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Defensins – innate immunity at the epithelial frontier

Fellermann, Klausa; Stange, Eduard F.b

European Journal of Gastroenterology & Hepatology: July 2001 - Volume 13 - Issue 7 - p 771-776
Review in Depth

Physical barrier function was formerly believed to play the major role in mucosal protection against luminal bacteria. This view has now been challenged by the discovery of specialized molecules that possess antimicrobial activity. More than 100 peptides have been identified so far, and the number is still growing. These peptides are distributed widely and conserved throughout phylogeny. The epithelial expression of antimicrobial peptides is of particular interest as many pathogens adhere to epithelial surfaces and may eventually invade the host. This rapidly acting defence system of innate immunity is already engaged before adoptive immune interactions take place. These antimicrobial peptides consist of constitutive and inducible forms, potentiating this barrier function in terms of an inflammatory response.

One important subgroup of antimicrobial peptides is the family of defensins, which are classified as alpha (α-) and beta (β-) defensins. Eight different peptides with varying antimicrobial properties have been identified. They are distributed widely in humans, and organ-specific expression patterns have been observed. Homologous peptides have been found in other mammals, vertebrates, invertebrates, insects and plants. The identification of α-defensins and their murine counterparts, cryptdins, in the small intestine prompted intensive research into epithelial antimicrobial defence.

aDepartment of Internal Medicine, Division of Gastroenterology, University of Lubeck, Lubeck, and bDepartment of Internal Medicine, Robert Bosch Krankenhaus, Stuttgart, Germany

Correspondence to Klaus Fellermann, Department of Internal Medicine I, Division of Gastroenterology, University of Lubeck, Ratzeburger Allee 160, 23538 Lubeck, Germany Tel: +49 451 500 6255; fax: +49 451 500 3645; e-mail: klaus.fellermann@medinf.mu-luebeck.de

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Introduction

Although the inner and outer surfaces of the human body are colonized by a multitude of pathogens, local or systemic infections occur only rarely. Numerous unspecific defence mechanisms provide an effective barrier that is not broken easily. For example, the skin is protected by the oily keratin layer, and the upper airways exhibit fimbriae with unidirectional transport. A special situation exists in the gastrointestinal tract, particularly in the colon with its massive bacterial load. Factors such as gastric acid, mucus production and propulsive peristalsis limit contact time with the mucosa and possible immune interaction. However, it became clear in the last decade that there are other counteractors to infection: the first to be unravelled was lysozyme. Since then, many other peptides and proteins that mediate defence have been characterized. They have been reviewed extensively [1–5], but their functional roles are just beginning to be elucidated. This review will focus on one family of cationic antimicrobial peptides (CAPs), the defensins, with a special focus on epithelial sites. (For comprehensive reviews, see references [6–9].)

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Genomics and structure

To date, eight different genes have been characterized. They typically have two short exons separated by a large intron. Four neutrophil peptides (HNP-1–4) have a short extra exon that encodes the leading peptide. Based on the organization of three intramolecular disulphide bonds between cysteine residues, they are termed alpha (α-) and beta (β-) defensins. HNP-1–4, human defensin 5 (HD-5) and human defensin 6 (HD-6) constitute the α-defensins. Two β-defensins have been identified: human beta defensin 1 (HBD-1) and human beta defensin 2 (HBD-2). The genomic clustering and their phylogenetic conservation make evolutionary development from one ancestral gene highly likely [10,11]. All known human defensins have been mapped to a locus on chromosome 8 p22-23.1 near the telomere region [11–13]. HBD-1 is located about 100–150 kb apart from HNP-1 [11], and 500–600 kb from HBD-2 [13]. The proposed organization is centromere–HBD-2–HBD-1–HNP–telomere, with the α-defensins HD-5 and HD-6 flanking the myeloic HNPs in a head-to-tail array [10]. So far, polymorphisms have been recognized only in the HBD-1 gene but their importance is unknown [14,15].

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Proteomics

Defensins are cationic, arginine-rich peptides consisting of 28–44 amino acids. The molecular weight varies from 3 to 5 kDa. They all share a typical tertiary structure despite differences in primary structure. Three antiparallel beta sheets are connected by loops, and a beta hairpin with hydrophobic properties protrudes orthogonally [16]. Six cysteine residues form three intramolecular disulphide bonds, which differ among defensins. In α-defensins, these are grouped C1–C6, C2–C4 and C3–C5, whereas in β-defensins, bonds occur between C1–C5, C2–C4 and C3–C6. The molecules are amphiphilic.

Defensins are synthesized as prepropeptides. The prosegment keeps the molecule in an inactive state; recently, matrilysin (MMP-7) was identified as the enzyme responsible for release of the mature defensin in mice [17]. In the human setting, the situation is less clear. HD-5 is stored in its proform in secretory granules of Paneth cells, and is processed to the mature peptide during or after release by cleavage C-terminally to an arginine [18]. Ghosh et al. favour trypsinogen-2 and postulate that activation takes place in the crypt lumen, where this enzyme is abundant [19].

Major isoforms of HD-5 are 32 and 39 amino acids in length, while HD-6 is predominated by its 32-amino-acid form [18]. In urine, several isoforms of HBD-1 with differing antimicrobial activity have been isolated, ranging from 36 to 47 amino acids in length [20]. The predominating isoforms were 40 or 44 amino acids long. For HBD-2, a 41-amino-acid naturally occurring isoform has been identified [21–23].

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Function and biological activities

Defensins have a broad antimicrobial spectrum ranging from Gram-negative to Gram-positive bacteria, mycobacteria, fungi and enveloped viruses. They disrupt microbial cell membranes by binding to the phospholipid-rich, negatively charged moiety, sometimes organized in multimeric complexes [24,25] (reviewed in [6]). However, their precise mode of action remains to be determined. Phagocytotic killing activity of leucocytes is attributed mainly to the high concentration of HNP-1–4. Although high concentrations are also cytotoxic for mammalian cells, lesser amounts promote growth in epithelial cells and fibroblasts [26], suggesting a role in wound-healing processes. Defensins are chemotactic for monocytes, polymorphonuclear leucocytes and T-cells [27–29]. Moreover, they seem to amplify acquired immune responses [30]. HNPs inhibit action of adrenocorticotropic hormone (ACTH) by reversibly binding to the ACTH receptor [31]. The biological function of the latter phenomenon is currently unknown, but diminished cortisol release during stress from infection is a rational explanation.

The in vitro antibacterial activity is in the micromolar range and has been very well characterized in the case of HD-5 [32]. It was effective against Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Candida albicans. Similar to the HNPs, the activity is inhibited by physiological salt concentrations, but is independent of the pH and is refractory to trypsin digestion. HBD-1 is highly active against Gram-negative bacteria at 100 μg/ml [33]. HBD-2 is 10 times more potent depending on the pathogen tested, but is less effective and bacteriostatic against Gram-positive strains [21].

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Distribution

The highest density of defensins is found in mammalian granulocytes [34,35]. HNP-1–4 make up about 10% of the total protein and are the most abundant in azurophilic granules mediating phagocytotic microbial killing. Their expression does not seem to be restricted to cells of myeloic origin, as HNPs have also been found in the placenta [36], inflamed colon [37] and saliva [38].

HD-5 and HD-6 are of epithelial origin and abundantly detectable in Paneth cell secretory granules [39–41]. A developmental expression pattern has been observed in the small intestine in accordance with the appearance of Paneth cells, whereas only HD-5 showed a transient colonic expression [42]. Possibly, these defensins may protect stem cells at the crypt base from microbial invasion. Moreover, HD-5 has been found in the female genital tract [43,44] (Table 1).

Table 1

Table 1

The more recent discoveries are the β-defensins. HBD-1 was first isolated from haemofiltrate [45] and HBD-2 from psoriatic scale [21]. Several other epithelial sites of synthesis have now been recognized. Northern blotting was positive for HBD-1 in the kidney and pancreas; other sites were only positive using the more sensitive RT-PCR analysis [46]. Furthermore, HBD-1 has been detected in the kidney, female reproductive tissue [20], salivary gland, pancreas [47], lung [33,48,49], gingival epithelium [50], skin [51] and colon [52] (Table 1). McCray et al. found a developmentally regulated pattern for HBD-1 in the lung, as formerly shown for epithelial α-defensins [48]. HBD-2 was expressed in the trachea, lung, bronchoalveolar lavage (BAL) fluid [22,49,53], skin [13], colon [52] and gingival tissue [23]. Using an RNA library, a broad epithelial distribution throughout many organs has been observed, with the highest amounts in the lung [22] (Table 1).

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Regulation and implications for disease

Little information is available about the regulation of epithelial α-defensins. Several nuclear factor of interleukin 6 (NF-IL 6) and activator protein 2 (AP-2) recognition sequences lie within the 5′-flanking region, implying a response towards pro-inflammatory stimuli [42]. Interestingly, a moderate two- to three-fold induction of defensin mRNA has been encountered in necrotizing enterocolitis paralleled by an increase in Paneth cells [54]. We showed recently that colonic biopsies disclose an up-regulated HD-5 and HD-6 expression in inflammatory bowel disease as well as diverticulitis. The cellular origin is unclear but may be attributable to metaplastic Paneth cells [55]. High interindividual variability of HD-5 and HD-6 expression was observed in the intestine, and increased levels were found in the duodenum in coeliac sprue [56].

HBD-2 is unique among the so far known human defensins, as the gene contains several binding sites for nuclear factor κB (NFκB) and activator protein 1 (AP-1), both involved in transcriptional regulation by pro-inflammatory cytokines, e.g. tumour necrosis factor α (TNFα) and lipopolysaccharide (LPS). These recognition sites are absent in the HBD-1 gene, which is constitutively expressed [46]. However, some regulatory control may still be possible as HBD-1 contains NF-IL 6 and interferon γ (IFNγ) consensus sites [11]. Increased levels of HBD-1 have been detected in urine and plasma in pyelonephritis [57] and in pregnant women [20]. Similar induction to HBD-2 has been observed for bovine homologues of β-defensins, named tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). LPS stimulated epithelial defensin response via the CD14 LPS-receptor, which has been identified on cells of epithelial origin [58,59]. This inducible pattern explains the finding of Liu et al. [13], who discovered HBD-2 only in inflamed skin, and resembles data with LAP in diseased bronchial and intestinal epithelium [60]. In fact, Becker and colleagues [61] have recently identified the activation pathway in tracheobronchial epithelium, where CD14 in conjunction with Toll-receptor mediates an LPS-driven HBD-2 response. Other activation pathways may be possible based on the fact that Gram-positive bacteria and yeasts are also capable of induction. Interestingly, mucoid Pseudomonas strains induce HBD-2, while non-mucoid forms do not [62]. The mechanism may be attributable to pathogen pattern recognition receptors, which identify specific surface determinants.

Another interesting observation is the impaired capacity of airway surface fluid (ASL) to kill microorganisms in cystic fibrosis (CF), which can be restored by reducing the salt concentration. It was postulated that defensins, expressed in respiratory epithelia and present in ASL, are inactivated in the high-salt environment of CF patients, and may account for recurrent pulmonary infections [63]. The relevance of HBD-1 for antimicrobial activity was elegantly shown by using antisense technology in a bronchial xenograft model. Abrogation of HBD-1 message resulted in decreased bacterial killing [33]. Singh et al. reported an increase of HBD-2 peptide in BAL fluid from CF patients and inflammatory lung disease [49]. As stimulated HBD-2 expression of epithelial cells did not differ among CF and non-CF patients, there seems to be no primary defect in defensin production. Moreover, HBD-2 protein is detectable in plasma and greatly increased in bacterial pneumonia [53]. It remains to be determined whether the peptide leaks into the plasma from the epithelial surface. Another inducible epithelial defensin with an interesting distribution pattern has been identified in cattle. Enteric β-defensin (EBD) is expressed in the distal small intestine as well as the colon, and raised levels were found upon infection with Cryptosporidium parvum [64]. No human homologue of this interesting defensin has been identified so far.

HBD-2 is up-regulated by TNFα, interleukin 1β (IL-1β), Gram-negative and to a lesser extent Gram-positive bacteria, and yeasts in keratinocyte cultures [21,49]. Frye et al. observed low levels of gastrointestinal HBD-1 expression, while HBD-2 was absent from uninflamed biopsies [56]. In epithelial cell lines of colonic origin, HBD-1 mRNA and peptide were present, whereas HBD-2 was only found upon interleukin 1α (IL-1α) stimulation or infection with enteroinvasive bacteria [52]. Experiments in a human fetal intestinal xenograft model resembled these in vitro findings of constitutive HBD-1 and inducible HBD-2 expression. We have addressed the question of whether the same occurs in the clinical setting. With RT-PCR analysis and immunohistochemistry, we found a constitutive colonic expression pattern for HBD-1. While the α-defensins were up-regulated in inflammatory bowel disease, including diverticulitis, HBD-2 was apparently a more frequent finding in ulcerative colitis than in Crohn's disease. This is striking as NFκB, a major inductor for HBD-2, is highly elevated in both entities [65,66].

Recent reports claimed that Helicobacter pylori stimulates HBD-2 expression in gastric epithelium in a human gastric epithelial cell line [67,68]. Only type-I strains carrying the cag A pathogenicity island were capable of activation, an event that possibly involved the NFκB pathway [67]. However, this was not specific to Helicobacter as Salmonella species showed the same response [68]. In fact, HBD-2 mRNA expression in the stomach is more often encountered in Helicobacter infection than in Helicobacter-negative cases, strengthening this observation (unpublished observations).

Thus, the available data may be interpreted as follows: organs with an epithelial lining have a unique defensin profile. The α-defensins HD-5 and 6 are to be found in the gastrointestinal tract, especially in the small intestine. HBD-1 is expressed constitutively and may confer a baseline function of host defence in the absence of inflammation. In the case of inflammation, HBD-2 is up-regulated, and the expression of other defensins may extend to other non-specialized cells counteracting the underlying trigger.

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Conclusions

Defensins comprise a new and interesting field of research. They not only confer unspecific defence at the body surface but are in part highly inducible in a specific manner. The induction is rapid in contrast to acquired immunity, and there is an increasing body of evidence suggesting that the aggressor elicits its own specific defensin profile. Further biological activities suggest that defensins link adaptive and innate immunity. Future investigations will address defects in defensin expression possibly causing currently unexplained disorders, such as the inflammatory bowel diseases. Another future focus is new pharmacological interventions enhancing innate defence in infectious diseases.

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References

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                              Keywords:

                              antimicrobial peptide; defensin; epithelial cell; innate immunity

                              © 2001 Lippincott Williams & Wilkins, Inc.