The Human Cathelicidin LL-37: a Multifunctional Peptide Involved in Infection and Inﬂammation in the Lung

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

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. [51] which was accompanied by processing of hCAP-18 to the Various squamous Frohm Nilsson et al. [52] active peptide LL-37 [33]. No data are available on the epithelia Body fluids Bronchoalveolar Agerberth et al. [8] regulation of hCAP-18/LL-37 expression in the lung. lavage fluid However, increased LL-37 levels have been demonstrated Nasal lavage fluid Tjabringa et al., this in tracheal aspirates of newborns during infection [23] and manuscript LPS and LTA were demonstrated to increase hCAP-18/LL- Seminal plasma Malm et al. [51] 37 expression in sinus epithelial cells [34], suggesting that Saliva Murakami et al. [53] Plasma Sorensen et al. [54] bacterial products may also regulate hCAP-18/LL-37 expression in the lung. hCAP-18/LL-37 is increased during inflammation [22–24]. Furthermore, in vitro studies showed that expression of 4. Antimicrobial activity of LL-37 hCAP-18/LL-37 is regulated by various mediators, and may be associated with cellular differentiation. This is demon- Most antimicrobial peptides were discovered by bio- strated by studies in human colon epithelium in which chemical isolation procedures guided by bioactivity screen- hCAP-18/LL-37 expression was demonstrated at the surface ing. These peptides were shown to display marked in vitro and upper crypts of normal human colon, but not in the and in vivo antimicrobial activity, putting them center stage deeper crypts or in epithelial cells of the small intestine [25]. in host defence against infection. Also, LL-37 was first In line with this, the differentiation-inducing agent sodium identified as a broad-spectrum antimicrobial peptide with butyrate increased hCAP-18/LL-37 expression. However, LPS-neutralizing activity [35]. LL-37 kills both gram- Schauber et al. demonstrated that the increased hCAP-18/ positive and gram-negative bacteria and fungi, and studies LL-37 expression and cell differentiation by short chain using negatively charged model membranes (mimicking fatty acids such as butyrate in human colonocytes may be bacterial outer membranes) suggest disintegration of the mediated via different intracellular pathways. While bacterial membrane and subsequent death by LL-37 via increased hCAP-18/LL-37 expression was suggested to be formation of a carpet-like layer over the membrane [36]. mediated via the MEK (mitogen-activated protein kinase Various studies have suggested an important role for LL-37 [MAPK]/extracellular signal-regulated kinase [ERK] in host defence against infection in vivo. Bals et al. showed kinase)-pathway, differentiation was suggested to be that pulmonary LL-37 overexpression in mouse airways mediated via activation of p38 [26]. Since butyrate exerts resulted in decreased bacterial load while systemic over- many of its effects through reversible inhibition of histone expression resulted in decreased mortality following deacetylases (HDAC) and thus affects expression of a bacterial challenge [37]. In addition, gene transfer of variety of genes, the authors explored whether a synthetic hCAP-18/LL-37 in a cystic fibrosis xenograft model HDAC inhibitor had similar activity as butyrate. The results restored bacterial killing to normal levels [38]. These showed that the HDAC inhibitor trichostatin A indeed also studies suggest that hCAP-18/LL-37 may protect against markedly increased hCAP-18/LL-37 expression in these bacterial lung infections. These conclusions are further cells. Since trichostatin A is a fungal product [27], this raises supported by mouse studies. Nizet et al. showed that mice the intriguing possibility that selected micro-organisms deficient in the cathelicidin CRAMP have a decreased affect LL-37 expression through an effect on HDAC capacity to control skin infections [14]. Analysis of hCAP- activity. Furthermore, the observation that not only 18/LL-37 expression in patients also provides microbial products and inflammation, but also cigarette (circumstantial) evidence for its importance in vivo. Atopic smoke affects histone acetylation [28], suggests that dermatitis patients, who suffer from frequent skin infec- smoking may affect pulmonary expression of hCAP-18/ tions, express low levels of LL-37 as compared to psoriasis LL-37. patients, who do not suffer from frequent skin infections Other studies revealed that bacterial products and [24]. Finally, Putsep et al. have suggested involvement of inflammation regulate hCAP-18/LL-37 expression. LL-37 in Kostmann disease, a severe congenital neutropenia 324 G. Sandra Tjabringa et al. / Pulmonary Pharmacology & Therapeutics 18 (2005) 321–327 characterized by recurrent infections and periodontal of cells at inflammatory sites [40]. Furthermore, defensins disease [39]. Neutrophil levels in these patients could be and LL-37 were demonstrated to chemoattract cells via restored by treatment with G-CSF. However, while G-CSF chemokine and formyl-peptide receptors, suggesting invol- restored neutrophil levels, patients still suffered from vement of antimicrobial peptides in cellular accumulation infections. Since patients neutrophils were shown to be [41,42]. In contrast to chemotactic activity, formyl-peptide deficient in LL-37, and no LL-37 could be detected in receptors were not involved in LL-37-induced activation of plasma and saliva despite G-CSF treatment, LL-37 was human monocytes and airway epithelial cells [16,18], and suggested to play a role in this disease. LL-37 levels were LL-37-induced maturation and release of IL-1b in LPS- restored in a patient who received a bone marrow transplant, primed monocytes was mediated via the P2X7 receptor [43]. and this patient recovered from infections and periodontal Interestingly, a very recent report showed that neutrophil disease. Collectively, these studies show that in addition to defensins may activate epithelial cells via similar P2 displaying antibacterial activity in vitro, LL-37 may play an receptor-nucleotide signalling pathways [44]. Finally, LL- important role in innate immunity in vivo. Whether such 37 was demonstrated to modulate dendritic cell differen- antimicrobial activity in vivo is exclusively the result of tiation and dendritic cell-induced T-cell polarisation [19]. direct killing of micro-organisms by LL-37, or is mediated Chemotactic activity towards a variety of cell types, via immunomodulatory effects of LL-37, remains to be including neutrophils, monocytes, T-cells [42] and mast determined. cells [45], and modulation of dendritic cell activity suggests that in addition to its role in innate immunity LL-37 may also be involved in adaptive immune responses. 5. LL-37 and inflammation In addition to acting as a chemoattractant, LL-37 may regulate the infiltration of inflammatory cells by activation Inflammation, including that in the lung, is characterized of airway epithelial cells. LL-37 was shown to activate the by an influx of inflammatory cells into the inflamed area. mitogen-activated protein kinases (MAPKs) extracellular Recruitment and activation of these cells is known to be signal-regulated kinases (ERK)1/2, JNK and p38, and to regulated in large part by inflammatory cytokines such as enhance the release of IL-8 [16,17]. MAPKs are involved in chemokines. Interestingly, it now appears that antimicrobial various cellular activities including proliferation, differen- peptides also act as inflammatory mediators by displaying tiation, cell survival, apoptosis and gene expression, cytotoxic, chemotactic and other cell-stimulating activities. suggesting that LL-37 may regulate these cellular events. Indeed, both defensins and LL-37 have recently been LL-37-induced release of the neutrophil chemoattractant demonstrated to display a variety of functions that are IL-8 by airway epithelial cells, which is ERK1/2-dependent, relevant to our understanding of the complex networks that may result in increased neutrophilic infiltration and an mediate inflammation. In addition to killing prokaryotic amplification loop of inflammation. LL-37-induced acti- cells, neutrophil defensins and LL-37 were demonstrated to vation of airway epithelial cells was suggested to be display cytotoxic activity towards eukaryotic cells at high mediated via transactivation of the EGFR, which concentrations, which may be reached upon degranulation involves activation of metalloproteinases, cleavage of

Fig. 2. Proposed mechanism for cellular activation by LL-37. Exposure of airway epithelial cells to LL-37 results in activation of a metalloproteinase (MP), possibly a member of the ‘a disintegrin and metalloprotease’ (ADAM) family. The identity of the structures involved in mediating this metalloproteinase activation remains to be established. The metalloproteinase/ADAM subsequently cleaves membrane-anchored EGFR-ligands which activate the EGFR resulting in MEK activation and gene transcription. Partly based on Ref. [16]. G. Sandra Tjabringa et al. / Pulmonary Pharmacology & Therapeutics 18 (2005) 321–327 325 membrane-anchored EGFR-ligands and activation of the Furthermore, LL-37 expression has been demonstrated to EGFR by these ligands [16] (Fig. 2). Interestingly, LL-37 be regulated in inflammatory disease, suggesting a role for displayed chemotactic activity at approximately 50 mg/ml, LL-37 in inflammatory process. Frohm et al. have shown whereas induction of epithelial IL-8 production was increased expression of the hCAP-18 gene (cathelicidin observed at approximately 10 mg/ml. These results indicate antimicrobial peptide; CAMP) in keratinocytes in inflamed that LL-37 displays direct and indirect effects on cell skin lesions of patients with psoriasis [22]. As outlined migration at concentrations that are in the same order of above, Ong et al. compared the expression of antimicrobial magnitude. peptides in patients with psoriasis, a Th1-dominated skin Recently LL-37 was suggested to be involved in wound disease, and atopic dermatitis, a Th2-dominated skin disease repair. Growth factors involved in the process of wound which is characterized by a susceptibility to skin infections. healing were shown to induce LL-37 expression [32], and Patients with atopic dermatitis showed lower skin hCAP-18/ hCAP-18/LL-37 is strongly expresed in healing skin LL-37 expression as compared to patients with psoriasis, epithelium [33,46]. A functional role for LL-37 in repair indicating that the type of inflammation, and inflammatory was suggested by the observation that anti-LL-37 antibodies cells that are involved, play an important role in the inhibit re-epithelialization of skin wounds [46]. Further- pathogenesis of disease [24]. more, Koczulla et al. demonstrated involvement of LL-37 in angiogenesis, which is mediated via the formyl-peptide receptor FPRL1 [15]. In vitro, LL-37 was shown to activate 6. Conclusion endothelial cells resulting in increased proliferation and formation of vessel-like structures, while in vivo cathe- In conclusion, LL-37 is a multifunctional peptide licidin (CRAMP)-deficient mice showed decreased vascu- displaying both antimicrobial activity and a variety of larisation during wound repair. These results suggest a functions related to inflammation, suggesting that LL-37 regulatory function for LL-37 in inflammation by mediating may regulate both infection and inflammation (Fig. 3). neo-vascularisation after tissue injury, and may also Therefore, LL-37 may form a template for the development implicate a role for LL-37 in the development of tumors of new anti-infectious agents. In addition, modulation of by facilitating blood supply to the tumor. proteinase activity with the aim to control processing of A variety of studies have demonstrated that the hCAP-18 to the active peptides, may be a novel approach in expression of antimicrobial peptides in patients with the treatment of infectious and inflammatory diseases. inflammatory disorders differs from that in control subjects. Synthetic and recombinant peptides based on the structure Agerberth et al. have shown that broncheoalveolar lavage of endogenous antimicrobial peptides are currently eval- fluid (BALF) from sarcoidosis patients contains enhanced uated for treatment of infectious and inflammatory diseases antibacterial activity as compared to healthy individuals. [47]. These include studies on MBI 226 and MBI 594AN, Sarcoidosis is a pulmonary disease characterized by a which are evaluated for treatment of catheter infections and interstitial pulmonary inflammation and a T-helper type 1 acne, respectively (Migenix, Vancouver, Canada). Devel- (Th1) cell response. In BALF from both healthy individuals opment of LL-37-based therapies should however, consider and sarcoidosis patients, various antimicrobial peptides the balance between antimicrobial and inflammatory were identified, including LL-37, and expression patterns activities. Therefore, studies such as those in human sweat differed between patients and healthy individuals [8]. showing that selected peptides derived from human LL-37

hCAP-18

Proteinase 3

Antimicrobial activity LL-37 LPS neutralisation

Angiogenesis Chemotaxis Cell death

Epithelial activation Epithelial • EGFR activation wound repair • MAPK activation • IL-8 release

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