Human Cathelicidin Antimicrobial Peptide LL-37

Human Cathelicidin Antimicrobial Peptide LL-37 Birgitta Agerberth1 and Gudmundur H. Gudmundsson2,3,* 1

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

2

Microbiology and Tumorbiology Center, Karolinska Institutet, S-171 77 Stockholm, Sweden

3

Biology Institute, University of Iceland, 101 ReykjavõÂ k, Iceland

* corresponding author tel: ‡354 525 5276, fax: ‡354 525 4886, e-mail: [email protected] DOI: 10.1006/rwcy.2002.1209.

SUMMARY

BACKGROUND

The antimicrobial peptide LL-37 is a single cathelicidin defense peptide of human origin. Cathelicidins are so far only found in mammals and contain a conserved pro region of the cathelin type and a C-terminal variant domain, which is liberated by processing enzyme(s) as a mature antimicrobial peptide. In humans the gene encoding LL-37 is expressed in various tissues and cells but most pronounced in polymorphonuclear granulocytes and epithelial cells. Epithelial cells are constantly exposed to bacteria and phagocytes are recruited to sites of bacterial infections to terminate the bacterial spread and eliminate the invader. Thus, LL-37 is an effector in the armament of immediate and early host defenses. In addition to bactericidal activity, LL-37 exhibits chemotactic activity for several types of inflammatory and immune cells. Thus LL-37 exhibits a dual adaptive roles, being involved in killing bacteria and additionally in recruiting host effector cells for enhancing early innate defenses and for initiating the adaptive axis of immunity.

Discovery

Cytokine Reference

The discovery of human cathelicidin was reported independently by three different research groups. The first report was based on a PCR strategy with primers directed towards the conserved cathelin pro region (Agerberth et al., 1995). By comparison of cDNA sequences for antimicrobial peptides of bovine origin it became clear that cathelin was a conserved pro region, functioning as a carrier of antimicrobial peptides. Based on this information and our characterization of the gene for the porcine antimicrobial peptide PR-39 (Gudmundsson et al., 1995) primers were directed to conserved regions between bovine and porcine sequences and used in PCR amplification (Agerberth et al., 1995). The amplified cDNA sequence was confirmed to encode cathelin and then used as a probe to screen a human bone marrow cDNA library. Several clones were isolated and characterized, containing the complete coding

Copyright # 2002 Published by Elsevier Science Ltd

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Birgitta Agerberth and Gudmundur H. Gudmundsson

sequence. By synthesizing the putative C-terminal peptide based on a predicted processing site, the antibacterial domain was recovered. Antibodies were raised against the peptide and later used as an important tool for isolation of the mature active peptide, LL-37. The second report on the identification of a cDNA encoding the precursor for LL-37 (human CAP18 or hCAP18) was based on a low stringency screen of a cDNA library from human bone marrow (Larrick et al., 1995). The cDNA probe used in the screen encoded rabbit CAP18 (18 kDa cationic antimicrobial protein). Earlier, the same research group had found that the CAP18 protein from rabbit granulocytes exhibited potent LPS-binding activity (Larrick et al., 1991). For the human counterpart, the C-terminal domain of hCAP18 was shown to be responsible for the LPS-binding activity (Larrick et al., 1995), i.e. the part that corresponds to the peptide LL-37. The third approach was based on the characterization of a unique protein derived from specific granules of human neutrophils. Information from partial amino acid sequence analysis of this protein made it possible to design primers, which were used to clone the corresponding cDNA from a library of chronic myeloid leukemia cells (Cowland et al., 1995).

Alternative names Concerning the discovery of LL-37 and its terminology it is important to emphasize the presence of the pro region, cathelin. The name cathelicidin was initially suggested by Zanetti's group (Zanetti et al., 1995) based on this conserved pro region. Cathelin was originally isolated and characterized as a cathepsin L inhibitor, hence the name (Ritonja et al., 1989). Later this activity was retracted (Lenarcic et al., 1993). However, cathelicidin was then already established as a name for this major family of mammalian antimicrobial peptides. LL-37 refers to the length of the mature peptide with 37 amino acid residues, starting with two Nterminal leucines (Gudmundsson et al., 1996). Initially, the putative active peptide was thought to be a 39 residue peptide and subsequently called FALL-39 after the first four N-terminal residues and the length. The rationale for selecting this peptide was a potential dibasic processing site, and a synthetic replica of this peptide exhibited antibacterial activity and was dependent on salt for potent activity (Agerberth et al., 1995). Antisera was raised against this putative peptide and used for isolation of the active mature peptide from degranulated

granulocytes (Gudmundsson et al., 1996). The characterized peptide was two amino acid residues shorter than the putative peptide or 37 residues and was renamed LL-37. The name hCAP18 (human cationic antimicrobial protein) refers to the relation to the rabbit homolog CAP18 (Larrick et al., 1995) that was originally characterized as a LPS-binding protein of 18 kDa. This name is now used for the complete precursor protein. Since the peptide and the pro form go hand in hand, some researchers have preferred to use the mixed term hCAP18/LL-37, when the processing stage is not known, as in immunohistochemistry and/ or by expression on the mRNA level. Today with the emerging picture of variant peptides connected to the cathelin carrier it seems logical to refer to the family as cathelicidins, and to specify the product in question based on the active peptide.

Structure Initial studies using an Edmundsson wheel plot revealed that the peptide could form an amphipathic  helix (Agerberth et al., 1995). Amino acid residues from position 11 (E) to 32 (V) were recognized to form a perfect amphipathic helix. The -helical conformation was later found to be critical for the antibacterial activity, hence the helical fold is connected to the activity (Johansson et al., 1998). We demonstrated that pH, anion, and peptide concentration are the main determinants for the conformation and the activity of LL-37 (Johansson et al., 1998). Thus, the conformation was found to depend on the microenvironment and was not induced upon contact with bacteria. Effects of the microenvironment are of interest concerning the activity of antimicrobial peptides. In the mucus of cystic fibrosis patients it was found that antibacterial activity was diminished and the decreased activity was correlated to increased salt concentrations (Smith et al., 1996). It is known that the activity of defensins is decreased at high salt concentrations, but it has not been established if improper folding is the reason for their decreased activity. However, it must be emphasized that intact epithelial cell surface defenses are based on a complicated network with many active components, which can also work in synergy or in an additive manner (Agerberth et al., 1999; Travis et al., 1999). The observation that antimicrobial peptides are clinically relevant has turned out to be of great importance. It has opened up the field and focused much deserved attention on both the peptides and immediate host defenses.

Human Cathelicidin Antimicrobial Peptide LL-37 3

Main activities and pathophysiological roles Most likely the peptides shield the epithelia from invading microbes. It was suggested previously that one function of peptide antibiotics could be to counterbalance the commensal microflora (Boman, 1991) and several recent lines of evidence confirm this view (Porter et al., 2002). In addition to cell surface protection, LL-37 is involved in the destruction of bacterial invaders since it is included as an active part of the neutrophil armament (Cowland et al., 1995; Gudmundsson et al., 1996). LL-37 might be an effector involved both in intracellular phagocytic killing as well as extracellularly, when phagocytes are recruited and degranulated at sites of infection or inflammation (Frohm et al., 1996). However, it has to be emphasized that the peptides most likely act in synergy with other components in vivo. During our studies on the expression of LL-37 in specific lymphocyte and monocyte populations we observed chemotactic activity for polymorphonuclear and CD4 T cells. This chemotaxis was also reported by J. J. Oppenheim's group, who showed that LL-37 was able to induce Ca2+ mobilization in monocytes (Yang et al., 2000a). The receptor mediating the chemotactic response to LL-37 was characterized as the formyl peptide receptor-like 1 (FPRL1) which is present on neutrophils, monocytes, and T cells (Yang et al., 2000b). The function of cathelin is still not clear although the protein is related to a family of thiol protease inhibitors such as kininogenin, cystatin, and spp24. Interestingly, the kininogenin precursor most similar to cathelin is a carrier of the potent vasodilator bradykinin, which is released when the precursor protein is cleaved by kallikrein (Hu et al., 1995). The only study on cathelin function indicates protease inhibition and chemotactic activity (Verbanac et al., 1993). However, detailed characterization has not yet been carried out and in the mean time its function remains uncertain. LL-37 has also been found to induce histamine release and affect intracellular Ca2+ mobilization in rat mast cells (Niyonsaba et al., 2001), indicating that mast cells are a target for LL-37. Furthermore, the same research group has shown rat mast cells to be chemotactic for LL-37, an effect mediated independently of the FPRL1 receptor. Instead, the protein phospholipase C signaling pathway is involved (Niyonsaba et al., 2002). Due to sequence variation between related peptides of different species it is of importance to confirm these results in a homologous

system and confirm that LL-37 has the same effect on human mast cells. Together, these observations demonstrate a dual function of LL-37 in host defense by killing bacteria and by recruiting inflammatory and immune effector cells.

GENE AND GENE REGULATION

Accession numbers For retrieving the gene sequences or cDNA use LL-37 as a search word in the Entrez (NCBI) database. cDNA: NM_004345 Gene: X96735

Chromosome location 3p21.3 is the chromosomal location for the CAMP gene encoding LL-37. To our knowledge this chromosomal region is not associated with sensitivity to infections. However, it is likely that such a situation will be demonstrable with reference to the mouse model.

Relevant linkages There are no available data connecting human disease and the location of the gene for LL-37.

Gene structure and evolution The structure of the gene encoding LL-37 (named CAMP for cathelicidin antimicrobial peptide) has been determined and the gene mapped to chromosome 3 (3p21.3) (Gudmundsson et al., 1995, 1996; Frohm et al., 1997). To our knowledge this region has not been mapped in relation to sensitivity for infections in humans. The gene consists of four exons, where exon 1±3 encode the signal peptide for export and the cathelin domain, and exon 4 encodes the processing site and the mature peptide LL-37. This organization is consistent with the general gene structure of all characterized cathelicidin genes (Gudmundsson et al., 1995; Zhao et al., 1995a,b; Pestonjamasp et al., 2001). Not only are the coding regions conserved between the cathelicidin genes; identities in introns and promoter regions are also pronounced, as in the genes encoding PR-39 and LL-37 (Gudmundsson et al., 1996). However, when comparing the noncoding sequences between the mouse gene CRAMP and

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human CAMP, only promoter sequences are conserved, indicating similar regulation (Gudmundsson et al., 1996; Pestonjamasp et al., 2001).

Regulatory sites and corresponding transcription factors Expression of antimicrobial peptides is sometimes regulated through the presence of microbial products, as has been shown for -defensin 2 (O'Neil et al., 1999). Toll-like receptor 2 has been found as a mediator in this response to bacterial lipoprotein, through a signal pathway involving NFB, thereby enhancing the potency of innate defenses (Birchler et al., 2001). Microbial activation has not been shown for LL-37. Since the expression of the gene encoding LL-37 is increased in inflammation, several groups have screened for direct cytokine induction. In skin, IL-1 has been found to upregulate the transcription of LL-37 in composite keratinocyte grafts (Erdag and Morgan, 2002). Currently it is not known if this induction reflects direct activation through the IL-1 receptor or a secondary effect. In addition, colocalization of IL-6 and LL-37 has also been reported (Frohm Nilsson et al., 1999). Due to regulatory elements in the promoter of the gene for LL-37, IL-6 has been considered a regulator of LL-37 (Gudmundsson et al., 1996). However, it is unclear if IL-6 is induced parallel to LL-37 or involved in the regulation of the LL-37-encoding gene. During our characterization of the gene encoding LL-37 (the CAMP gene) we identified several promoter elements that could be of interest for the regulation of the gene. The binding sites of potential interest were for the transcriptional factors NF-IL6 (nuclear factor for IL-6 expression) and APRF (acute phase response factor), now known as STAT3. NF-IL6 is a member of the C/EBP family of transcription factors with a basic-leucine zipper domain involved in DNA binding and dimerization. The C/EBP transcription factors are engaged in several cellular responses including growth, differentiation, immune, and inflammatory processes with relation to various diseases (Ramji and Foka, 2002). According to current terminology NF-IL6 is equivalent to C/EBP . Interestingly, in a mouse deficient in the related C/EBP", expression of the mouse cathelicidin was virtually absent in bone marrow and a binding site for C/EBP" was identified in both the human and mouse promoters (Verbeek et al., 1999). Other potential binding sites for regulatory factors were found in the promoter of the mouse cathelicidin gene related to GM-CSF (granulocyte-monocyte

stimulating factor), NFB, IFN (interferon ), and IFN responses (Pestonjamasp et al., 2001). However, the functional relevance of these binding motives remains to be established. Since different levels of expression seem to occur for the cathelicidin genes one can assume that several different regulatory pathways are involved. The complete picture certainly includes several regulatory factors and/or different combinations, depending on the cell type. In cells like granulocytes and epithelial cells the expression of the LL-37 gene is included in the differentiation program. The gene encoding LL-37 is expressed in pro-granulocytic cells of the bone marrow and in epithelial cell lines, where the expression is induced with sodium butyrate a wellknown inducer of differentiation (Hase et al., 2002). According to Hase et al., the expression of the gene encoding LL-37 is intrinsic in cellular epithelial differentiation (Hase et al., 2002). However, parallel induction is possible and we have indications that differentiation can be separated from LL-37 expression. With respect to gene regulation, detailed information is lacking for genes of antimicrobial defenses. However, it is clear that the regulation of genes encoding antimicrobial peptides follows different routes. -Defensins are mainly found in phagocytic cells, while -defensins are expressed in epithelia, where -defensin 1 (HBD-1) is constitutively expressed, while HBD-2 is inducible, with activation through NFB (O'Neil et al., 1999). LL-37 is constitutively expressed by both phagocytes and epithelial cells. The variant antimicrobial peptides are likely an adaptation to different microbial challenges, making it harder for the microbe offender to escape. By using different regulatory pathways the system is not as vulnerable as it would be if one main switch were used. The upregulation of innate effectors such as antimicrobial peptides is a process that could be harnessed in preventive medicine to hinder bacterial invasion at epithelial surfaces. In the gut butyrate is a possible inducer that can be utilized for this purpose (Hase et al., 2002).

Cells and tissues that express the gene The main sites of expression for the gene encoding LL-37 are epithelial cells and phagocytes, but the highest concentrations are found in granulocytic neutrophils (Frohm et al., 1997; Sorensen et al., 1997). Both epithelial cells and phagocytes are located at strategic sites for host defenses, i.e. occurring

Human Cathelicidin Antimicrobial Peptide LL-37 5 immediately on the epithelial surfaces and soon thereafter by recruited leukocytes. In lungs the peptide and the pro form have been detected in epithelial cells (Bals et al., 1998; Agerberth et al., 1999) and epithelial expression has also been observed in the gastrointestinal tract (Bals et al., 1998; Frohm Nilsson et al., 1999; Islam et al., 2001; Hase et al., 2002). Borregaard's group detected the unprocessed pro form of LL-37 (hCAP18) in plasma and after developing an enzyme-linked immunosorbent assay (ELISA) estimated its concentration to be 1.12 mg/mL plasma (Sorensen et al., 1997). Interestingly, the pro form of LL-37 is present at several fold higher concentration in plasma than other proteins derived from the specific granules of neutrophils. Later we demonstrated that not only blood neutrophils produce LL-37, but also B cells, NK cells,  T cells, and monocytes (Agerberth et al., 2000). It is therefore conceivable that some of the pro-LL-37 is derived from these mononuclear cells. The presence of the pro form of LL-37 in plasma provides the capacity of a rapid activation process that must be an advantageous response to infection. Only one proteolytic cleavage would be needed for releasing a bactericidal defense molecule or an LPS scavenger. However, it must be emphasized that the details of this activation process are far from clear since the signal for the processing enzyme and the role of apolipoprotein AI as a binding protein to LL-37 in plasma are unknown (Wang et al., 1998; Sorensen et al., 1999). In our original report on the characterization of the LL-37 cDNA we detected expression in testis by using commercially available northern blots (Agerberth et al., 1995). A detailed study showed expression of the gene to be mainly localized to the epithelia of the epididymis (Malm et al., 2000). In addition, high levels of the pro form for LL-37 (hCAP18) was detected in seminal plasma and was associated with spermatozoa (Malm et al., 2000).

PROTEIN

Accession numbers For retrieving the protein sequence use LL-37 as a search word in the Entrez (NCBI) database. Protein: P49913

Description of protein Concerning the human cathelin pro region there are no available data on the protein. The active peptide

LL-37 is a 37 residue peptide with two leucines at the N-terminus and forms an -helical amphipathic structure that is dependent on the microenvironment for folding and activity (Johansson et al., 1998).

Discussion of crystal structure There are no available data on the crystal structure.

Important homologies The cathelin pro region is related to the cystatin family of thiol protease inhibitors that includes: kininogenin, cystatin, and spp24 (Hu et al., 1995). However, it is unclear if cathelin has a function in protease inhibition as the related proteins. Variant peptides are connected to the cathelin region and LL-37 is related to the amphipathic -helical peptides that are linked to the cathelin region in other mammalian species.

Posttranslational modifications One can assume that the signal peptide is cleaved off from the precursor of LL-37 during the process of cotranslational delivery to the lumen of the rough endoplasmic reticulum. Originally when cathelin was isolated and characterized from porcine leukocytes, the N-terminus was found to be blocked (Ritonja et al., 1989). The N-terminal glutamine had been converted, yielding pyrroglutamate. This glutamine is conserved in all the known cathelicidins so it suggests a similar modification in all cathelicidins. To liberate the active peptide LL-37, proteinase 3 was identified as the processing enzyme by Borregaard's group. This processing is connected to exocytosis, and no detectable cleavage was observed after phagocytosis, indicating an extracellular function for LL-37 (Sorensen et al., 2001). In contrast the bovine and porcine cathelicidins are processed by elastase (Panyutich et al., 1997; Scocchi et al., 1992).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce The main cell types where the peptide LL-37 is located are epithelial cells and neutrophilic granulocytes. In addition to the epithelia of lung and colon

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the peptide has been found to be expressed in the epithelia of mouth, tongue, esophagus, vagina, cervix, and epididymid (Frohm Nilsson et al., 1999; Malm et al., 2000). In blood the peptide has also been located in B cells, NK cells,  T cells and monocytes (Agerberth et al., 2000).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Expression of LL-37 is found upregulated in connection to inflammation most clearly in the skin (Frohm et al., 1997) at both mRNA and protein level. The elicitors for this increased expression during inflammation are unknown. The downregulation of LL-37 expression in Shigella infections has been noted, and in vitro Shigella plasmid DNA can mediate this effect (Islam et al., 2001).

RECEPTOR UTILIZATION The receptor mediating the chemotactic response to LL-37 was characterized as the formyl peptide receptor-like 1 (FPRL1) which is present on neutrophils, monocytes, and T cells (Yang et al., 2000b). Several other antimicrobial peptides have been shown to exhibit chemotactic activity. However, different human peptides have distinct host cell affinity (Yang et al., 2000a).

IN VITRO ACTIVITIES

In vitro findings The main activity characterized so far for LL-37 is killing of bacteria. LL-37 has broad-spectrum activity against gram-positive and gram-negative bacteria, while the antifungal activity is limited. The most detailed study concerning bactericidal activities has been performed by Lehrer's group (Turner et al., 1998). For the protection of the skin it is notable that the most common skin pathogen, Streptococcus group A, is very sensitive to LL-37 (Dorschner et al., 2001). Furthermore, activity has also been detected against herpes simplex virus types 1 and 2. However, the antiviral activity is limited against this type of virus (Yasin et al., 2000).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Studies by our group showed LL-37 to be induced in skin epithelia during inflammation (Frohm et al., 1997). In psoriatic lesions and in contact dermatitis this upregulation most likely has adaptive value to prevent infections in the ruptured skin. Notably, bacterial infections are surprisingly uncommon in psoriatic lesions. Expression of LL-37 has also been demonstrated at wound edges at sites of sterile incisions (Dorschner et al., 2001), however the peptide is also delivered to the wound fluid by incoming granulocytes (Frohm et al., 1996). Consequently, high concentrations of LL-37 are found in the wound crust (Dorschner et al., 2001). Furthermore, the peptide is very active against group A Streptococcus, one of the commonest and most invasive skin pathogens (Dorschner et al., 2001). Thus, LL-37 seems to be a key element in cutaneous innate immunity. In this context it is of interest that vernix caseosum, a white creamy substance rich in lipids and present on the skin surface of newborn infants, contains several antimicrobial polypeptides, including LL-37 (Marchini et al., 2002; Yoshio et al., 2002) . It is tempting to conclude that LL-37 contributes to strengthening of the skin barrier and thereby prevents microbial colonization of the infant skin. In the colon, as in lungs, a complex defense system operates where LL-37 is expressed in epithelial cells (Bals et al., 1998; Islam et al., 2001). However, the situation in the colon is totally different from that in lungs. In the lung a bacteria-free microenvironment is the aim, while colon epithelia provide an important surface for communications with the commensal microflora. The bacterial load in the colon is overwhelming and as already mentioned peptides like LL-37 could be involved in the regulation of this microflora. We have also observed an important relation between surface defenses and pathogens of the gastrointestinal tract. During Shigella or Vibrio infections expression of the antimicrobial peptides LL-37 and the defensin HBD-1 was downregulated (Islam et al., 2001). This downregulation might represent an immune escape strategy used by pathogens in order to overcome surface defenses. As part of a successful invasion pathogenic bacteria not only affect the expression of antimicrobial peptides,

Human Cathelicidin Antimicrobial Peptide LL-37 7 they have also evolved resistance to these peptides by utilizing efflux pumps (Neisseria) (Shafer et al., 1998) or by modifying lipid A by acylation (Salmonella) (Guo et al., 1998). Preventing downregulation or enhancing antimicrobial peptide expression could thus provide an alternative treatment in protecting human and animal stocks against bacterial intruders. Notably, using sodium butyrate treatments, symptoms of shigellosis were reduced in a rabbit experimental model (Rabbani et al., 1999). Butyrate, a well-known inducer of differentiation in colon epithelial cells, has indeed been shown to enhance the expression of LL-37 (Hase et al., 2002).

knockout mouse model will certainly be an important tool to determine the general contributions of cathelicidins in resistance to infections and inflammatory responses. The importance of other antimicrobial peptides in the gastrointestinal tract came from the matrilysin knockout mouse. The matrilysin protease is necessary for cleavage and generation of defensins in the gut (the cryptdins). The matrilysin-deficient mouse exhibited increased sensitivity to orally administered bacteria (Wilson et al., 1999). Together these knockout mice models indicate the importance of antimicrobial peptides in immunity.

Species differences

Endogenous inhibitors and enhancers

An interesting evolutionary pattern emerges when the known mammalian cathelicidin sequences are compared. As usually for related genes, changes due to point mutations are observed in homologous domains. These changes are seen in the cathelin encoding exons, and in sequences encoding peptides that have common origin such as the amphipathic linear peptides, including LL-37. In some species rapid divergence has occurred through gene duplications and shuffling of exon 4, connecting totally unrelated peptides to cathelin and thus enhancing the variation profoundly. This predicted evolutionary pattern has occurred in the porcine genome, where more than 10 cathelicidins with various different peptides are found (Lehrer and Ganz, 2002). Through analyses of different cathelicidins this divergence seems lineage- and species-specific which indicates events parallel to mammalian evolution. One may predict that the evolutionary radiation reflects an adaptation to variant microbial challenges. Furthermore, in species that only have one cathelicidin gene, like mouse, rat, and human, the peptide domain is of the linear -helical amphipathic type. This suggests that the original peptide version was amphipathic and -helical or that there are evolutionary constraints on this peptide structure due to other functions, where receptor binding is included.

Knockout mouse phenotype Interestingly, gene-deficient knockout mice lacking the cathelicidin CRAMP, a structural and functional homolog to LL-37, are much more sensitive to group A Streptococcus infections in the skin, resulting in necrotic skin lesions and lower efficiency of bacterial clearance (Gallo et al., 1997; Nizet et al., 2001). This

In plasma LL-37 is bound to ApoA-1 with a Kd in the range of 0.6±2.4 mM and thus work as a scavenger for the cytotoxic effect of LL-37 (Wang et al., 1998; Sorensen et al., 1999).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects There is very limited information on the exact concentration of the peptide in normal and diseased tissues. The concentration of the pro form in normal plasma was found to be 1.12 mg/mL (Sorensen et al., 1997).

Role in experiments of nature and disease states The association between antimicrobial peptides and a human disease was originally observed based on studies of cystic fibrosis. This indicated the importance of antimicrobial peptides in immunity (Smith et al., 1996). This finding was first reported by the group of M. Welsh and is now considered a milestone in research on antimicrobial peptides. In cystic fibrosis (CF), the CFTR (cystic fibrosis transmembrane-conductance regulator) protein, a chloride channel, is mutated leading to abnormal airway surface fluid and a failure of CF epithelia to kill

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bacteria (Smith et al., 1996). Which mechanisms are involved is still under debate, but it is now accepted that antimicrobial peptides, including LL-37, are of importance for lung surface defenses. Increased salt concentrations have been suggested as an important denaturating factor; indeed salt affects individual antimicrobial peptides/proteins in addition to diminishing their synergistic and/or additive action (Singh et al., 2000). LL-37 is expressed in lung epithelial cells and alveolar macrophages (Agerberth et al., 1999). Increased concentrations are found in aspirates of newborn infants during lung infection(s) (SchallerBals et al., 2002). Furthermore, it is of interest that in a xenograft model antimicrobial activity could be restored after exposure of CF xenografts to an adenovirus expressing the gene encoding LL-37 (Bals et al., 1999).

IN THERAPY

Toxicity LL-37 like most antimicrobial peptides has effect on eukaryotic cells but at concentrations that are higher than the effective antimicrobial concentrations (Johansson et al., 1997). Whether this cytotoxic effect becomes problematic during the phase of upregulation in inflammation has not been documented.

ACKNOWLEDGEMENT The authors would like to thank Eirikur Steingrimsson for critical reading of the manuscript.

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