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Originally published online as doi:10.1189/jlb.0704409 on January 3, 2005

Published online before print January 3, 2005
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(Journal of Leukocyte Biology. 2005;77:476-486.)
© 2005 by Society for Leukocyte Biology

Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins

Jürgen Harder1 and Jens-Michael Schröder

Clinical Research Unit at the Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Germany

1 Correspondence: Clinical Research Unit, Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 7, D-24105 Kiel, Germany. E-mail: jharder{at}dermatology.uni-kiel.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
Patients with psoriasis, a chronic, hyperproliferative and noninfectious skin disease, suffer surprisingly fewer cutaneous infections than would be expected. This observation led us to the hypothesis that a local "chemical shield" in the form of antimicrobial proteins provides psoriatic skin with resistance against infection. We subsequently began a systematic analysis of in vitro antimicrobially active proteins in psoriatic-scale extracts. A biochemical approach with rigorous purification and characterization combined with antimicrobial testing identified a number of mostly new human antibiotic peptides and proteins. In this review, we will focus on the most prominent antimicrobial proteins in psoriatic-scale extracts, which we identified as the S100-protein psoriasin, human ß-defensin 2 (hBD-2), RNase 7, lysozyme, and human neutrophil defensin 1–3. Apart from these cutaneous, antimicrobial proteins, only a few others, including hBD-3, have been characterized. A great number of minor antimicrobial proteins await further structural characterization.

Key Words: innate immunity • psoriasis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
Skin is in permanent contact with microorganisms but usually free of signs of infection. It is commonly believed that the physical barrier with its lipids as well as the slightly acidic pH near 5.0 is responsible for this phenomenon. However, a number of questions have not yet been answered convincingly. For example, why is it that the number and composition of the cutaneous microflora remain almost constant under physiologic conditions (near 105 microorganisms/cm2), despite the presence of many favorable conditions (nutrients such as amino acids, lipids, fatty acids, and salts and an advantageous ambient temperature) for the growth of microorganisms? Such conditions in vitro may lead to a bacteria doubling-time of as low as 20–30 min. The activity of neutrophils or macrophages cannot explain this phenomenon, as healthy skin does not contain an abundant population of these phagocytes. An attractive hypothesis that explains the nearly constant number and composition of the physiologic skin flora is that of an innate epithelial chemical defense shield. Such an epithelial chemical defense is common in multicellular organisms such as plants and insects, which do not have an adaptive immune [1 ].

In 1987, Michael Zasloff [2 ] discovered magainins in frog skin. These are a family of 3–4 kDa antimicrobial peptides lacking cysteine bridges with an {alpha}-helical structure in solution. They have been shown to exhibit potent antimicrobial activity against bacteria and fungi [2 ]. Subsequently, a number of other skin-derived antimicrobial peptides were discovered in different frog species [3 ]. Later studies revealed that mammals are also able to protect their cutaneous surfaces by expressing antimicrobial proteins. Antimicrobial peptides of the cathelicidin family are found in a variety of mammals: For example, PR-39 is expressed on the cutaneous surface of the pig [4 ], and mouse skin expresses CRAMP (a homologue to the human LL-37) [5 ]. The in vivo relevance of antimicrobial peptides in cutaneous host defense has been demonstrated in a mouse model. Mice deficient in the expression of CRAMP were more susceptible to skin infections caused by group A Streptococcus (GAS), and GAS mutants resistant to CRAMP produced more severe skin infections in normal mice [6 ].

To gain more insight into the relevance of antimicrobial proteins in human skin defense, we began with a biochemical approach using high-performance liquid chromatography (HPLC) to systematically identify the principal antimicrobial proteins participating in cutaneous defense. We hypothesized that lesional psoriatic skin might be a rich source of antimicrobial proteins, as patients with psoriasis suffer fewer cutaneous bacterial infections than would be expected [7 ]. As antimicrobial peptides of metazoan origin are mostly cationic [1 ], we used a heparin-affinity column to selectively trap cationic antimicrobial proteins. Heparin-bound proteins of psoriatic scales were then further separated by reversed-phase (RP)-HPLC. Figure 1 shows a typical preparative RP-HPLC chromatogram of psoriatic-scale peptides eluted from a heparin-affinity column. Each fraction (30 µl) was lyophilized, dissolved in 5 µl 0.01% (v/v) aqueous acetic acid, and tested for microbicidal activity against E. coli using the radial diffusion assay system [8 ] or microdilution test systems [9 ]. The use of bacteria other than E. coli in the biological read-out system revealed different antimicrobial profiles (Fig. 2 ). This observation could be explained by the presence of multiple, different antimicrobial peptides, which exhibit different antimicrobial spectra—from more microbe-specific to broad-spectrum antimicrobial peptides and proteins.



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  		Figure 1. Psoriatic-scale extracts contain a broad spectrum of antimicrobial proteins. Psoriatic-scale extracts were applied to a heparin-affinity column, and the bound material was further separated by C8 RP-HPLC (RP-8). Bound proteins were eluted using a linear gradient of increasing acetonitrile concentrations and recorded at 215 nm. The resulting chromatogram is shown. Aliquots (30 µl) of HPLC fractions were tested for bactericidal activity against Escherichia coli using the radial diffusion assay system. The diameter of the clearing zones is shown (bars). The identified antimicrobial proteins are indicated: E, Elafin; H1, human neutrophil defensin 1–3 (HNP-1–3); H2, human ß-defensin 2 (hBD-2); H3, hBD-3; L, lysozyme; P, psoriasin; R, RNase7.

 


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  		Figure 2. Microorganism-dependent antimicrobial activity of psoriatic-scale extracts, which were applied to a heparin-affinity column, and the bound material were further separated by RP-8. Bound proteins were eluted using a linear gradient of increasing acetonitrile concentrations and recorded at 215 nm. The resulting chromatogram is shown. Aliquots (30 µl) of HPLC fractions were tested against the indicated microorganisms in a microdilution assay system. HPLC fractions with antimicrobial activity are indicated by the bars. S. aureus, Staphylococcus aureus; P. aeruginosa and P. aerug., Pseudomonas aeruginosa; C. albicans, Candida albicans; A. baumannii, Acinetobacter baumannii; Prop. acnes, Propionibacterium acnes; Strep. pyogenes, Streptococcus pyogenes; Strep. pneumoniae, Streptococcus pneumoniae.

 
It has been shown that antimicrobial peptides can demonstrate synergy with each other [10 , 11 ]. Therefore, it should be kept in mind that evaluating the antimicrobial activity of a single antimicrobial protein will most likely underestimate its overall effectiveness in an in vivo situation where the synergistic interaction of diverse antimicrobial proteins takes place and increases the total antimicrobial activity.

In the following paragraphs, we describe those antimicrobial peptides and proteins that have been detected and purified from psoriatic-scale extracts so far and represent quantitatively dominating antimicrobial peptides.


   hBD-2
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
When the fractions from a RP-8-HPLC of psoriatic scale-derived proteins eluted from a heparin affinity column were screened for bactericidal activity against E. coli, various fractions revealed strong activity (Fig. 1) . Electrospray ionization-mass spectrometry (ESI-MS) analyses of an antimicrobial fraction eluting at 40% acetonitrile (Fig. 1) revealed the presence of a 4328-Da peptide, corresponding to the 41 residues form of hBD-2 [12 ], which exhibits antimicrobial activity primarily against Gram-negative bacteria such as E. coli and P. aeruginosa. It is also active against the yeast C. albicans but not against Gram-positive S. aureus, against which it only demonstrates bacteriostatic activity at very high concentrations [12 13 14 15 ].

hBD-2 represents one of the major constituents of heparin-binding, psoriatic-scale proteins. A crude estimate by using ultraviolet absorbance of purified material indicated a mean of 10–50 µg/g psoriatic scales. Assuming that the density of skin extract is 1 g per ml, the concentrations of hBD-2 can be estimated at 2–10 µM, which is in agreement with a previous report that estimated the concentration of hBD-2 in interleukin (IL)-1{alpha}-induced epidermal cultures to be 3.5–16 µM [15 ]. These concentrations are similar to those used in in vitro antimicrobial studies of hBD-2. Given that the aqueous intercellular space between the keratinocytes in the epidermis is small and keeping in mind that hBD-2 is expressed locally, it is likely that the local in vivo concentrations of hBD-2 are much more higher than the minimum bactericidal concentrations calculated from in vitro studies. The capacity of hBD-2 to kill bacteria in vivo has been demonstrated in a mouse gene therapy study with hBD-2-transfected tumor cells. Following a bacterial infection, mice with hBD-2-bearing tumors bore fewer viable bacteria than controls [16 ].

In normal skin, hBD-2 immunoreactivity is localized to the uppermost layers of the epidermis and/or stratum corneum [17 ]. This is in concordance with recent studies showing an up-regulation of hBD-2 in cultured primary keratinocytes, which had been induced to differentiate by high calcium concentrations [18 , 19 ]. HPLC analysis revealed the presence of hBD-2 in extracts as well as in the supernatants of cultured, primary keratinocytes (own unpublished results). This suggested that hBD-2 is stored in and secreted by keratinocytes. Whereas secretion of hBD-2 by epithelial cells has been described in various reports, it is not clear whether hBD-2 may also act as an intracellular, antimicrobial peptide, acting against invading microorganisms [20 , 21 ]. A recent finding demonstrated that in IL-1{alpha}-stimulated epidermal keratinocytes, hBD-2 is stored in the lamellar bodies [22 ]. These vesicles release their contents during differentiation of the keratinocytes and after barrier disruption, into the intercellular space of the outer epidermal layers.

Expression of hBD-2 in keratinocytes is effectively induced by IL-1{alpha}, IL-1ß, or P. aeruginosa [12 , 15 , 23 , 24 ]. The mechanism of hBD-2 induction by contact with microorganisms is not yet clear. It is most likely mediated by pathogen-associated molecular patterns (PAMPs), which bind to pattern recognition receptors on the surface of keratinocytes. Eligible receptors could be Toll-like receptors (TLRs), which are structurally related to the Drosophila Toll receptor and have been reported to be expressed in keratinocytes [25 26 27 28 ].

Whereas hBD-2 is up-regulated in psoriatic lesions, its expression is significantly less or even absent in the lesional skin of patients with atopic dermatitis [29 ]. One reason for the lack of hBD-2 induction in atopic skin could be low amounts of hBD-2-inducing, proinflammatory cytokines or hBD-2-inducing microbial PAMPs. Another explanation could be the presence of T helper cell type 2 (TH2) cytokines (e.g., IL-4, IL-13) in this disease. TH2 cytokines have been reported to inhibit tumor necrosis factor {alpha} (TNF-{alpha})-mediated hBD-2 induction in vitro. Therefore, their predominance in atopic lesional skin might block the induction of antimicrobial proteins such as hBD-2 and hBD-3 [29 , 30 ]. One may speculate that a diminished expression of antimicrobial proteins might be one reason why patients with atopic dermatitis often suffer from recurrent skin infections.

Epithelial cells other than keratinocytes also express hBD-2. However, nonepithelial cells exhibit much lower levels of expression of hBD-2 or do not express it at all. Recent investigations showed a limited expression of hBD-2 mRNA in monocytes/macrophages and dendritic cells. The induction of higher levels of expression can only be achieved in monocytes and alveolar macrophages by interferon-{gamma} (IFN-{gamma}) and/or lipopolysaccharide in a dose- and time-dependent manner [31 ]. Although immunocytochemistry demonstrates immunoreactive hBD-2 in freshly isolated blood monocytes and alveolar macrophages, the in vivo relevance of this observation is not clear [31 ]. These cells are likely a minor or even negligible source of hBD-2 in psoriatic skin.


   hBD-3
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
The third member of the hBD family, hBD-3, can be detected in RP-8-HPLC fractions off heparin affinity column-bound, psoriatic scale-derived proteins (Fig. 1) . The hBD-3 protein was first isolated from psoriatic scales [32 ], and the hBD-3 gene was also independently discovered based on bioinformatics and functional genomic analyses [33 , 34 ]. hBD-3 was found to be a powerful peptide antibiotic exhibiting a broad spectrum of antimicrobial activity against various Gram-negative and Gram-positive bacteria as well as fungi at low micromolar concentrations. It is interesting that hBD-3 was also revealed to be active against multiresistant S. aureus and vancomycin-resistant Enterococcus faecium [32 , 33 , 35 36 37 ].

Nuclear magnetic resonance (NMR) solution structure analysis of hBD-3 and the other ß-defensins has revealed a similar tertiary structure with a short helical segment preceding a three-stranded, antiparallel ß-sheet. The surface charge density of each defensin is markedly different; the surface of hBD-3 is significantly more basic. NMR data suggest that hBD-3 forms a symmetrical dimer through strand ß2 of the ß-sheet [38 ]. This hypothesis is further supported by electrophoretic separation in Tricine gels in the presence of urea, where the mobility of hBD-3 was found to correspond to that of a dimer (10 kDa) [32 ]. The capacity of hBD-3 to form stable dimers together with its high positive charge might be an explanation as to why it exhibits stronger antimicrobial activity than hBD-2 (especially against Gram-positive bacteria such as S. aureus) [38 ]. Structural characterization of several ß-defensins has confirmed that the presence and location of disulfide bonds are conserved within the family (Cys1-Cys5, Cys2-Cys4, Cys3-Cys6) [38 ]. It is interesting that the disulfide connectivity does not seem to be important for antimicrobial activity [39 ].

Keratinocytes are the likely source of hBD-3 in psoriatic lesions. Tissue studies have revealed that hBD-3 mRNA is expressed throughout the epithelia of many organs and in some nonepithelial tissues. Skin, gingival keratinocytes, tonsils, esophagus, trachea, placenta, adult heart, skeletal muscle, and fetal thymus comprise the major hBD-3 mRNA-expressing tissues [19 , 32 , 33 ]. Several studies also revealed that proinflammatory cytokines induce the expression of defensins: When comparing the induction of hBD-2 and hBD-3 in keratinocytes at a transcriptional level, IL-1 was found to be the strongest inducer of hBD-2, whereas IFN-{gamma}, which does not induce hBD-2, is the most powerful hBD-3-inducing cytokine [19 , 30 , 33 ]. As had been observed in relation to hBD-2, contact with bacteria was found to induce the expression of hBD-3 in keratinocytes and tracheal epithelium [19 , 32 , 33 ]. The mechanism of hBD-3 induction through contact with bacteria has not yet been elucidated. The bacterial products, cellular receptors, and signal cascades involved in all remain to be determined.


   RNase 7
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
{alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
Screening of RP-8-HPLC fractions off heparin affinity column-bound, psoriatic scale-derived proteins revealed a major polar 20-kDa protein (by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses). This was found to exhibit antimicrobial activity against E. coli (Fig. 1) . ESI-MS analyses of this HPLC fraction revealed a mass of 14,546.06 Da, corresponding to ribonuclease 7 (RNase 7), a highly basic protein (pI: 9.80), which bears four disulfide bridges [40 ]. RNase 7 has sequence similarity with members of the human RNase A superfamily, among them, RNase 2 [eosinophil-derived neurotoxin (EDN)], RNase 3 [eosinophil-cationic protein (ECP)], and RNase 5 (angiogenin). In contrast to the inducible ß-defensins, RNase 7 is highly abundant in healthy skin and has been identified as one of the principal cationic proteins of healthy human skin [40 ].

The antibacterially active RNase A member ECP is found in eosinophils and neutrophils [41 ] and apparently not in epithelial cells. In contrast, RNase 7 is expressed in various cells of epithelial origin including trachea, tonsils, pharynx, tongue, and salivary glands, as well as in renal cells and the thymus [40 ]. It is not yet known whether leukocytes also produce RNase 7; however, neither neutrophils nor monocytes contain RNase 7 transcripts (our unpublished results).

RNase 7 exhibits broad-spectrum antimicrobial activity against Gram-negative bacteria (P. aeruginosa and E. coli), Gram-positive bacteria (S. aureus and P. acnes), and the yeast C. albicans. It is remarkable that the number of colony-forming units of P. aeruginosa and E. coli decreases by five orders in the presence of RNase 7 at a concentration of 2–5 µM. Of particular interest, a similar efficacy against a Vancomycin-resistant strain of E. faecium was seen at a concentration of only 20 nM [40 ]. Therefore, it may be said that RNase 7 represents on a per-molar base, the most potent and efficacious human antimicrobial protein known so far.

Other members of the RNase A superfamily, in addition to RNase 7, have been reported to have antimicrobial properties. ECP was shown to exhibit antimicrobial activity against S. aureus and E. coli, with activity (50% lethal dose, 0.5–1 µM) [42 ] similar to that observed for RNase 7. Human angiogenin (RNase 5) exhibits microbicidal activity at low micromolar concentrations against Streptococcus pneumoniae and the yeast C. albicans [43 ].

RNase 7 is constitutively produced in healthy human skin. The amount isolated per gram of psoriatic-scale material (10–25 µg/g) was found to be higher than that isolated from normal stratum corneum extracts (4–8 µg/g). It was therefore suggested that the expression of RNase 7 might be inducible, despite the high level of constitutive expression in healthy skin. The proinflammatory cytokines IL-1ß, IFN-{gamma}, and to a lesser degree, TNF-{alpha} have been shown to induce RNase 7 mRNA expression [40 ]. As has been found in the case of hBD-2 and hBD-3, it was shown that bacteria can also up-regulate the expression of RNase 7 [40 ]. As of yet, the molecular mechanism of bacteria-mediated RNase 7 induction in keratinocytes remains unclear. As mentioned above, it has been reported that TLRs are expressed in keratinocytes. As the signal transduction pathways for TLRs and IL-1 have many common components, it could be speculated that activation of TLRs through bacteria and their products might induce RNase 7. However, this hypothesis remains to be proven.

The concurrent, antimicrobial activity and RNase activity of RNase 7 gave rise to the speculation that the enzymatic activity is required for the antimicrobial activity. It has been shown in the case of ECP that RNase activity is not essential for antibacterial activity [44 ]. It remains to be determined whether this also proves to be the case for RNase 7.

The exact role and physiological function of all known members of the human RNase A superfamily are not yet apparent. The antiviral activity of EDN and ECP suggests that these eosinophil-derived RNases may contribute to an eosinophil-mediated antimicrobial and antiviral defense system [45 ]. Angiogenin (RNase 5) exhibits angiogenic as well as microbicidal activity and may contribute to systemic responses to infection [43 ]. It remains to be determined whether RNase 7 exhibits physiological functions other than that of an epithelial antimicrobial protein.


   {alpha}-DEFENSINS HNP-1–3
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
N-terminal protein sequencing of an antimicrobial-active fraction eluting at low acetonitrile concentrations (Fig. 1) revealed the sequence to be AXYXRIPAXI, which is identical to the amino acid sequence of the neutrophil {alpha}-defensin HNP-1. (X stands for a blank seen for cysteines as residues.) ESI-MS analyses revealed the presence of three peptides with masses at 3442, 3371, and 3486 Da, which correspond to the masses of the three {alpha}-defensins HNP-1, HNP-2, and HNP-3, respectively (Fig. 3 ). The amino acid sequences of HNP-1, -2, and -3 are identical except for a single amino-terminal amino acid, which is altered in HNP-3 [46 ]. As a result of their high abundance in neutrophilic granulocytes, they were termed HNPs. HNP-1–3 constitute greater than 30% of the protein content of azurophil granules and exhibit a wide spectrum of antimicrobial activity against various bacteria, fungi, and viruses [46 ]. The antimicrobial features of HNP-1–3 together with their high abundance in neutrophilic granulocytes indicate that these antimicrobial peptides participate in the nonoxidative killing of phagocytosed bacteria in neutrophils. The important role of neutrophil {alpha}-defensins in killing of phagocytosed microbes has been demonstrated in patients suffering from specific granule deficiency (SGD) [47 ]. Polymorphonuclear leukocytes (PMN) from patients with SGD display a nearly complete deficiency of defensins. SGD patients suffer from frequent and severe bacterial infections, suggesting that the profound deficiency of PMN microbicidal defensins may contribute to the clinical manifestations of these disorders [47 ].



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  		Figure 3. Identification of human {alpha}-defensins HNP-1–3 in psoriatic scales. A RP-8-HPLC of heparin-bound, psoriatic-scale extracts was performed (Fig. 1) . A HPLC fraction with antimicrobial activity, which eluted at low acetonitrile concentration, was analyzed by quadrupol-time-of-flight-ESI-MS. The raw data revealed two peak series of threefold and fourfold charges (upper panel). The processed data revealed masses of 3442.3, 3371.5, and 3486.1 Da (lower panel), which corresponded to the masses of HNP-1, HNP-2, and HNP-3, respectively.

 
To date, it is believed that HNP-1–3 are mainly (or exclusively) produced in neutrophils and not in other cells. The detection of HNP-1–3 in other cell preparations has been explained as a consequence of contamination with neutrophils, a situation recently found in the case of CD8 T cells [48 , 49 ]. Therefore, it would be assumed that the high amounts of HNP-1–3 found in psoriatic-scale extracts may originate from infiltrating neutrophils. There are, however, some findings that contradict the hypothesis that neutrophils are the only source of HNPs in lesional psoriatic skin: First, epidermal neutrophil infiltrates are generally seen in psoriatic skin only in the acute phase. The scale material we used for our analyses was collected mainly from patients with chronic plaque psoriasis, where neutrophils are seen uncommonly in lesional skin. Second, if the HNP-1–3 found in lesional psoriatic-scale extracts is derived from neutrophils, it would also be expected to find high amounts of other neutrophil-derived proteins. Typical marker proteins include those for neutrophil azurophilic and specific granules, such as myeloperoxidase and lactoferrin. However, we detected only minimal (close to negligible) levels of immunoreactive myeloperoxidase and lactoferrin or their fragments. This supports the hypothesis that chronic plaque-psoriasis scale extracts contain only minimal amounts of proteins derived from contaminating neutrophils. Future experiments should elucidate a possible alternative cellular source of HNP-1–3 in chronic plaque-psoriasis scales. It has yet to be determined whether HNP-1–3 are also present in extracts of healthy skin and whether primary keratinocytes might be a cellular source of these peptide antibiotics.


   ELAFIN
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
ESI-MS analyses of an antimicrobial fraction eluting between HNP-1–3 and hBD-3 (Fig. 1) revealed the presence of two peptides with masses of 5999 Da and 6226 Da. These peptides were identified as the skin-derived serine protease inhibitor Elafin (also known as skin-derived antileukoproteinase) [50 , 51 ] and its 59 amino acid (aa)-containing precursor. Further purification of Elafin and the 59 aa precursor by cation exchange HPLC followed by C2C18-RP-HPLC led to a loss of bactericidal activity against E. coli in Elafin-containing HPLC fractions. This suggested that natural Elafin and the 59 aa precursor are not antimicrobially active against E. coli in vitro. It has recently been shown that a recombinant form of Elafin (containing 57 aa), which is identical with the major form isolated from psoriatic scales, exhibited only growth-inhibiting but not bactericidal properties against three different P. aeruginosa strains [52 ]. In concordance with the natural Elafin isolated from psoriatic scales, no bactericidal activity against E. coli of this recombinant 57 aa-containing form could be detected [52 ]. In another study, it was reported that the full-length, 95 aa-containing synthetic Elafin precursor exhibited killing activity against P. aeruginosa (93% killing by 2.5 µM Elafin) and weak activity against S. aureus (48% killing by 25 µM Elafin) [53 ]. The antimicrobial activity of the N-terminal domain (residues 1–50) and the C-terminal domain (residues 51–95) showed far less activity [53 ]. These studies suggest that full-length Elafin (95 residues) is required to achieve optimal bacterial killing, whereas the short, processed form of Elafin seems to play no important role as an antimicrobial protein. However, as we did not find the unprocessed 95-aa form of Elafin in psoriatic skin, it remains to be determined whether this form contributes to cutaneous bacterial defense. The observation that Elafin expression in keratinocytes and other epithelial cells is induced upon contact with bacteria and proinflammatory cytokines strengthens the hypothesis that Elafin may serve as a cutaneous defense factor that is active in inflammatory conditions [52 , 54 ]. It is interesting that the potential of Elafin to act in vivo against microbial infection has been demonstrated in a mouse model, where adenoviral-mediated transfection of the lung with Elafin protected against injury and infection as a result of P. aeruginosa [55 ]. It remains to be shown whether its anti-infective activity in vivo originates from its antimicrobial or from its protease-inhibiting properties.


   LYSOZYME
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
C2C18-RP-HPLC (results not shown) readily separated a protein that coeluted with hBD-2 upon RP-8-HPLC of psoriatic-scale extracts. This protein demonstrated antimicrobial activity against E. coli and S. aureus in the radial diffusion assay system. ESI-MS analyses revealed a pure protein showing a mass of 14,693 Da, which corresponds to the mass of lysozyme, a finding confirmed by Edman sequencing. In many independent experiments in our laboratory using a different, natural skin-derived lysozyme, we could confirm that lysozyme indeed exhibits antimicrobial activity at low micromolar concentrations against E. coli and S. aureus (unpublished results). Lysozyme has been detected in many body fluids and epithelia including skin [56 ]. Furthermore, lysozyme is present in the primary and secondary granules of neutrophils, where it participates in the killing of ingested microorganisms [57 ]. The cellular source of the psoriatic scale-derived lysozyme is not clear; however, as was discussed above, the absence of other neutrophil-derived proteins suggests that the purified lysozyme comes from keratinocytes rather than from neutrophils.

Although the muramidase activity (cleaveage of the glycosidic bond between N-acetylmuramic acid and N-acetyl glucosamine in the bacterial peptidoglycan) of lysozyme has been implicated in its antibacterial activity (especially against Gram-positive bacteria), it has also been shown that the bactericidal potency of lysozyme is not only a result of muramidase activity but also of its cationic and hydrophobic properties [58 , 59 ]. Furthermore, it has been demonstrated that the bactericidal effect of lysozyme is synergistically enhanced with hBD-2 and hBD-3 [36 , 60 ]. The relevance of lysozyme as an antimicrobial factor in vivo has been demonstrated in transgenic mice models. Increased production of lysozyme in transgenic mice led to enhanced bacterial killing in the lung [61 , 62 ]. In constrast, lysozyme-deficient mice showed increased susceptibility to Klebsiella pneumoniae infection [61 , 62 ].


   PSORIASIN (S100A7)
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 
Analysis of RP-8-HPLC fractions for bactericidal activity against E. coli identified prominent E. coli-killing activity eluting at high acetonitrile concentrations (Fig. 1) . The protein responsible for this bactericidal activity represented the principal component of the heparin affinity column-bound, psoriatic scale-derived proteins. Biochemical characterization of the protein revealed that it is identical to the NH2 terminally acetylated form of the S100 protein psoriasin (accession number M86757). Psoriasin is also known as S100A7, psoriasin 1 (accession numbers NM_002963 and BC034687), and formerly, as the S100A7 variant c [63 ]. Psoriasin was originally discovered in psoriatic skin lesions by a proteomics strategy as a new Ca2+-binding S100 protein of unknown biological function [64 ]. Of interest, keratinocytes appear to secrete psoriasin as their main E. coli-specific bactericidal compound in vitro and in vivo. This may explain the natural resistance of skin against colonization by E. coli [65 ].

S100 proteins are believed to mediate a variety of functions in eukaryotic cells including differentiation, cell-cycle progression, intracellular Ca2+ signaling, and cytoskeletal membrane interactions as well as playing a role in leukocyte chemotaxis [66 , 67 ]. A few studies have also indicated that S100 proteins may play a putative role in innate host defense: A Ca2+-binding heterodimeric complex of two S100 proteins (MRP8 and MRP14), known as calprotectin, exhibits selective biostatic activity at high concentrations against C. albicans [68 , 69 ]. Furthermore, a short C-terminal peptide fragment of calgranulin c, a minor calgranulin in neutrophils, has demonstrated bactericidal activity against Gram-negative bacteria [70 ]. Psoriasin, which is produced by keratinocytes but not by neutrophils should therefore be added to the list of antimicrobial S100 proteins.


   OTHER HUMAN SKIN-DERIVED ANTIMICROBIAL PEPTIDES AND PROTEINS
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
CONCLUSION
 REFERENCES
 
In the following section, we will discuss some other antimicrobial proteins of potential importance for cutaneous defense. Using our biochemical approach, we have not yet isolated these antimicrobial proteins from psoriatic-scale extracts. This could be a result of a low abundance of these proteins in psoriatic skin, susceptibility to protease degradation, or in case of dermcidin, exclusive production in sweat glands and an anionic characteristic that precludes binding to the heparin column.

LL-37/hCAP-18
An antimicrobial peptide of potential importance in human skin defense is the cathelicidin LL-37/hCAP-18 [71 ]. Cathelicidins contain an N-terminal cathelin domain and an antimicrobial C-terminal domain. Humans have only one cathelicidin gene. Its product, hCAP-18, is present in the secondary (specific) granules of neutrophils, and its C-terminal antimicrobial peptide, LL-37, is liberated as one of the major constituents through cleavage by proteinase 3. LL-37/hCAP-18 is expressed by many cells, including epididymis, spermatids, various lymphocytes, epithelial cells, and in particular, keratinocytes (for review, see ref. [72 ]). Keratinocytes express hCAP-18 at sites of inflammation [73 ]. Mature LL-37 peptide as well as processed forms of LL-37 are present in sweat [74 , 75 ]. Although our biochemical approach has not yet identified LL-37 and its processed forms as major antimicrobial proteins in psoriatic scales, immunodot blot analysis has detected increased amounts of LL-37 in skin biopsies of psoriasis patients [29 ]. In contrast to psoriasis, decreased expression of LL-37 (as well as hBD-2, hBD-3) has been detected in skin biopsies of patients suffering from atopic dermatitis [29 , 30 ]. These data suggest that the low expression of antimicrobial peptides may, at least in part, account for the increased susceptibility of patients with atopic dermatitis to skin infection. The relevance of cathelicidins in cutaneous host defense has been demonstrated in a mouse model. Mice deficient in the expression of the cathelicidin CRAMP (the mouse homologue to the human LL-37) were more susceptible to skin infections caused by GAS, whose mutants resistant to CRAMP resulted in more severe skin infections in normal mice [6 ].

Dermcidin
Dermcidin is a novel, anionic antimicrobial peptide, which is produced and secreted exclusively by human eccrine sweat glands [76 , 77 ]. Dermcidin is proteolytically cleaved, resulting in dermcidin 1 (DCD-1), a 47-aa-containing peptide that exhibits antimicrobial activity against a variety of bacteria including E. coli, Enterococcus faecalis, S. aureus, as well as C. albicans. DCD-1 is found in sweat in antimicrobially efficacious concentrations of 1–10 µg/ml. Its antimicrobial activity is not affected by the low pH value and high salt concentrations of human sweat. This indicates that sweat glands may have a function in the innate immune responses of the skin by secreting antimicrobial proteins such as DCD-1 and the cathelicidin LL-37.

Adrenomedullin
Adrenomedullin is a pluripotent, 52-aa peptide with numerous physiological roles, including vasodilation, renal homeostasis, hormone regulation, neurotransmission, and growth modulation (for review, see ref. [78 ]). Adrenomedullin mRNA and protein expression have been detected in keratinocytes of the epidermis and hair follicles, as well as in cells of the eccrine and apocrine sweat glands and sebaceous glands [79 ]. Adrenomedullin exhibits high antimicrobial activity against E. coli [minimum bactericidal concentration (MBC), 1.5 µg/ml] and moderate activity against S. aureus (MBC, 25 µg/ml) [80 ]. It is interesting that adrenomedullin seems to be effective in killing P. acnes (MBC, 25 µg/ml). This suggests that adrenomedullin may play a role in the skin disease Acne vulgaris, where there is an association with hypercolonization by P. acnes. Adrenomedullin is secreted by keratinocytes in vitro (35 fmol/106 cells/12 h), but it is not clear whether concentrations within the antimicrobial range of 0.1–10 µg/ml are reached in vivo [80 ].

Neutrophil gelatinase-associated lipocalin (NGAL)
The NGAL, also called human neutrophil lipocalin, is a 25-kDa protein initially isolated from the specific granules of human neutrophils [81 , 82 ]. It has been shown that NGAL exhibits bacteriostatic activity through its ability to bind bacterial ferric siderophores [83 ]. It has been shown that the addition of only 5 µM NGAL to E. coli led to a 20-fold growth inhibition [83 ]. The low expression of NGAL in healthy skin is increased in skin disorders, which are characterized by dysregulated epithelial differentiation. These include psoriasis, pityriasis rubra, and squamous cell carcinoma [84 ]. Expression of NGAL in human keratinocytes is up-regulated by IL-1ß, insulin growth factor-I, and transforming growth factor-{alpha} [85 ].

hBD-1
Several reports indicate that hBD-1 is produced and secreted at microbicidal concentrations from epithelia of the genito-urinary tract [86 87 88 ]. hBD-1 protein, as yet, has not been isolated from human skin. However, using in situ hybridization and immmunohistochemistry, hBD-1 mRNA and peptide were found to be expressed consistently in skin samples from various body sites and localized to the suprabasal keratinocytes, sweat ducts, and sebaceous glands of human skin [17 , 89 ]. In vitro induction of keratinocyte differentiation by increasing the concentration of calcium was associated with up-regulation of hBD-1 gene expression [19 , 90 , 91 ]. This may explain why the hBD-1 peptide shows a stronger expression in more differentiated terminal layers of human skin (Malpighian layer, stratum corneum) [17 ]. In contrast to hBD-2 and hBD-3, gene expression of hBD-1 in keratinocytes is not markedly induced by proinflammatory cytokines such as IL-1ß, TNF-{alpha}, and IFN-{gamma} or by bacteria such as P. aeruginosa [19 ].

Only a few studies have investigated the antimicrobial spectrum of hBD-1. Recombinant and natural forms of hBD-1 (36, 39, and 42 aa) exhibit salt-sensitive antimicrobial activity against various laboratory and clinical strains of E. coli at micromolar concentrations (0.3–10 µM). The 36-aa form was found to retain antimicrobial activity even in normal urine [88 ]. In addition to activity against E. coli, Singh et al. [13 ] reported that concentrations of 1–10 µg/ml of a hBD-1 preparation derived from a recombinant baculovirus are able to kill P. aeruginosa. The dose required to kill 50% of P. aeruginosa was found to be 1 µg/ml and 100 ng/ml for hBD-1 and hBD-2, respectively [13 ]. In other studies, only minor antimicrobial activity of native hBD-1 was detected, resulting in the killing of only a few microorganisms [87 ]. As of yet, no studies have reported activity of hBD-1 against pathogenic Gram-positive bacteria such as S. aureus.

As also shown for hBD-2, hBD-1 exhibits chemotactic activity for cells stably transfected with the CC chemokine receptor 6 (CCR6). As CCR6 is preferentially expressed by immature dendritic cells and memory T cells, these data suggest that hBD-1, like hBD-2, may function through interaction with CCR6 to recruit immature dendritic cells and memory T cells to cutaneous sites of microbial invasion [92 ].

hBD-4
The fourth member of the hBD family, hBD-4, was initially identified by screening the human genome database [93 ]. Synthetic hBD-4 revealed antimicrobial activity at micromolar concentrations against P. aeruginosa and Staphylococcus carnosus. Inducible gene expression of hBD-4 was detected in primary keratinocytes [19 ], but so far, nothing is known about the expression of the hBD-4 peptide in human skin. Attempts have to date failed to isolate the hBD-4 peptide from psoriatic-scale extracts as well as from healthy human skin-derived stratum corneum extracts. Therefore, further investigations need to be performed to determine the levels of expression of the hBD-4 peptide in skin and the role of hBD-4 in the chemical skin defense system.

Chemokines as antimicrobial factors
More recently, some chemokines have been identified as antimicrobially active peptides. Screening of a total of 30 human chemokines identified 17 human chemokines that exhibit antimicrobial activity in vitro. These include macrophage-inflammatory protein-3{alpha} [MIP-3{alpha}; CC chemokine ligand 20 (CCL20)], regulated on activation, normal T cell expressed and secreted (RANTES; CCL5), and truncated forms of the platelet-derived chemokine connective tissue activating peptide III but not IL-8 [94 , 95 ].

As MIP-3{alpha} (CCL20) and RANTES (CCL5) have been reported to be secreted by keratinocytes [96 ] and to be up-regulated in psoriatic keratinocytes [97 , 98 ], we investigated the contribution of these chemokines to the total antimicrobial activity of the psoriatic-scale extracts. Purification of the chemokines was performed using the same procedure as used for purification of cationic antimicrobial peptides. We screened RP-8-HPLC fractions of heparin-bound, psoriatic scale-derived peptides and proteins for immunoreactive RANTES (CCL5) and MIP-3{alpha} (CCL20) by the use of specific enzyme-linked immunosorbent assays. We were able to identify immunoreactive RANTES and MIP-3{alpha} in RP-8-HPLC fractions that eluted close to RNase 7 or hBD-3 (our unpublished results). The amounts present in HPLC fractions were estimated to be in a range of 20–50 ng/ml in separate RP-HPLC fractions. Total amounts that were present in these fractions were 20- to 50-fold less than has been determined to be necessary to achieve antimicrobial activity in vitro [95 ]. Therefore, we concluded that these chemokines do not contribute to the in vitro antimicrobial activity present in HPLC fractions and are therefore also unlikely to contribute to in vivo direct (leukocyte-independent), antimicrobial activity.

hBD-2 exhibits chemokine receptor CCR6-mediated chemotactic properties for immature dendritic cells and memory T lymphocytes [92 ]. Although the natural CCR6 ligand MIP-3{alpha} (CCL20) is nearly 20-fold more potent than hBD-2, the amounts of hBD-2 produced by skin keratinocytes are much higher than that estimated for MIP-3{alpha} in psoriatic skin (ref. [99 ] and our unpublished results), supporting a possible role for hBD-2 as a CCR6 ligand in vivo.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
REFERENCES
 
We have identified psoriatic-scale extracts as a unique source of human-inducible antibiotic peptides and proteins, among them, psoriasin, hBD-2, and RNase 7, as by far, the quantitatively dominating antimicrobial peptides. These peptides and proteins can also be found at much lower concentration in healthy skin, where they are expressed focally, thereby suggesting local induction. In addition to these major antimicrobial peptides and proteins, there is now ample evidence (as shown by the high number of RP-8-HPLC fractions with antimicrobial activity; Figs. 1 and 2 ) that human skin has the capacity to produce additional, as-yet uncharacterized antimicrobial peptides and proteins with more or less broad-spectrum activity against various bacteria and fungi. Future work will focus on these peptides and proteins and the mechanism of their induction and relevance in vivo.


   ACKNOWLEDGEMENTS
 
This work has been supported by Deutsche Forschungsgemeinschaft, SFB 617. We thank Dr. Paul A. J. Russo for help with the manuscript.

Received July 18, 2004; revised October 26, 2004; accepted December 10, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 hBD-2
 hBD-3
 RNase 7
 {alpha}-DEFENSINS HNP-1-3
 ELAFIN
 LYSOZYME
 PSORIASIN (S100A7)
 OTHER HUMAN SKIN-DERIVED...
 CONCLUSION
 REFERENCES
 

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