importance of small cationic peptides that serve as cleavage product LL-37 in infectious and inflammatory endogenous antibiotics. These so-called antimicrobial processes in the lung. peptides act as effector molecules of innate immunity, and are mainly produced by phagocytes and epithelial cells. Whereas most antimicrobial peptides were identified based 2. Structure, processing and expression on their antimicrobial activity, they have recently been shown to display a variety of additional and partly The 18-kDa hCAP-18 is the only human member of the unexpected activities. These findings have suggested a cathelicidin family of antimicrobial peptides known to date. role for antimicrobial peptides in infection, immunity and Like most antimicrobial peptides, cathelicidins are also wound repair. The two main families of small, cationic produced as inactive preproproteins (Fig. 1). The signal antimicrobial peptides that have been identified in peptide is removed and the resulting precursor is character- humans are the defensins and the cathelicidins. Various ized by a conserved N-terminal domain and a variable human defensins have been identified, but it appears that cationic C-terminal domain. This C-terminal domain contains the active peptide that can be released from the * Corresponding author. Tel.: C31 71 526 2950; fax: C31 71 526 6927. precursor protein by the action of serine proteinases. E-mail address: [email protected] (P.S. Hiemstra). Neutrophil-derived proteinase 3 was identified as the main 1094-5539/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2005.01.001 322 G. Sandra Tjabringa et al. / Pulmonary Pharmacology & Therapeutics 18 (2005) 321–327 Fig. 1. Structure and cleavage sites of hCAP-18, and hCAP-18 and LL-37 levels in nasal lavage fluid derived from healthy individuals by washing the nasal cavity with PBS, as determined by Western blot analysis. Each lane represents nasal lavage fluid derived from an individual donor (labeled 1–3). serine proteinase responsible for extracellular cleavage of The structure and activities of cathelicidins vary between hCAP-18 released by neutrophils, resulting in the release of species. In cattle and pigs, cleavage of cathelicidins has the antimicrobial peptide LL-37 [5]. LL-37 derives its name been demonstrated to be mediated by elastase. The from its primary structure: a 37-amino acid peptide with two variability in the C-terminal domain between members of leucine residues at its N-terminus. A recent study on hCAP- the cathelicidin family results in an heterogeneous group of 18 present as a precursor protein in high concentrations in peptides with a variety of functions. The pig cathelicidin seminal plasma revealed that LL-37 may not be the only PR-39 is a proline- and arginine-rich antimicrobial peptide, cathelicidin peptide that is derived from processing of the that has been demonstrated to be involved in a variety of precursor. It was found that this hCAP-18, largely derived activities, including wound repair, metastasis of tumours, from the epididymis, is cleaved by a serine proteinase that is inflammation, chemotaxis and angiogenesis (reviewed in produced by the prostate (gastricsin) in the slightly acidic [3]). Like LL-37, the mouse cathelicidin-related antimicro- environment of the vagina [6]. Cleavage by gastricsin was bial peptide (CRAMP) forms an amphipatic a-helical found to result in the release of ALL-38, an alternative peptide. Therefore, studies using CRAMP-deficient mice cleavage product of hCAP-18 which differs from LL-37 by that show the importance of cathelicidins in host defence an additional N-terminal alanine residue. LL-37 and ALL- against skin infection [14] and angiogenesis [15] are 38 have similar antimicrobial activities. Another very relevant to our understanding of the role of LL-37. LL-37 recent study showed that human sweat contains proteinases has antimicrobial, LPS-neutralizing, chemotactic, and that process LL-37 into several smaller antimicrobial angiogenic activities, has been shown to activate airway peptides [7]. Interestingly, the pro-inflammatory activity epithelial cells and mononuclear phagocytes [3,16–19] and of these LL-37-derived peptides, as assessed by their ability was suggested to be involved in wound repair. In addition, to induce IL-8 release from keratinocytes, was lower Zaiou et al. recently showed that the N-terminal domain of compared to that of LL-37. Whereas LL-37 has been hCAP-18, which shares homology with the cystatin family isolated from human bronchoalveolar lavage fluid [8] and is of cysteine proteinase inhibitors, displays antimicrobial readily detected in nasal lavage fluid (Fig. 1), it is unknown activity and inhibits proteinase activity, suggesting that also whether airway secretions also contain other hCAP-18- the N-terminal domain is involved in host defence [20]. derived peptides such as ALL-38 or the smaller hCAP-18/LL-37 was first identified in neutrophils, and peptides detected in human sweat. It needs to be noted subsequently demonstrated to be also expressed in mono- that ALL-38 can not be distinguished from LL-37 by the cytes, T-cells, mast cells, keratinocytes in inflamed skin and Western blot technique used in the analysis of e.g. nasal various squamous epithelia (Table 1). Various studies have lavage fluid (Fig. 1). demonstrated hCAP-18/LL-37 in the human airways. In situ Airway secretions contain various serine proteinase hybridization studies of the conducting airways showed LL- 37/hCAP-18 expression in the surface epithelia and in the inhibitors such as a1-proteinase inhibitor, but also relatively submucosal glands [21], and hCAP-18/LL-37 was detected high concentrations of locally produced secretory leukocyte in bronchial alveolar lavage fluid (BALF) [8] and nasal proteinase inhibitor (SLPI) and elafin [9–11].These lavages (Fig. 1). proteinase inhibitors are inactivated by oxidants present in cigarette smoke and produced by inflammatory cells, suggesting that certain conditions may restrict the ability of these inhibitors to control hCAP-18 processing. How- 3. Regulation of hCAP-18/LL-37 expression ever, if and how these inhibitors contribute to the regulation of hCAP-18 processing in airway secretions is unknown. Whereas neutrophils produce the majority of their Interestingly, in addition to their potential contribution to granule content during their development in the bone the control of the generation of hCAP-18 derived anti- marrow, the epithelial expression of antimicrobial peptides microbial peptides, both SLPI and elafin also display at the site of infection and inflammation is either antimicrobial activity themselves [12,13]. constitutive or inducible. Several studies have shown that G. Sandra Tjabringa et al. / Pulmonary Pharmacology & Therapeutics 18 (2005) 321–327 323 Table 1 In pharyngeal cells, hCAP-18/LL-37 expression was Presence of hCAP-18/LL-37 in various cell types and body fluids induced after H. influenzae infection [29], and hCAP-18/ Cell type/tissue/secretion Reference LL-37 expression in nasal mucosa was found to be higher in Leukocytes Neutrophils Cowland et al. [48] rhinitis [30]. In contrast, Islam et al. showed that hCAP-18/ Monocytes Agerberth et al. [49] (mRNA) LL-37 expression in Shigella spp infected individuals was B-cells Agerberth et al. [49] decreased, and Shigella infected epithelial cell cultures were gd T-cells Agerberth et al. [49] shown to express decreased hCAP-18/LL-37 levels [31]. NK cells Agerberth et al. [49] Finally, growth factors that are involved in the process of Mast cells Di Nardo et al. [50] Epithelial Lung Bals et al. [21] (mRNA) wound healing were shown to induce hCAP-18/LL-37 cells Inflamed skin Frohm et al. [22] expression in keratinocytes [32], and increased expression Colon Islam et al. [31] of cathelicidins was demonstrated in cutaneous wounds, Epididymis Malm et al.