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5 Antimicrobial Skin Peptides And Proteins


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Dscripción de los péptidos antimicrobianos de la piel

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5 Antimicrobial Skin Peptides And Proteins

  1. 1. Cell. Mol. Life Sci. 63 (2006) 469–486 1420-682X/06/040469-18 DOI 10.1007/s00018-005-5364-0 Cellular and Molecular Life Sciences © Birkhäuser Verlag, Basel, 2006 Review Antimicrobial skin peptides and proteins J. M. Schröder* and J. Harder Department of Dermatology, University-Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 7, 24105 Kiel (Germany), Fax: +49 431 597 1592, e-mail: Received 9 August 2005; received after revision 21 October 2005; accepted 16 November 2005 Online First 16 January 2006 Abstract. Human skin is permanently exposed to mi- also focally expressed in skin in the absence of inflam- croorganisms, but rarely infected. One reason for this mation. This suggests that non-inflammatory stimuli of natural resistance might be the existence of a ‘chemical endogenous and/or exogenous origin can also stimulate barrier’ consisting in constitutively and inducibly pro- AMP synthesis without inflammation. Such mediators duced antimicrobial peptides and proteins (AMPs). might be ideal ‘immune stimulants’ to induce only the Many of these AMPs can be induced in vitro by proin- innate antimicrobial skin effector molecules without flammatory cytokines or bacteria. Apart from being ex- causing inflammation. pressed in vivo in inflammatory lesions, some AMPs are Key words. Innate immunity; antimicrobial peptides; antimicrobial proteins; skin infection; keratinocytes; psoriasis; atopic dermatitis. Introduction strategy is the existence of an intact physical barrier con- sisting in the stratum corneum in skin and mucus in mu- As a barrier organ human skin is always in contact with cosa. Both desquamation and secretion of mucus lead to the environment and is covered with a characteristic mi- a permanent renewal of these body surfaces and simulta- croflora [1]. Resident microorganisms, which surpris- neous elimination of the microorganisms adhering to ingly are present in rather stable numbers and composi- these layers. Infiltration of microorganisms into the living tion, always grow as small colonies at the surface of the epidermis will be inhibited by the lipid barrier present in horny layer (stratum corneum) and within the uppermost the uppermost parts of stratum corneum [2, 3]. Although stratum corneum layers. The composition varies qualita- it has been suggested for a long time that the physical bar- tively and quantitatively, depending on localisation, be- rier is the sole component protecting skin from infection, tween 102 and 107 microorganisms/cm2. there are a number of hints that this is not the case. Bac- With respect to the capability of microorganisms to di- teria produce a number of enzymes that can degrade both vide under optimal conditions (nutrient-rich conditions, lipids and proteins and therefore should make them capa- higher temperature, humidity, ions) within 20 min, it ble of overcoming the physical barrier. Another strategy might be an enigma that healthy body surfaces usually to protect the skin from infection would be phagocytosis do not show excessive microbial growth and no signs of of the invading microorganisms. Although it is commonly infection. This unexpected phenomenon might be ex- believed that active elimination of microorganisms is plained by considering skin as a defense organ, where possible only by professional phagocytes, there does ex- particular strategies have evolved to protect the skin from ist ultrastructural evidence that keratinocytes have the ca- infection. One of the most important elements of this pacity to phagocytose C. candida albicans [4]. In contrast to skin keratinocytes, where phagocytosis of microorgan- * Corresponding author. isms represents a rare event, in mucosa cells phagocyto-
  2. 2. 470 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins sis of microorganisms is a more common phenomenon Table 1. Skin-derived AMPs. [5]. Despite these findings it is doubtful that active pha- Name Antimicrobial activity Chemotactic Induced under gocytosis of microorganisms by keratinocytes is of major for inflammatory importance for maintaining skin integrity, because Gram+ Gram– Fungi conditions phagocytosis is a long-lasting process and thus efficacy is very low. All of these observations led to the hypothesis Lysozyme + + – ? – that, apart in addition to the physical barrier, a ‘chemical Dermcidin + + + ? – barrier’ of the skin should also exist. This could consist in Psoriasin (+) + ? T cells, + chemicals produced in the uppermost parts of the skin that neutrophils control growth of bacteria at body surfaces and inhibit in- RNase 7 + + + ? + fection. Because healthy skin does not contain any blood- hBD-1 (+) + ? T cells, – derived leukocytes, such as neutrophils, which contain a dendritic cells number of bactericidal compounds [6], epithelial cells might be the source of ‘chemical barrier’ compounds. hBD-2 (+) + + T cells, + dendritic cells, There is ample evidence, that in plants, which do not con- mast cells tain any adaptive immune system, mainly antimicrobial hBD-3 + + + T cells, + peptides and proteins (AMPs) such as defensins and dendritic cells, thionins represent the effector molecules (reviewed in monocytes [7]). These are often produced in the uppermost, environ- LL-37 + + + neutrophils, + ment-exposed layers of various plant organs [8], suggest- monocytes, ing that these molecules may play a role as effector of T cells, mast the epithelial defense system to protect surfaces from in- cells fection. Apart from plants, invertebrate epithelia produce AMPs, as demonstrated in Drosophila melanogaster, where contact with microbes produces local induction of AMPs [9]. This observation favours the hypothesis that mal cells in granular layers and malpighian cells [12, epithelial production of AMPs represents a general phe- 15]. Pilosebaceous follicle cells and hair bulb cells as nomenon which suggests effector molecules in an epithe- well as all parts of eccrine sweat glands were also posi- lial defense system. Since 1987, findings show that ver- tive for lysozyme. tebrates might also use antimicrobial peptides as part of a Although lysozyme is mainly a Gram-positive bacteria ‘chemical defense system’ to protect skin from infection. (e.g. S. aureus)-killing antimicrobial peptide [16] it is Zasloff isolated from the skin of the African clawed frog also active against Gram-negative bacteria, e.g. Escheri- Xenopus laevis a broad spectrum antimicrobial peptide chia coli [17] or Pseudomonas aeruginosa [18], suggest- he termed ‘magainin’ [10], which is also stored as an ac- ing that it might function in healthy skin to control growth tive, processed peptide in large granules within the neu- of bacteria. However, the contribution of lysozyme to cu- roepithelial glands of the skin and is secreted upon stim- taneous defense is still unclear because lysozyme is ex- ulation with noradrenalin [11]. pressed exclusively in the cytoplasm, and immunoreac- Based on these observations in frog skin and findings from tive lysozyme is absent in the stratum corneum [12] and insects and plants, it is no surprise that human skin can also skin washing fluid [19]. produce AMPs. Indeed, in the past 10 years a number of skin-derived AMPs have been discovered in human skin material using a biochemical approach and table 1 summa- Antimicrobial peptides of sweat rizes the skin-derived AMPs discussed in this review, to- gether with their major biological properties. The eccrine sweat gland is one of the major cutaneous ap- pendages, and is a secretory as well as an excretory organ [20]. Its principal function is thermoregulation during ex- Lysozyme, a human healthy skin-derived posure to a hot environment or physical exercise. In addi- antimicrobial protein. tion to this function, sweat may have also a role as an in- nate defense system controlling skin bacterial flora by re- The first antimicrobial protein found in human skin was leasing antimicrobial peptides. lysozyme [12, 13], which originally was discribed as ‘bacteriolytic activity’ in nasal secretions by Alexander Fleming [14], suggesting that human nasal epithelial Dermcidin glands also secrete antimicrobial compounds. In skin Dermcidin appears to be the principal sweat antimicro- lysozyme is mainly located in the cytoplasm of epider- bial peptide that is constitutively produced exclusively
  3. 3. Cell. Mol. Life Sci. Vol. 63, 2006 Review Article 471 Figure 1. RP-HPLC of a healthy stratum corneum (A) and lesional psoriatic scale extracts (B). Heparin-bound material of extracts obtained from 100 g of pooled healthy stratum corneum or from 7 g of lesional psoriatic scales were separated by preparative wide-pore RP-8-HPLC and 10-ml aliquots of each fraction were tested for E. coli-cidal activity in the radial-diffusion assay system. Bars represent the diameter of the clearing zone in the assay system. Note the difference in the antimicrobial activity pattern and the increased activity in psoriatic scale extracts, despite the use of 14-fold less amounts of material. H2, hBD-2; H3, hBD-3; L, lysozyme; P, psoriasin; R, RNase7. by eccrine gland cells [21, 22]. It is expressed as a 9.3- Cathelicidin LL-37 in sweat kDa precursor, that is proteolytically cleaved, resulting Apart from dermcidin, the human cathelicidin LL-37 is in dermcidin 1 (DCD-1), a 47-aa-containing peptide also expressed in eccrine glands and duct cells [24]. Im- that shows antimicrobial activity against Staphylo- munoreactive LL-37 is localized in both the eccrine se- coccus aureus, Enterococcus faecalis, E. coli as well as cretory glands and ducts, where it is found to be diffusely C. albicans at low micromolar concentration (1–10 µg/ located in the cytoplasm of the secretory gland, and also ml) [22]. Its activity is not affected by low pH and located in the ductal epithelium of the ducts – in contrast increased salt concentrations, conditions that could oc- to dermcidin, which is located in the secretory glands, but cur in sweat in vivo [22]. In sweat, concentrations of not in the ducts [24]. Interestingly, among traces of the 1–10 mg/ml DCD-1 – depending on the donor – were CAP-18-precursor and traces of the mature LL-37 a pre- found [22]. dominant 14-kDa form of LL-37 was found in sweat, as Patients with atopic dermatitis have recurrent bacterial or shown by Western blot analyses [24]. In another study it viral skin infections, an observation that led to the hy- was shown that enhanced antimicrobial activity exists in pothesis that patients with atopic dermatitis may have a normal human sweat and is the consequence of process- reduced amount of DCD peptides in sweat. Using sur- ing of LL-37 to previously unknown, naturally occurring face-enhanced laser desorption ionisation time-of-flight cathelicidin peptides [25]. High-performance liquid chro- mass spectrometry and enzyme-linked immunosorbent matography (HPLC) separation of sweat samples from assay (ELISA) it could be shown that the amount of sev- different individuals showed remarkable consistency in eral DCD-derived peptides in sweat of patients with the elution profile of antimicrobial molecules. Analyses atopic dermatitis is significantly reduced [23]. Further- of HPLC fractions by immunodetection against LL-37 more, atopic dermatitis patients with a history of bacter- suggested that some of these less abundant, yet appar- ial and viral skin infections were found to have signifi- ently potent, antimicrobial molecules were similar to LL- cantly less DCD in their sweat. In support of these find- 37. Investigation of antimicrobial activity and identifica- ings, in healthy subjects, sweating leads to a reduction of tion of new peptides generated from synthetic LL-37 viable bacteria on the skin surface, but this does not oc- showed that further processing occurs by a serine pro- cur in patients with atopic dermatitis [23], indicating that tease present in sweat. The resulting 31, 30 and 20 res- reduced expression of DCD in sweat of patients with idues containing LL-37 peptide fragments exhibited en- atopic dermatitis may be important. hanced antimicrobial activity against skin pathogens such as S. aureus and C. albicans [25].
  4. 4. 472 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins RNase 7 is a major AMP of healthy skin The relative high abundance of RNase 7 in skin, its in- Analyses of healthy person’s stratum corneum extracts ducibility by contact with bacteria and its preference for for AMPs revealed strong activity for both Gram-negative killing Gram-positive Enterococci may indicate a major and Gram-positive bacteria [our unpublished results]. At- role of this AMP in preventing skin infection by Gram- tempts to identify the principal AMPs by biochemical ap- positive gut bacteria. proaches revealed a 14.5-kDa protein [26]. Amino acid sequence analyses and molecular cloning of the corre- sponding complementary DNA (cDNA) showed that this Psoriasin (S100A7), an E. coli-cidal protein protein, RNase 7, is similar to members of the RNase A of healthy skin superfamily, among them, RNase 2 [eosinophil-derived neurotoxin (EDN)], RNase 3 [eosinophil-derived cationic Certain species of bacteria die rapidly on the surface of protein (ECP)] and RNase 5 (angiogenin) [27, 28]. Elec- human skin, whereas other species do not [31]. This dis- trospray-ionisation mass spectrometry (ESI-MS) analy- tinction can be readily demonstrated by exposure of the ses revealed the presence of four disulfide bridges [26]. fingertips to either E. coli or S. aureus in a humid atmos- RNase 7 is a highly basic protein (pI: 9.80) that represents phere for 30 min; the surface of a petri dish filled with nu- one of the major heparin-binding proteins in healthy skin- trient agar is then touched with the bare fingertips, and derived stratum corneum extracts. We were able to isolate the dish is incubated overnight. A S. aureus print of the 4–8 mg/g stratum corneum [26]. In contrast, from psori- fingertips is produced, but no viable E. coli are trans- atic-scale material 10–25 mg/g RNase 7 could be recov- ferred [19]. This surprising observation led to the hy- ered [29], suggesting that RNase 7 might be inducible, pothesis that healthy human skin might constitutively despite the high level of constitutive expression in healthy produce a defense chemical that could preferentially and skin. Indeed, the proinflammatory cytokines interleukin very effectively control the growth of E. coli. Such factor (IL)-1b, interferon (IFN)-g and to a lesser degree also would also explain why E. coli is rarely found to colonise tumour necrosis factor (TNF)-a induced RNase 7 mes- skin [1]. senger RNA (mRNA) expression in keratinocytes. Apart Biochemical analyses of E. coli-killing activity in extracts from primary cytokines, primary keratinocytes treated of a healthy person’s stratum corneum identified psoriasin with heat-killed bacteria also induced RNase 7 [26]. As (S100A7) as a principal E. coli-cidal antimicrobial protein ye—t, the molecular mechanism of bacteria-mediated in healthy skin stratum corneum extracts [19]. Reversed- RNase 7 induction in keratinocytes remains unclear. phase (RP)-HPLC analyses of a healthy person’s washing fluid (fig. 2) together with ESI-MS analyses of HPLC fractions revealed psoriasin (MW: 11.366; the N-acetyl- RNase 7 is a broad-spectrum antimicrobial protein ated, terminal methionine missing form of S100A7c) as Natural skin-derived RNase 7 exhibits a broad spectrum an additional principal protein constituent [19], indicating of antimicrobial activity against both Gram-negative bac- teria (E. coli and P aeruginosa), and Gram-positive bac- . teria (Propionibacterium acnes, S. aureus) and the yeast C. albicans. Remarkably, the number of colony-forming units decreases by five orders at concentrations in the low micromolar range. It is of particular interest that for un- known reasons RNase 7 is extremely effective at killing a vancomycin-resistant strain of Enterococcus faecium al- ready at 20 nM concentrations [26]. Thus – on a molar base – RNase 7 represents one of the most potent and ef- ficacious human antimicrobial proteins known so far. RNase 7 is not the only member of the RNase A family that shows antimicrobial activity: it has been demon- strated that the eosinophil-derived RNase ECP exhibits antimicrobial activity against S. aureus and E. coli with Figure 2. RP-HPLC of healthy skin washing fluid. Pooled washing fluid obtained from forearm skin of 50 persons by rinsing 2 cm2 of activity in the low micromolar range – similar to that of each person with 2 ml of buffer was concentrated using protein-free RNase 7 [30]. Also, human angiogenin (RNase 5) is ac- Amicon YM2 filters and separated with an increasing acetonitrile tive at low micromolar concentrations against Strepto- gradient at a micropore C2C18-RP-HPLC column. Aliquots of each coccus pneumoniae and the yeast C. albicans [27]. As yet fraction were analysed by ESI-MS. The major absorbing peak at 215 nm indicated by the arrow revealed an exact mass of 11,366 Da, there is no evidence that stratum corneum obtained from corresponding to the N-terminally acetylated, methionine-missing healthy donors also contains these other RNases at con- sequence of psoriasin, as shown by MS/MS analyses of tryptic di- centrations sufficient to be microbiocidal – if at all. gests. Reproduced courtesy of [19].
  5. 5. Cell. Mol. Life Sci. Vol. 63, 2006 Review Article 473 that psoriasin is released to the body surface. A detailed masses for S100A15 protein and its modified forms, we biochemical analysis of (pooled) healthy stratum cor- found no clear evidence for a high abundance of the pro- neum-derived psoriasin, however, revealed psoriasin to be tein – if any – in healthy stratum corneum extracts. ESI- heterogeneous with the 11.366-Da form as the predomi- MS analyses yielded several ions that could correspond nant psoriasin form in these extracts [19]. to truncated forms of S100A15, but this observation Sequencing of the human S100 gene cluster has iden- needs further confirmation [unpublished results]. tified five copies of S100A7-like genes (S100A7a – S100A7e) [32], thus showing evidence of gene duplica- tion during primate evolutionary history. We found that Psoriasin is a focally expressed AMP only the S100A7c variant (as 11.366-Da form) can be re- In skin, psoriasin is focally expressed (fig. 3) [19] and re- covered from skin-washing fluid, suggesting that it is re- leased from keratinocytes, particularly in areas where leased from keratinocytes. In contrast, various other, not- high bacterial colonization is well documented, such as yet-characterized psoriasin variants and post-translation- the uppermost parts of hair follicles (fig. 3A) and nose ally modified and mutated psoriasin forms could be skin (fig. 3C). On the other hand, dry areas of the skin, isolated only from skin extracts [19], suggesting that such as lower leg skin (fig. 3B) show a rather ‘patchy’ these are stored within the keratinocytes. staining for psoriasin. It is interesting to note that apart Recently, in an effort to identify psoriasis-associated from keratinocytes, sebocytes, the lipid-secreting cells of genes, a novel gene, named S100A15, with high homol- sebaceous glands, also showed immunoreactive psoriasin ogy to the S100A7 was discovered, which is also overex- (fig. 3C), suggesting that psoriasin is possibly also se- pressed in psoriasis [33]. Like psoriasin, S100A15 in situ creted together with lipids. Indeed, when healthy skin is hybridisation experiments revealed staining of epidermal first washed with buffer and then with acetone (to extract layers of psoriatic skin, suggesting a similar function as skin lipoids), psoriasin can be recovered from both aque- psoriasin. As yet, however, its biological function is not ous and acetone extracts [19]. These data indicate that the clear. When we looked at ESI-MS analyses of crude pso- highly hydrophobic psoriasin is also stored in the lipid riasin preparations for ions corresponding to calculated layer of healthy skin. Figure 3. Immunohistochemical analyses reveal psoriasin to be expressed focally. There is strong staining (red) in the uppermost epider- mal layers of the hair follicles and surrounding epidermis of the cheek (A), focal expression in the uppermost parts of the lower leg epi- dermis (B) and strong immunoreactivity in the suprabasal keratinocytes of the epidermis and in the sebaceous glands (SG) of nose skin (C). Note the absence of psoriasin in eccrine sweat glands (ES). Scale bars, 200 mm. Reproduced courtesy of [19].
  6. 6. 474 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins Unlike defensins and cathelicidin LL-37, psoriasin seems to kill E. coli by a different mode of action, because ultra- structural analyses of psoriasin-treated E. coli revealed no signs of perforation. Interestingly, the antimicrobial activ- ity of psoriasin, which exists in aqueous solution as a non- covalent dimer that contains two half Zn2+-binding sites and two Ca2+-binding EF-hands per molecule [34], is sen- sitive towards treatment with Zn2+, but not Ca2+, Mg2+, Fe2+ or Mn2+. This suggests that sequestration of Zn2+ is the mechanism how psoriasin kills E. coli [19]. This hypothe- sis is supported by experiments, which show that the Zn2+ chelator TPEN also kills E. coli, and that TPEN and psori- asin have additive effects [19]. The exact mechanism by which psoriasin kills E. coli, however, is yet unknown. It has been speculated that Zn2+ deprivation ultimately affects Zn2+-dependent enzymes; Zn2+ as well as Cu2+are essential transition metal ions for functional E. coli superoxide dis- mutase [35]. This enzyme has high sensitivity to chelators Figure 4. Psoriasin is secreted in vivo on the body surface. Stan- of divalent cations [36] and is almost exclusively synthe- dardized areas of various body locations on healthy volunteers sized in the aerobic stationary phase in the periplasm of were rinsed with buffer to determine the concentration of psoriasin many Gram-negative bacteria [37], suggesting that these present on the skin. Data represent the median concentration of bacteria have a particular need to defend themselves psoriasin/cm2 in all people for each body site. Reproduced cour- tesy of [19]. against oxidative damage by endogenously generated reac- tive oxygen species [38]. Zn2+ as well as Cu2+ comparti- mentalisation could, therefore, be a critical step in bacter- The focal expression of psoriasin seen upon immunohis- ial defense in oxidative stress; psoriasin might exploit tochemistry indicates that local concentrations of psori- compartmentalisation in its antimicrobial activity. asin released to the skin surface may also depend on the A few other studies indicated involvement of S100 proteins body site. Indeed, in support of figure 3B, only small in innate host defense; calprotectin, a non-covalent het- amounts of psoriasin were found at areas with dry skin, erodimer of S100A8 and S100A9, shows Zn2+-sensitive, C. and high amounts at places where bacterial colonization albicans-selective biostatic activity [39, 40]. Furthermore, mainly occurs (fig. 4) [19]. a short C-terminal peptide fragment of S100A12 has Gram-negative bacteria-killing activities [41]. Many S100 proteins are expressed in epithelial tissues Psoriasin, the principal E. coli-cidal AMP (reviewed in [42]), supporting the hypothesis that these of healthy skin proteins are also involved in epithelial defense. In vitro, psoriasin shows antimicrobial activity preferen- tially against E. coli, with a LD90 (lethal dose that achieves a CFU reduction of 90%) near 0.5 mM [19]. Inducible epithelial antimicrobial peptides Whereas there is also bactericidal activity for P. aerugi- nosa and S. aureus at much higher concentrations (LD90 Although a number of inducible antimicrobial peptides >30 mM), psoriasin is far less bactericidal for the com- have been found in plants, in insects and also in cattle (re- mensal Staphylococcus epidermidis [19]. Psoriasin an- viewed in [43, 44]), it is not clear whether human epithe- timicrobial activity can be observed at neutral and acidic lia produce inducible AMPs. The first inducible AMPs pH, as well as at increased concentrations of salt [19], as found in cattle were the b-defensins ‘tracheal antimicro- has been seen at healthy skin surfaces and upon condi- bial peptide TAP’ [45] and structurally very similar ‘lin- tions, when sweat is evaporating. Inhibition of psoriasin gual antimicrobial peptide, LAP’ [46]. These peptides by a specific and neutralizing monoclonal antibody in were discovered in the course of asking why mammalian vivo and in vitro confirmed that psoriasin is the principal mucosa, despite hosting a constant epithelium-specific E. coli-bactericidal component of healthy skin [19]. Pre- microbial flora, is normally not infected. Both TAP and treatment of healthy skin with neutralizing psoriasin an- LAP can be induced in cultured bovine tracheal epithelial tibodies also resulted in an increase of commensals, sug- cells by heat-killed bacteria, bacterial lipopolysaccharide gesting that a permanent psoriasin ‘film’ at skin surfaces or TNF-a [45, 46]. In situ hybridisation studies revealed might also contribute to growth control of the commen- that LAP mRNA is widely expressed in numerous ep- sal flora. ithelia at a low level, but upregulated in infected skin and
  7. 7. Cell. Mol. Life Sci. Vol. 63, 2006 Review Article 475 lung [47]. These studies promised that inducible AMPs, no evidence so far for the presence of truncated forms of in particular b-defensins, could also exist in humans, hBD-2 in psoriatic scale extracts [our unpublished re- although until 1997 no experimental evidence was avail- sults], although hBD-2 is susceptible towards cathepsins able and although a non-inducible human b-defensin, [53]. A crude estimate indicated amounts of 10–50 mg/g hBD-1, had already been discovered (as a trace peptide psoriatic scales, suggesting concentrations of 2–10 mM in from human hemofiltrate obtained from patients with skin [29], which is in agreement with estimated concen- end-stage renal disease) [48]. trations of hBD-2 in IL-1a-induced epidermal cultures, With the observation that in cattle inducible epithelial where concentrations of 3.5–16 mM were estimated [54]. peptide antibiotics are only detectable in inflammatory Given that the aqueous intercellular space between the lesions, we hypothesised that a rich source of human ep- keratinocytes in the epidermis is small and hBD-2 is ex- ithelial inducible antimicrobial peptides should be in- pressed locally, it is very likely that local concentrations flammatory tissue material. The non-infectious inflam- of hBD-2 are much higher in vivo than the minimum bac- matory skin disease psoriasis is characterised by inflam- tericidal concentrations calculated from in vitro studies matory lesions and hyperproliferation of epidermis, [29, 54]. resulting in massive production of scales [49]. Surpris- ingly, patients with psoriasis rarely suffer from skin in- fections [50]. Therefore, it was suggested that within the hBD-2 is a preferentially Gram-negative skin lesions, antimicrobial peptides are generated, and bacteria-killing AMP therefore psoriatic scales might represent a rich source of human-inducible AMPs. Indeed, several novel human in- Natural, psoriatic scale-derived hBD-2 shows preferen- ducible AMPs could be purified from this human mater- tial antimicrobial activity against Gram-negative bacteria ial, among them the human b-defensins hBD-2 and hBD- such as nonmucoid and mucoid strains of P aeruginosa . 3 (reviewed in [29]). and E. coli (LD90, 10 mg/ml), less activity against C. albi- cans (LD90, 25 mg/ml) and only bacteriostatic activity against S. aureus at 100 mg/ml [51]. In insect cells, gen- Human b-defensin-2, hBD-2, the first human erated recombinant hBD-2 showed bacterial killing of E. inducible defensin coli, E. faecalis, P aeruginosa and S. aureus [29, 54–56]. . Synthetic hBD-2 exhibited antimicrobial activity against When acidic lesional psoriatic scale extracts were sepa- various periodontal bacteria [57, 58]. Activity of hBD-2 rated by heparin affinity chromatography followed by depends on ion composition and is sensitive to the con- preparative RP-HPLC, it is a striking observation that, centration of NaCl. The ability of hBD-2 to inhibit bacte- when compared with heel stratum corneum extracts, pso- rial growth diminishes when salt concentration is in- riatic scale extracts – despite the 14-fold lower amount of creased from 20 to 150 mM [55–60], suggesting that scale material – show much more bactericidal activity in hBD-2 will be unable to kill bacteria in serum or at skin all HPLC fractions and a different activity pattern (fig. 1). surface covered with evaporated sweat. Some bacteria, This observation supports the idea that lesional psoriatic e.g. Burkholderia cepacia, are surprisingly resistant to scales contain various inducible AMPs not present or not hBD-2 [61]. Most likely, alteration of the bacterial mem- present in such amounts in healthy stratum corneum. brane structure is important. It has been suggested that In order to selectively isolate AMPs (which strongly bind the spirochete Treponema denticola is resistant to hBD-2 to bacteria) from psoriatic scales, a scale extract was ap- because it lacks lipopolysaccharide (LPS) in the mem- plied to an E. coli affinity column generated by linking E. brane [62]. But other reasons could also be important: coli to sepharose [51]. By RP-HPLC of E. coli affinity group A Streptococci (GAS) producing streptococcal column-bound material, we then identified as a principal inhibitor of complement (SIC) have been reported to bactericidal component a b-defensin-like 4328-Da pep- be resistant towards a number of cationic antimicrobial tide eventually termed hBD-2 [51]. HBD-2 is – like other peptides, including hBD-2 by binding of SIC towards defensins – a cationic peptide. Although the shape and AMPs [63]. charge distribution of hBD-2 are similar to other de- fensins, X-ray analyses revealed that an additional a-he- lical region distinguishes this defensin topologically from HBD-2 is a locally induced AMP other mammalian a- and b-defensin structures. HBD-2 Epidermis of human skin comprises multiple kerati- forms non-covalent dimers with a quarternary octameric nocyte layers in various differentiation stages. Both the arrangement [52]. stratum corneum and the granular layer consist of termi- The mature 41 residues containing hBD-2- peptide repre- nally differentiated keratinocytes involved in superficial sents one of the major constituents of heparin-binding formation of the physical barrier [64] and contain so- proteins in lesional psoriatic scale extracts [29]. There is called lamellar bodies, which represent lipid-rich secre-
  8. 8. 476 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins tory granules. HBD-2 immunoreactivity is localized to for antimicrobial activity the disulfide bridges in hBD-3 the uppermost layers of the epidermis and/or stratum are not important [74]. corneum. On a subcellular level, hBD-2 is stored in lamellar bodies of stimulated keratinocytes of the spinous layer of the epidermis, suggesting that hBD-2 is released HBD-3 is a broad-spectrum peptide antibiotic with the lipidlike contents of lamellar bodies [65]. In- terindividual and site-specific differences in intensity of In contrast to hBD-2, HBD-3 is a broad-spectrum antimi- immunostaining were observed, and the pattern of pep- crobial peptide. Investigations with synthetic material re- tide localization was seen to be rather focal [66] – similar vealed salt-insensitive and potent activity against many po- to the staining pattern of psoriasin [19], suggesting that tentially pathogenic Gram-negative and Gram-positive hBD-2 is locally induced. HBD-2 is also seen in the strat- bacteria and fungi, including methicillin-resistant strains ified epithelia of the oral cavity as well as in gingival of S. aureus (MRSA) and vancomycin-resistant Entero- cells. Also, here it is predominantly localized in supra- coccus faecium (VRE) [69]. When in vitro activities of basal stratified epithelium and in upper epithelial layers hBD-3 alone or combined with lysozyme, metronidazole, [67]. Whereas hBD-2 transcript expression is frequently amoxicillin and chlorhexidine were investigated against higher in non-keratinised or ortho-keratinised epithe- several oral bacteria, hBD-3 showed bactericidal activity lium, immunostaining for hBD-2 is more intense in hy- against all of the bacterial species tested [75]. Synthetic perkeratinised epithelium [68]. Upon culture of oral epi- hBD-3 has been reported to efficiently kill Burkholderia thelial cells, hBD-2 peptide is seen only in differentiating cepacia [72]. However, a systematic study that tests bacte- involucrin-positive cells upon stimulation with proin- ricidal properties of synthetic hBD-3 against Gram-posi- flammatory cytokines or bacterial products [67]. tive cocci and Gram-negative fermentative and nonfer- mentative rods showed that all strains were highly or inter- mediately susceptible to hBD-3, except B. cepacia [76]. Human b-defensin-3 The mechanism by which hBD-3 kills S. aureus is not yet known. Ultrastructural analyses of hBD-3-treated S. au- Because hBD-2 is not an antibiotic for Gram-positive reus have revealed signs of perforation of the peripheral bacteria, it has been hypothesised that keratinocytes may cell wall, with explosion-like liberation of the plasma produce other inflammation-induced antimicrobial com- membrane within 30 min and bacteriolysis (fig. 5). The pounds that are active against S. aureus. In one study, morphological effects [69] resemble those seen when S. hBD-3 was identified in lesional psoriatic scales using a aureus is treated with penicillin [77]. biochemical approach, and based on protein sequence HBD-3 exhibits chemotactic activity on monocytes and data, it was cloned from keratinocytes [69]. Following the CCR6-transfected HEK 293 cells [72, 74]. This activity hypothesis that all defensin genes are located on chromo- some 8p22-23 and analysing the DNA sequence from a contig containing the hBD-2 gene also turned up the hBD-3 gene [70]. Two other, independent studies used bioinformatics to identify the hBD-3 gene [70–72]. HBD-3 is a highly basic antimicrobial peptide (pI 10.08). When cation exchange-HPLC analyses are performed with psoriatic scale extracts, hBD-3 elutes just before RNase 7, which represents the lastly eluting protein in a salt gradient [our unpublished results]. Unlike hBD-2 electrophoretic separation of hBD-3 in Tricine-urea, SDS polyacrylamide gel electrophoresis (PAGE) reveals a sin- gle band at 9 kDa corresponding to a noncovalent dimer, as confirmed by nuclear magnetic resonance (NMR), analyses [69, 73]. The connectivity of the cysteine bridges in most of the defensins known so far has been predicted, but not experimentally proven. Recent analyses of chemically synthesized topological analogs of hBD-3 with predefined disulfide connectivities, including the (presumably) native b-pairing, have revealed similar an- Figure 5. Morphology of hBD-3-treated S. aureus. Transmission tibiotic properties for differently folded hBD-3 species. electron micrographs of S. aureus incubated in 10 mM phosphate buffer for 2h (A) or treated with synthetic hBD-3 for 30 min (B) or Interestingly, hBD-3 species with substitutions of all Cys 2 h (C and D) are shown. Bars represent 0.1 mm. Reproduced cour- residues still are antimicrobially active, suggesting that tesy of [69].
  9. 9. Cell. Mol. Life Sci. Vol. 63, 2006 Review Article 477 strongly depends on the topology of the disulfide bridges monella typhimurium) and Gram-positive bacteria (Lyste- in hBD3. The absence of any disulfide bridge abolishes ria monocytogenes, S. aureus, Staphylococcus epider- the chemotactic activity of hBD-3, suggesting that a de- midis and vancomycin-resistant Enterococci) at micro- fined three-dimensional (3D) structure stabilized by disul- molar concentrations [87]. Although in a previous report fide bonding is required for productive binding to and ac- yeast species of Candida appear to be resistant to LL-37 tivation of CCR6 receptor. In contrast to chemotatic activ- [87], a recent study reported Candida-cidal activities of ity, the bactericidal activity of hBD-3 remains unaffected LL-37 [88]. by the absence of any disulfide bridge [74]. Unlike many defensins, LL-37 is active against several HBD-3 mRNA is expressed throughout the epithelia of bacteria in high salt conditions [87]. LL-37 is not highly many organs and in some non-epithelial tissues. Tran- selective to prokaryotic cells, whereas tripomastigotes of scripts were found in skin, tonsils, gingival keratinocytes, the protozoan parasite Trypanosoma cruzi and peripheral esophagus, trachea, placenta, adult heart, skeletal muscle leukocytes are susceptible in vitro to micromolar concen- and fetal thymus [69, 71, 72]. trations of this peptide, this cytotoxic activity is inhibited by serum components [89]. Apart from the C-terminal hCAP18 peptide LL-37 the The human cathelicidin hCAP-18/LL-37 cathelin domain of hCAP18 is also antimicrobially active as demonstrated with recombinant cathelin, which shows Cathelicidins are bipartite molecules with an N-terminal growth inhibition of S. aureus and S. epidermidis (MIC of cathelin domain (= cathepsin L inhibitor) and an antimi- 32 mM) [90]. crobial C-terminal domain. Humans apparently have only Investigations of hCAP18 processing in neutrophils indi- one cathelicidin gene, originally found to be expressed in cate that the propeptide is cleaved to generate the antimi- bone marrow [78–80]. Its product, hCAP-18, is present as crobially active peptide LL-37 in exocytosed material of one of the major proteins in the secondary (specific) these cells, liberated by proteolytic processing coincident granules of neutrophils [81]. This protein is stored as a with degranulation and secretion [91]. Experimental evi- precursor that requires additional processing to yield its dence shows that proteinase 3 is solely responsible for C-terminal antimicrobial peptide, LL-37. Although the processing the hCAP18 propeptide after exocytosis in neutrophil is the major cellular source of hCAP-18, it is neutrophils [91]. Further studies of hCAP18 have shown also expressed in other blood cells that are involved in in- cleavage of epididymal-derived hCAP18 in seminal flammatory and immune responses, including natural plasma by the prostate-derived protease gastricsin (pep- killer (NK) cells, gdT cells, B cells and monocytes [82]. sin C) in the presence of vaginal fluid of low pH generat- In addition, this gene is widely expressed in skin, epithe- ing antimicrobially active ALL-38, suggesting that pro- lia of the airways, mouth, tongue, esophagus, intestine, cessing of hCAP18 by gasticsin at low pH represents a cervix and vagina, in epididymis and in testis (reviewed mechanism to prevent infection after sexual intercourse in [83]). [92]. The detection of proteolytically processed catheli- Upregulation of this human cathelicidin gene has been cidin in skin keratinocyte extracts indicates also that ker- demonstrated in inflammatory skin disorders, whereas in atinocytes have the enzymatic machinery necessary for normal skin no induction was found [84]. In situ hybri- generation of hCAP18 C-terminal peptides. Analysis of dization and immunohistochemistry showed the tran- stratum corneum from normal and virally infected skin script and peptide to be located in keratinocytes through- has shown that cathelicidin present in this compartment is out the epidermis of the inflammatory regions [84]. On a processed to a mature 5-kDa form that is presumably LL- subcellular level, lamellar granules were identified to 37 or a smaller processed cathelicidin form such as KS- store hCAP-18/LL-37 [85]. The content of lamellar bod- 30 or RK-31 [25, 85, 93]. ies is released within 30 min after disruption of the per- The in vivo relevance of cathelicidins in cutaneous host meability barrier [86], a timing consistent with antimi- defense has been demonstrated in a mouse model. Mice crobial peptide production and release following injury. A deficient in the expression of the cathelicidin CRAMP further study showed that LL-37 is expressed in healthy (the mouse homolog to the human LL-37) were more sus- skin in eccrine glands and ductal epithelium [24]. ceptible to skin infections caused by group A Streptococ- cus (GAS). GAS mutants resistant to CRAMP resulted in more severe skin infections in normal mice [94]. LL-37 is a broad-spectrum AMP LL-37 represents a highly cationic peptide with a +6 net charge at neutral pH. It is a membrane-active amphi- Possible immune functions of skin-derived AMPs pathic a-helical peptide that shows a wide spectrum of antimicrobial activity. In vitro it inhibits the growth of Several reports suggest that a number of peptides and a variety of Gram-negative (E. coli, P aeruginosa, Sal- . proteins exhibit not just antimicrobial but also immuno-
  10. 10. 478 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins regulatory properties (for reviews see [95, 96]). b-De- lialisation. Moreover, decreased levels of LL-37 are asso- fensins were shown to be chemotactic for dendritic cells ciated with chronic wounds such as chronic ulcers [103]. and memory T cells by using the chemokine receptor LL-37 could contribute to cutaneous wound vascularisa- CCR6, which is shared by the chemokine MIP3a/LARC/ tion as well, because application of LL-37 results in an- CCL20 [97]. The b-Defensins hBD-1 and hBD-2 are se- giogenesis in two model systems. This peptide stimulates lectively chemotactic only for CCR6-expressing cells, in- proliferation of cultured human umbilical vein endothe- cluding iDCs and resting memory CD4CD45RO, as well lial cells (HUVECs) and causes endothelial sprouting in as some CD8 T lymphocytes. Although the dose neces- a hamster model system, biological activities that appear sary to elicit half-maximum responses in these cells was to be mediated by interaction of LL-37 with FPRL1 ex- found to be nearly 20-fold higher than the dose of the nat- pressed on endothelial cells [104]. ural CCL-20, which is in the nanomolar range, this find- ing might be of high relevance in vivo, because hBD-2 represents one of the major peptides in inflamed skin Regulation of AMP expression in skin such as psoriatic lesions, where it is present at micromo- lar concentrations [29]. Apart from being an attractant for Some antimicrobial peptides such as hBD-1 are virtually immature dendritic cells [74], hBD3 is also chemotactic constitutively expressed in skin keratinocytes. HBD-1 for monocytes, which do not express CCR6, suggesting it mRNA and hBD-1 immunoreactivity were found to be uses an additional chemotaxin receptor. On the basis of consistently expressed in skin samples from various body usage of chemokine receptors and the tertiary structural sites and were localized to the suprabasal keratinocytes, similarities between defensins and chemokines [98], sweat ducts and sebaceous glands of human skin [66, the defensins were considered to be ‘microchemokines’ 105], suggesting that hBD-1 represents a rather constitu- which act on cells of the adaptive immune system [99]. tively expressed antimicrobial peptide. The expression of Thus, b-defensins might also contribute to regulation of hBD-1 in suprabasal, more differentiated areas of the skin host adaptive immunity against microbial invasion under (malpighian layer, stratum corneum) suggest that differ- inflammatory conditions. entiation regulates hBD-1-expression. In vitro differenti- Defensins have also considerable immunological adju- ation of keratinocytes can be induced by increasing the vant activity, and linkage of b-defensins (or selected che- concentration of Ca2+, conditions which led to upregula- mokines) to an idiotypic lymphoma antigen has yielded tion of hBD-1 gene expression [106–108]. In contrast to potent antitumor vaccines [96, 99]. The functional over- many other antimicrobial peptides, gene expression of lap between defensins and chemokines is reinforced by hBD-1 is not markedly induced by proinflammatory cy- reports that some chemokines have antimicrobial activi- tokines such as IFN-g, IL-1b or TNF-a, or by bacteria ties [100]. However, although showing similarity in ac- such as P aeruginosa [108]. . tivity and overall tertiary structure, the evolutionary rela- tionship between defensins and chemokines remains to be determined. HBD-2 is induced by differentiation, IL-1 In contrast to b-defensins, LL-37/hCAP-18 is chemotac- and P. aeruginosa tic for neutrophils, monocytes and T cells in the micro- In contrast to hBD-1, hBD-2 expression is markedly in- molar range, but not for dendritic cells. Thus, these an- creased in the skin surrounding inflamed regions, but is timicrobial peptides have distinct, host-target cell spectra. not detectable in adjacent noninflamed regions, support- The chemotactic activity of LL-37 is mediated by formyl ing the hypothesis that hBD-2 is inducibly expressed in peptide receptor-like 1. The capacities of b-defensins and vivo. HBD-2-induction in skin keratinocytes only occurs cathelicidin LL-37 to mobilize various types of phago- in differentiated cells. Whereas basal cell layers of the cytic leukocytes, immature dendritic cells and lympho- epidermis and a basal cell carcinoma cell line (KB) al- cytes provide evidence for their participation in alerting, ways lack hBD-2-immunoreactivity [66], in cultured pri- mobilizing and amplifying innate and adaptive antimi- mary keratinocytes conditions that facilitate differentia- crobial immunity of the host (reviewed in [101]). tion – such as culture of keratinocytes in the presence of Besides its direct antimicrobial and chemotactic function, high concentrations of Ca2+– increases background hBD- LL-37 also has multiple roles as a mediator of inflamma- 2 expression [108, 109], suggesting that sufficient differ- tion, influencing diverse processes such as cell prolifera- entiation is a prerequisite of hBD-2 production in skin. tion and migration, immune modulation, wound healing, Genomic analyses of the hBD-2 promoter region revealed angiogenesis and the release of cytokines and histamine several putative transcription factor binding sites, includ- (reviewed in [102]). By promoting re-epithelialisation of ing nuclear factor kappa B (NFkB), activator protein 1 healing skin, LL-37 could play a role in repair of dam- (AP-1), AP-2 und NF-IL-6, which are known to be in- aged tissue. High levels of LL-37 are present upon volved in the induction and regulation of inflammatory wounding, and an anti-LL-37 antibody inhibits re-epithe- responses [60, 110]. For induction of hBD-2 in skin ker-
  11. 11. Cell. Mol. Life Sci. Vol. 63, 2006 Review Article 479 atinocytes and respiratory tract epithelial cells under in- ing that keratinocyte-derived IL-1 may also contribute to flammatory conditions, IL-1a, IL-1b or P aeruginosa . the induction of hBD-2. Thus, induction of hBD-2 may have proven to be the most effective in vivo-relevant stim- predominantly occur through IL-1 signalling [112]. In uli, whereas in these cells other bacteria and cytokines vivo the indirect way via mononuclear phagocyte-derived such as TNF-a or IL-6 have little or no ability to induce IL-1 appears to be the most relevant induction pathway in hBD-2 [51, 54, 108, 111–113]. Recently, the Th1 cell- cutaneous inflammation, because keratinocytes can only derived cytokine IL-22 has been identified as a potent produce the inactive IL-1b precursor, and the pool of stimulus for hBD-2 and hBD-3 expression in keratino- stored IL-1a is not released [113]. cytes, where expression of the IL-22 receptor was upreg- ulated by IFN-g [114]. Direct bacterial induction of hBD-2 by a novel pathway? Toll-like receptors: major pathogen-recognition- A number of reports indicate that bacteria can also di- receptors for hBD-2-induction? rectly stimulate epithelial cells for production of hBD-2 – The induction of host antimicrobial molecules following a pathway that might represent an ancient, cytokine-inde- binding of pathogen components to pathogen pattern pendent pathway possibly also used by invertebrates and recognition receptors such as CD14 and the Toll-like re- plants. In oral epithelial cells (or cell lines) of different ceptors (TLRs) is a key feature of innate immunity. Ep- origin, hBD-2-transcription is induced directly by various ithelia represent important environmental interfaces bacteria (and its cell wall products), e.g. P aeruginosa, . where LPS recognition pathways may be important in E. coli, S. aureus, S. epidermidis and Fusobacterium nu- inducing hBD-2. Indeed, in tracheobronchial epithelial cleatum (an oral commensal bacterium), but not or far cells, hBD-2 is induced by a LPS preparation, ultimately less by Streptococcus pyogenes and Porphyromonas gin- activating NFkB through a CD14-dependent mechanism givalis (an periodontal pathogen) [123–125]. In a study [111]. This might be possible also in skin keratinocytes, with respiratory tract epithelial cells a clinical isolate of a although this is still an open question. Induction of hBD- mucoid phenotype of P aeruginosa turned out to be the . 2 via TLR-4 was seen in some studies [115–118], most powerful inducer of hBD-2 in these cells [60] and in whereas others observed no hBD-2-induction in ker- primary skin keratinocytes [our unpublished results]. The atinocytes with LPS [112]. In other studies, no TLR4 ex- observation that only this mucoid P aeruginosa strain in- . pression was seen in normal keratinocytes, but it was seen duces hBD-2 at a low bacteria/epithelial cell ratio support in HaCaT keratinocytes [119, 120]. TLR-2 may also me- the hypothesis that apart from known common TLR-acti- diate hBD-2 induction. HEK293 cells transfected with vating ligands such as LPS, peptidoglycan and flagella TLR-2 respond to stimulation with bacterial lipoprotein filament structural protein, most likely other, yet-to-be- by hBD-2 production [121]. In one study, commercial determined ‘pathogen-associated molecular patterns LPS preparations could stimulate epidermal ker- (PAMPs)’, are responsible for hBD-2 induction. Kinetic atinocytes to produce hBD-2 and IL-8, and the LPS re- analyses of bacteria-induced hBD-2 production indicate sponse was inhibited with monoclonal antibody (mAb) involvement of multiple distinct signalling pathways. One specific for TLR2, but not for CD14 or TLR4. Repurified study has revealed that NFkB is neither essential nor suffi- LPS and lipid A did not stimulate epidermal ker- cient for hBD-2 induction. E.g. hBD-2 regulation by F nu- . atinocytes, whereas peptidoglycan from Gram-positive cleatum occurs via p38 and Jun-N-terminal kinase (JNK), bacteria and yeast cell wall particle induced b-defensin-2 while phorbolester (which is a powerful b-defensin inducer and IL-8 production. Thus, epidermal keratinocytes ex- in keratinocytes [108]) induces hBD-2 via the p44/42 ex- press functional TLR2, but not CD14 or TLR4. And the tracellular signal-regulated kinase pathway [124]. ‘LPS’ response of epidermal keratinocytes shown in sev- eral previous studies was suggested to be mediated by TLR2-dependent recognition of non-LPS bacterial com- Growth factors are major hBD-3 inducers ponents contaminating commercial LPS preparations Although hBD-3 represents an inducible b-defensin, its [122]. expression is regulated in a different manner than expres- sion of hBD-2. In skin-derived keratinocytes as well as in gingival keratinocytes, hBD-3 mRNA is moderately in- IL-1 is the major endogenous inducer of hBD-2 duced by TNF-a, whereas INF-g is a more powerful The expression of hBD-2 in skin keratinocytes was pre- hBD-3-inducing cytokine [69, 126, 127]. Growth factors viously found to be IL-1 dependent [113]. Only the IL-1R of major importance in wound healing, insulin-like antagonist inhibited the hBD-2 expression in epidermal growth factor-I (IGF-I) and transforming growth factor-a culture that has been stimulated either with supernatants (TGF-a), induce the expression of hBD-3 in human ker- of LPS-treated mononuclear cells or LPS alone, suggest- atinocytes. This induction occurs by transactivation of the
  12. 12. 480 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins epithelial growth factor (EGF) receptor [112]. In this a consensus vitamin D response element (VDRE) in the process the EGF receptor is activated by membrane- hCAP-18/LL-37 promoter that was bound by the vitamin bound ligands such as TGF-a released through a metallo- D receptor (VDR). Interestingly, induction of the hCAP- protease-dependent process, which may lead to a rapid 18/LL-37 homolog in murine cells was not observed. localized activation of the receptors by low ligand con- This was due to the absence of the VDRE in the murine centrations. Interestingly, proinflammatory cytokines hCAP-18/LL-37 homolog promoter [132]. such as IL-1 and IL-6 may modulate the transactivation of the EGF receptor, leading to a further increased expres- sion of hBD-3 during inflammation, even though these Implications of AMPs in skin diseases cytokines did not induce hBD-3 expression directly [112]. The observation that AMPs are expressed in the upper- Like b-defensins in cattle and hBD-2, hBD-3 is also in- most parts of the epidermis in skin may suggest a protec- duced when epithelial cells come into contact with bacte- tive function of these effector molecules as part of a ria [69, 72, 128]. This induction occurs at various levels, ‘chemical barrier’ in skin infection and inflammation. It suggesting dependency on the virulence state, a hypothe- is therefore interesting to speculate that a defect in this sis supported by the observation that a mucoid clinical iso- protective system may lead to recurrent local infections, late of P aeruginosa, as seen for hBD-2 [60] and RNase- . which in turn may lead to inflammation. On the other 7 [26], is the strongest microbial inducer of hBD-3 [69]. hand, it is possible that overexpression of AMPs in skin may lead to increased protection against skin infection. This hypothesis is supported by a disease concomitance Downregulation of b-defensin expression seen in patients with psoriasis, a non-infective inflamma- Whereas several studies reveal mechanisms of b-defensin tory skin-disease, which unexpectedly rarely suffer from induction in epithelial cells by various stimuli, there is infectious skin diseases [50]. Our biochemical analysis of only little information about downregulation of hBD in- antimicrobial peptides in psoriatic scale extracts have duction. Whereas in one study LPS-dependent hBD-2 in- shown the presence of hBD-2, RNase 7 and psoriasin as duction in a human airway cell line can be inhibited by principal antimicrobial proteins. Moreover, hBD-3 and corticosteroids [129], another study failed to identify in- several other as yet not structurally characterized antimi- hibitory effects of dexamethason upon hBD-2 induction crobial peptides were identified (fig. 1) [29]. in bronchial epithelial cells, but observed inhibition of In contrast to psoriasis, patients with atopic eczema often hBD-3 induction [130]. In skin keratinocytes all-trans suffer from skin infection with S. aureus. In acute and retinoic acid dose-dependently downregulated hBD-2, chronic lesions of these patients, a decreased expression hBD-3 and hBD-4 induction by various stimuli, suggest- of hBD-2, hBD-3 and LL-37 has been observed [127, ing that these therapeutics may have yet unrecognised 133]. It is believed that the expected induced expression side effects by downregulating innate epithelial defense of antimicrobial peptides in inflamed atopic lesions is in- effector molecule expression [108]. hibited as a result of elevated Th2 cytokines in atopic skin. Indeed, it could be shown that Th2-cytokines such as IL-4 and IL-13 suppress the cytokine-mediated induc- Regulation of cathelicidin LL-37 expression tion of hBD-2 and hBD-3 [127, 133]. The lack of these in- The cathelicidin hCAP18/LL-37 also represents an in- ducible antimicrobial peptides, however, could be also in- ducible AMP that is produced by skin keratinocytes in terpreted by the lack of major inducers in skin, such as vivo upon wounding and in inflammation [84, 103]. In an IL-1b, or the rather selective endogenous ‘defensin in- in vitro model, the highest hCAP18 levels are attained at ducer’ IL-22 [114]. 48 h post-injury, declining to pre-injury levels upon Skin-derived antimicrobial peptides may also play a role wound closure [103]. Apart from infiltrating neutrophils, in other skin diseases. In lesional and perilesional epithe- hCAP18 is detected in the epithelium migrating over the lium of patients with acne vulgaris, a marked upregula- wound bed, suggesting that hCAP18/LL-37 induction tion of hBD-2 has been observed that might be the con- in keratinocytes depends on wound-healing processes sequence of inflammation [134]. An upregulated ex- [103]. In support of this hypothesis, it was found that pression of hBD-2 was also seen in superficial folliculitis IGF-I induces the expression of hCAP-18/LL-37 in cul- [135]. The expression of LL-37 in keratinocytes of tured human keratinocytes [131]. patients with condyloma acuminatum and verruca vul- Another major inducer of hCAP-18/LL-37 might be garis was found to be increased, suggesting a role of 1,25-Dihydroxyvitamin D3. 1,25-Dihydroxyvitamin D3 this cathelicidin in cutaneous papillomavirus infection and three of its analogs induced expression of hCAP-18/ [93]. Furthermore, LL-37 is able to inactivate vaccinia LL-37 [132]. The induction was observed in immortal- virus, which has implications for eczema vaccinatum ized keratinocytes and several other cells. It occurred via [136].
  13. 13. Cell. Mol. Life Sci. Vol. 63, 2006 Review Article 481 Decreased levels of antimicrobial peptides and proteins produced within the living epidermis upon stimulation. have been found to be associated with burns and chronic The stimulus could be wounding, which may include dis- wounds. In skin, hBD-2 is only produced by kerati- ruption of the physical barrier, or contact with pathogenic nocytes, suggesting that lack of keratinocytes in chronic bacteria or bacterial products and/or endogenous proin- ulcera or dead keratinocytes, as in burn wounds, may re- flammatory cytokines and growth factors. sult in a lack of hBD-2 [137, 138]. This suggests a host de- A number of peptides and proteins with antimicrobial ac- fense defect within the burn wound and a therapeutic role tivity of different protein families have been described in for antimicrobial peptides in the management of burn skin. The relative importance of each in protecting skin wounds [138]. Indeed, a transient cutaneous adenoviral from infection or actively defending against infection gene therapy with hCAP-18/LL-37 was found to be effec- is currently speculative. One would expect that skin-de- tive for the treatment of burn wound infections [139]. rived antimicrobial peptides in vivo should be present in These observations allow us to speculate that other recur- amounts that have been seen to be microbicidal in vitro. rent skin infections may be associated with a dysregula- This has been demonstrated in part by in vivo experi- tion of antimicrobial peptide and protein production ments in mice for cathelicidins [94] and in humans for caused by lack of induction, inhibition by Th2 cytokines psoriasin [19]. or by pathogenic bacteria that may either inactivate these Although there is currently no report describing the rela- peptides [140] or interfere with induction. tive abundance of currently known antimicrobial peptides and proteins of different classes in healthy and inflamed skin, a rough estimate of proteins present in skin extracts General conclusions and future directions after RP-HPLC by silver staining, together with ESI-MS analyses for verification of its structure, indicates, that in Findings of the past 10 years clearly show, that apart from healthy skin-derived stratum corneum extracts psoriasin the physical defense shield with the formation of stratum represents the most abundant AMP, followed by RNase 7 corneum, skin epidermal cells also have the capacity to and lysozyme (fig. 6a). Whereas hBD-2 was isolated only mount a ‘chemical barrier’. This barrier includes pre- in low amounts, hBD-3 as yet wasn’t detected. hCAP-18/ formed compounds present at the uppermost layers of the LL-37 as well as proteolytic fragments thereof have not skin (including the stratum corneum), as well as newly yet been identified in the extract, suggesting that LL-37 synthesised antimicrobial peptides and proteins that are and its fragments represent rather a trace AMP in healthy Figure 6. SDS-PAGE analyses of proteins in extracts of stratum corneum and psoriatic scales, after RP-HPLC. 10 ml-aliquots of HPLC frac- tions (as shown in fig. 1) were analysed in a Tricine/SDS/urea-PAGE-system [69], and proteins and peptides were silver stained. Note in healthy pooled stratum corneum extract (A) psoriasin (P), RNase 7 (R) and lysozyme (L) as predominant antimicrobial proteins, confirmed by ESI-MS-analyses (data not shown). The 4-kDa band of a hydrophobic peptide eluting behind psoriasin does not show identity with the cathelicidin LL-37 and belongs to an unrelated peptide. In psoriatic scale extracts (B) psoriasin (P) represents again the most abundant an- timicrobial protein. The second most abundant antimicrobial peptide is HBD-2 (H2), followed by lysozyme (L) and RNase 7 (R). Again, in HPLC fractions where LL-37 or truncated products would be expected, neither silver-stained bands nor ESI-MS analyses revealed a pep- tide that would correspond to LL-37.
  14. 14. 482 J. M. Schröder and J. Harder Antimicrobial skin peptides and proteins skin that is present in amounts only detectable with sen- sitive immunochemical methods. The faint band of a 4- kDa peptide, which is reproducibly seen upon RP-HPLC fractions with elution time corresponding to LL-37, shows upon ESI-MS analyses an exact mass of 4037, which does not fit with the mass of LL-37 or any mass of the LL-37 fragments found in sweat [25], suggesting that it belongs to a different peptide. HPLC analyses of psori- atic scale extracts with sensitive silver staining of protein bands (fig. 6b) and identification of the currently known AMPs by ESI-MS analyses revealed again psoriasin as the most abundant AMP, now followed by hBD-2 and RNase 7, lysozyme, HNP-1-3 and elafin [141]. Although HNP-1-3 may originate from infiltrating neutrophils (leukocytes, which are very common in active psoriasis lesions), the absence of myeloperoxidase and lactoferrin in these extracts make it possible that HNP-1-3 also orig- inates from keratinocytes [141]. HBD-3 can also be de- tected, but it is not found as a major peptide [141]. Again, LL-37 has not been identified with these methods [141]. Thus, one may conclude that host defense of healthy hu- man skin is mediated, in part, by a ‘chemical barrier’, which consists in several AMPs that are expressed at dif- ferent parts of the epidermis, which comprises various Figure 7. Putative model of a protective function of keratinocyte- keratinocyte layers with multiple states of differentiation. derived antimicrobial peptides for microwounds to prevent inflam- Some of these AMPs are constitutively produced in the mation. Bacterial growth on intact skin with an intact physical bar- uppermost areas of the epidermis, where they are stored rier will be controlled by bacteriostatic and bactericidal compounds at the skin surface (A, left). Once the physical barrier is disturbed in lamellar bodies and then secreted upon final differen- [e.g. loss of stratum corneum (A, center)], bacteria (or bacterial tiation (being now present at the surface of fully differen- products) have access to living epidermal keratinocytes, which may tiated keratinocytes that finally will become stratum cor- now produce and secrete inducible antimicrobial peptides. When neum). microorganisms induce proinflammatory cytokines and come into contact with keratinocytes and immune cells in deeper areas of the Psoriasin, the most abundant and secreted antimicrobial skin, inflammatory cells are recruited, and inflammation begins (B). protein of healthy skin, is bactericidal by essential trace element zinc deprivation [19]. We suspect that at skin sur- faces, microbial growth is mainly controlled by essential trace element deprivation, in particular also iron (by si- lished reports. However, our own, unpublished observa- derophore-binding lipocalin 2) [131], and physical re- tions indicate the existence of such compounds. moval from body surfaces by desquamation. In the case of extensive wounding of the epithelium or a In microwounds, which can occur from physical stress by high load of pathogenic bacteria, which cannot be con- disruption of the physical barrier, the skin may use an (in- trolled by epidermis-derived AMPs, inflammatory reac- ducible) antimicrobial defense strategy with induction of tions would be initiated by epidermal recognition of path- keratinocyte-derived AMPs but not proinflammatory me- ogenic bacteria, which may mediate the production of diators, as outlined in figure 7. On intact skin with an in- proinflammatory cytokines, initially by keratinocytes, tact physical barrier, bacterial growth will be controlled and then followed by immune cells, eventually resulting by bacteriostatic and bactericidal compounds (e.g. psori- in leukocyte tissue infiltration of the skin (fig. 7B). The asin and RNase 7) at the skin surface (left side of fig.7A). model shown in figure 7A would occur without any visi- Once the physical barrier is disturbed (middle portion of ble signs and might therefore never even have attracted fig. 7A), bacteria (or bacterial products) have access to notice. living epidermal keratinocytes. These may now produce The focal expression of various AMPs in healthy skin and secrete inducible antimicrobial peptides, which, without any visible signs of inflammation suggests that, when present in sufficient amounts or activity, would stop indeed, conditions may exist which cause AMP induc- infection – without inflammation. This model, however, tion in the absence of proinflammatory cytokines or would implicate the existence of microbial products that growth factors produced during wound healing and that induce antimicrobial peptides without inducing proinflam- microbes present at skin surfaces may facilitate antimi- matory mediators – a hypothesis not yet proven by pub- crobial peptide induction without inflammation. A re-
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