Lecture

1. © 2005 Nature Publishing Group http://www.nature.com/natureimmunology ARTICLES
Antimicrobial psoriasin (S100A7) protects human skin
from Escherichia coli infection
Regine Glaser1,3, Jurgen Harder1,3, Hans Lange2, Joachim Bartels1, Enno Christophers1 &
¨ ¨
Jens-Michael Schroder1
¨
Human healthy skin is continuously exposed to bacteria, but is particularly resistant to the common gut bacterium Escherichia
coli. We show here that keratinocytes secrete, as the main E. coli–killing compound, the S100 protein psoriasin in vitro and
in vivo in a site-dependent way. In vivo treatment of human skin with antibodies to psoriasin inhibited its E. coli–killing
properties. Psoriasin was induced in keratinocytes in vitro and in vivo by E. coli, indicating that its focal expression in skin may
derive from local microbial induction. Zn2+-saturated psoriasin showed diminished antimicrobial activity, suggesting that Zn2+
sequestration could be a possible antimicrobial mechanism. Thus, psoriasin may be key to the resistance of skin against E. coli.
Epithelial surfaces harbor a complex and abundant ensemble of high concentrations of E. coli (such as anogenital and neonatal skin)
microbes, which surprisingly rarely lead to infections. The stratum are usually not infected with this gut bacterium.
corneum of skin, which is a desiccated and nonviable layer of flattened Here we describe the identification of the 11–kilodalton (kDa)
epidermal cells, acts as a physical barrier that separates the body from metal ion–binding S100 protein psoriasin (S100A7) as the principal
the environment. This barrier is difficult for microorganisms to E. coli–killing antimicrobial protein of healthy human skin present on
penetrate and explains at least in part the rare infection rate. the skin surface and focally expressed in healthy skin keratinocytes.
Human skin is capable of mounting its own battery of innate The finding that bacterial challenge induces psoriasin indicates that
immune effector molecules produced by the uppermost epidermal cell psoriasin is involved as a local host defense molecule protecting healthy
layers of human skin. Some of these molecules, including lysozyme1, skin, and possibly other body epithelia, from infection by E. coli.
secretory leukoprotease inhibitor or antileukoprotease2, RNase 7
(ref. 3) and dermcidin4, are constitutively produced by healthy skin RESULTS
keratinocytes1–3 or sweat duct cells4. Other antimicrobial peptides, Psoriasin kills E. coli
such as human b-defensin 2 (HBD-2)5 and HBD-3 (ref. 6) and the Certain species of bacteria die rapidly on the surface of human skin,
cathelicidin LL-37 (ref. 7), are locally induced in skin keratinocytes whereas other species do not10. This distinction can be readily
after infection or inflammation. The functional importance of anti- demonstrated by exposure of the fingertips to either E. coli or
microbial peptides in innate cutaneous host defense is demonstrated Staphylococcus aureus in a humid atmosphere for 30 min; the surface
by the increased susceptibility to infection with Group A streptococcae of a Petri dish filled with nutrient agar is then touched with the bare
of mice with knockout of the mouse ortholog of the human fingertips and the dish is incubated overnight. An S. aureus print of
cathelicidin gene CAMP8. Deficiency in expression of the antimicro- the fingertips is produced, but no viable E. coli are transferred
bial peptides HBD-2 and LL-37 in skin lesions of patients with atopic (Supplementary Fig. 1 online).
dermatitis may account for the high occurrence of Gram-positive skin To address the hypothesis that compounds with preferential E. coli
infections in those patients9. bactericidal activity are secreted, we analyzed fluids obtained after
Although human skin is capable of producing various antimicrobial washing the skin (skin-washing fluids) and extracts of the stratum
proteins with a broad spectrum of activity, many different bacterial corneum of healthy people for antimicrobial activity and consistently
strains survive on the skin of the fingertips, whereas Escherichia coli is found high titers of E. coli–killing activity from both sources (data not
rapidly killed10. We therefore hypothesized that healthy human skin shown). Most of the antimicrobial activity bound to a heparin-affinity
might constitutively produce a defense chemical that could preferen- column. Separation of stratum corneum extracts by preparative C8
tially and very effectively control the growth of E. coli. Such a factor reversed-phase high-performance liquid chromatography (RP-HPLC)
would explain why E. coli is rarely found to colonize skin11 and would and analysis of RP-HPLC fractions for bactericidal activity identified
offer an explanation as to why skin regions that are often exposed to prominent E. coli–killing activity eluting at 60% acetonitrile (Fig. 1a).
1Clinical Research Unit of the Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Germany. 2Institute of Biochemistry, University of Kiel,
Germany. 3These authors contributed equally to this work. Correspondence should be addressed to J.-M.S. (jschroeder@dermatology.uni-kiel.de).
Published online 28 November 2004; doi:10.1038/ni1142
NATURE IMMUNOLOGY VOLUME 6 NUMBER 1 JANUARY 2005 57

2. ARTICLES
Further purification with ‘micro-cation’
exchange HPLC produced three 280-nm a b 0.4 80
Acetonitrile (%)
absorbing peaks (Supplementary Fig. 2 2 100 0.3
Acetonitrile (%)
online). All fractions had an 11-kDa band 40
A280
by tricine SDS-PAGE analysis (data not 50 0.2
0
shown), suggesting a single compound.
0.1
Quadrupole time-of-flight electrospray- 0
ionization mass spectrometry analyses 0.0
showed a molecular mass at 11,366 Da for 0 10 20 30 40
A215
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
Time (min)
the principal antimicrobial protein (Supple- 1
mentary Fig. 2 online) and, in addition,
many ions corresponding to 11-kDa proteins
antimicrobial activity
with slightly different masses. We achieved
Relative
final purification of the principal E. coli–
killing protein by C2-C18 ‘micro’ RP-HPLC
(Fig. 1b, top and middle). Tricine SDS-
0
PAGE analysis of the antimicrobially active
15 20 25 30 0 10 20 30 40
fraction identified a protein migrating at
Time (min) Time (min)
11 kDa (Fig. 1b, bottom). Edman sequencing
failed in all experiments, indicating the pre-
kDA
sence of blocked N termini.
antimicrobial activity
14
Peptide mapping with subsequent Edman
Relative
sequencing, mass mapping and mass spectro- 10
metry sequencing of the principal E. coli–
killing protein (Supplementary Fig. 2 online)
identified a protein sequence that fitted
unambiguously with the predicted sequence
15 20 25 30 MW
of an N-terminally acetylated form of the Time (min)
Ca 2+-binding S100 protein psoriasin, also
called S100A7 or psoriasin 1 or, formerly, Figure 1 Identification of psoriasin as an E. coli–killing protein. (a) Top, preparative C8 RP-HPLC of
S100A7 variant c12. pooled healthy human heel stratum corneum extracts. Bottom, E. coli–killing activity of HPLC fractions.
Mass mapping experiments, mass spectro- The fraction containing the highest titer of E. coli–killing activity (top, downward arrow) was separated
metry sequencing and Edman sequencing of further. (b) Top, final purification of E. coli–killing psoriasin by C2-C18 RP-HPLC. Middle and bottom,
the bactericidal activity coincides with the main ultraviolet-absorbing peak (middle) giving a single
peptide fragments of minor 11-kDa proteins, band at 11 kDa by SDS-PAGE (bottom). MW, molecular weight marker.
which were found in pooled stratum cor-
neum extracts but not in washing fluid,
indicated the presence of post-translationally modified psoriasin phosphate buffers in which the pH varied between 5.5 and 7.4 or the
forms, gene variants12 and mutated psoriasin forms (data not sodium chloride varied between 10 and 150 mM and found that
shown), which need to be further analyzed biochemically and biolo- psoriasin was active at low and neutral pH and at salt concentrations
gically. The mean recovery of the principal 11,366-Da psoriasin variant present in sweat, which were estimated to be in the range between 18
was estimated by ultraviolet peak integration to be 43 mg psoriasin per and 60 mM NaCl14 (Fig. 2b,c).
gram of normal heel stratum corneum (n ¼ 8).
Zn2+ inhibits the antimicrobial activity of psoriasin
Psoriasin preferentially kills E. coli To investigate how psoriasin kills E. coli, we examined the morpho-
To study the spectrum of bacteria that are susceptible to psoriasin, we logical changes of E. coli strain ATCC 35218 using transmission
investigated the purified natural psoriasin for microbicidal activity electron microscopy after exposure to high bactericidal concentrations
using the microdilution antimicrobial test system13. Psoriasin showed of psoriasin, but found no signs of cell wall damage (Supplementary
potent microbicidal activity against several strains of E. coli, including Fig. 3 online), indicating that psoriasin kills E. coli by a mechanism
American Type Culture Collection (ATCC) strain 35218 (90% lethal different from that of many cationic antimicrobial peptides15.
dose, 0.5 mM; Fig. 2a) and ATCC strains 11303, 12021, 12022 Calprotectin, a Ca2+-binding heterodimeric complex of the S100
and 11775 and the clinical isolates 3402 and B 336 (data not proteins MRP8 and MRP14 (also known as calgranulin A and B,
shown). There was far less antimicrobial activity against S. aureus respectively)16,17, shows Zn2+-sensitive C. albicans–selective biostatic
(ATCC 6538), Pseudomonas aeruginosa (ATCC 15442) and Staphylo- activity18,19. To test the possibility that the S100 protein psoriasin kills
coccus epidermidis (ATCC 14990; Fig. 2a). We detected no activity E. coli by sequestering Zn2+, we preincubated psoriasin with micro-
against a clinical isolate of Candida albicans at concentrations up to molar concentrations of ZnSO4 before the antimicrobial assay and
17.6 mM (data not shown). found a dose-dependent inhibition of its antimicrobial activity at
To further support the idea of a possible antimicrobial function for nearly equimolar or higher concentrations of Zn2+ (Fig. 2d). Although
psoriasin in vivo and to show that psoriasin is also active at skin psoriasin is a Ca2+-binding protein12, preincubation with other
surface conditions, we did antimicrobial assays in buffers that were bivalent ions such as Ca2+ or Fe2+ neither inhibited nor increased
similar in pH and ionic composition to that of skin surfaces or sweat antimicrobial activity when tested in the microdilution assay system
(10 mM phosphate buffer, pH 7.4, served as control). We tested (Supplementary Fig. 3 online).
58 VOLUME 6 NUMBER 1 JANUARY 2005 NATURE IMMUNOLOGY

3. ARTICLES
a 100 b 106
E. coli
105 Control
E. coli (CFU / ml)
P. aeruginosa
Psoriasin
Killing (%)
(50 µg / ml)
50 S. aureus 104
S. epidermidis
103
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
0 102
0.1 0.4 1.6 6.4 25.6 5.5 6.0 7.4
pH
Psoriasin (µM)
c d
4 100
E. coli (× 106 CFU / ml)
Control
3 80
Psoriasin Psoriasin
(50 µg / ml)
10 µg / ml
Killing (%)
2 60
20 µg / ml
1 40
0 20
0 10 20 40 60 100 150
NaCI (mM)
0
0 2.5 5 10
Zn2+ (µM)
Figure 2 Psoriasin shows antimicrobial activity preferentially against E. coli in
various salt and pH conditions. (a) Antimicrobial activity of purified skin-derived
psoriasin against E. coli (ATCC 35218), S. aureus (ATCC 6538), P. aeruginosa
(ATCC 15442) and S. epidermidis (ATCC 14990) in the microdilution assay system.
e
100
(b) Antimicrobial activity of natural psoriasin against E. coli (ATCC 35218), tested
at various pH levels in the microdilution assay system. (c) Antimicrobial activity of
80
psoriasin against E. coli (ATCC 35218), tested at various salt concentrations.
Control, bacteria in 10 mM phosphate buffer, pH 7.4 (without psoriasin). (d) Zn2+ Buffer
Killing (%)
60
inhibits the bactericidal activity of psoriasin in a dose-dependent way. Psoriasin
was pretreated with various concentrations of ZnSO4 (horizontal axis) and then Psoriasin
(5 µg / ml)
40
was analyzed for remaining bactericidal activity against E. coli (ATCC 35218)
in the microdilution assay system. (e) Additive E. coli–killing activity of psoriasin
and the Zn2+ chelator TPEN. Various concentrations of TPEN (horizontal axis) with 20
psoriasin or with buffer (as a control) were analyzed for killing activity against E. coli
(ATCC 35218) in the microdilution assay system. Data are the mean 7 s.d. of one 0
0 0.5 1 2
representative experiment of three, each done in triplicate. TPEN (µM)
To further address the hypothesis that Zn2+ sequestration could be a the lipid-rich sebaceous glands (Fig. 3a). Skin obtained from other
mechanism by which psoriasin kills E. coli, we investigated whether sites such as the lower leg (Fig. 3b), the cheek (Fig. 3c) and the scalp
TPEN (NNN¢N¢-tetrakis(2-pyridylmethyl)ethylene diamine), a tran- (Fig. 3d) had fairly ‘patchy’ staining for psoriasin. In contrast to the
sient metal chelator known to induce cellular Zn2+ deprivation20, sebaceous glands, eccrine sweat glands did not stain for psoriasin
showed similar effects. TPEN did have E. coli–killing activity (Fig. 2e). (Fig. 3c). Around the hair follicle, immunoreactivity was detected in
Furthermore, incubation of TPEN together with psoriasin resulted in the epidermis close to the upper part of the hair follicle (Fig. 3d).
enhanced and additive rather than synergistic E. coli–killing activity These results indicate the largest amounts of psoriasin were in the
(Fig. 2e). Thus, psoriasin kills E. coli by sequestration of Zn2+. epidermis of the face and the scalp as well as in sebocytes, the lipid-
secreting cells of sebaceous glands, suggesting that psoriasin is possibly
Psoriasin is focally expressed in healthy skin secreted together with sebum lipids.
Psoriasin, originally discovered in psoriatic skin lesions as a Ca2+-
binding S100 protein of unknown biological function21,22, is upregu- Psoriasin is bactericidal in vivo
lated in epidermis of psoriasis lesions. To investigate the distribution of Next we investigated the hypothesis that psoriasin might be an
psoriasin in healthy skin in vivo, we used immunohistochemistry. With important host defense component of healthy human skin in vivo
a psoriasin monoclonal antibody (mAb) that stained keratinocytes in protecting it from infection by E. coli. Forearm skin (0.5 cm2) or, as a
lesional psoriatic epidermis as expected, but not when the mAb was control, skin covered with an adhesive tape was inoculated with a
pretreated with natural psoriasin (data not shown), there was intense suspension containing increasing numbers (1 Â 101 to 1 Â 106) of
intracytoplasmic psoriasin staining in keratinocytes of healthy skin colony-forming units (CFU) of E. coli (ATCC 35218). After exposing
from the nose in the upper, more differentiated Malpighian epidermal skin for 2 h under occlusion, we determined the number of remaining
layers, with weaker staining of keratinocytes in the suprabasal layers CFU. The killing rate was over 99.8% for all test concentrations of
(Fig. 3a). Intense psoriasin immunoreactivity was present in cells of E. coli applied on human skin in vivo (Fig. 4a).
NATURE IMMUNOLOGY VOLUME 6 NUMBER 1 JANUARY 2005 59

4. ARTICLES
E. coli–killing activity in vitro and in vivo (Supplementary Fig. 4
a b online). Antimicrobial activity was not inhibited when equivalent
concentrations of an irrelevant mAb of the same IgG isotype
(ovomucoid-specific mAb OM30-15) were applied to the skin before
bacteria (Supplementary Fig. 4 online). Having established that the
mAb HL15-4 neutralized the antimicrobial effect of psoriasin in vitro
and in vivo, we used this approach to test the function of psoriasin in
protecting skin from E. coli colonization in various people. The
application of mAb HL15-4 to skin from healthy donors (n ¼ 8)
c d
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
before inoculation with E. coli resulted in a substantial increase in
E. coli growth (Fig. 4b).
To investigate whether psoriasin is secreted in vivo, we analyzed
pooled skin-washing fluid obtained from a total of 930 cm2 of healthy
human skin (50 donors) for released proteins by RP-HPLC–
electrospray ionization mass spectrometry analyses. We detected 12 mg
of the 11,366-Da psoriasin variant as the principal E. coli–killing
protein present in the skin-washing fluid derived from all donors
(Supplementary Fig. 5 online).
For quantitative analyses and to determine how psoriasin is secreted
in vivo at different skin sites, we developed a psoriasin-specific
enzyme-linked immunosorbent assay (ELISA) using mAbs generated
Figure 3 Psoriasin is focally expressed in human skin and some adnexal against purified natural 11,366-Da psoriasin (Supplementary Fig. 4
structures. The localization of psoriasin was assessed with a mAb to online). To determine the amount of psoriasin present at various skin
psoriasin. (a) There is strong immunoreactivity (red staining) in the
surface sites of healthy human donors, we used washing fluid obtained
suprabasal keratinocytes of the epidermis (E) and in the sebaceous glands
(SG) of nose skin. (b,c) There is focal psoriasin expression in the uppermost from 0.5-cm2 areas that had been rinsed with 250 ml of 10 mM sodium
part of lower leg epidermis (b) and cheek epidermis (c), but not in eccrine phosphate buffer, pH 7.4. Psoriasin amounts at skin surfaces depended
sweat glands (ESG). HF, fair follicle. (d) The uppermost epidermal layers of on the donor (n ¼ 8), the skin area and previous washings for body
the hair follicles and the surrounding epidermis also show positive staining. care. Psoriasin amounts at the skin surface were mainly in the range of
Scale bars, 200 mm. 5–20 ng/cm2, with peak amounts up to 100 ng/cm2 in skin areas where
microbe densities were high and where body surface areas were rich in
sebaceous glands (Fig. 5).
The in vitro antimicrobial activity of psoriasin was sensitive to Zn2+ Psoriasin, which is known to strongly bind to epidermal-type fatty
(Fig. 2d). We therefore preincubated human skin in vivo with various acid–binding protein23, eluted at a high acetonitrile concentration
concentrations of Zn2+ before applying E. coli. We found a dose- from RP-HPLC columns (Fig. 1a), and we found immunoreactivity,
dependent increase in bacteria that survived (Supplementary Fig. 4 in addition to in keratinocytes, in lipid-rich sebaceous glands (Fig. 3a),
online), whereas Ca2+, as seen in vitro, had no effect. suggesting that psoriasin represents a very hydrophobic protein. To
To further confirm that psoriasin contributes to the E. coli–killing investigate if psoriasin is soluble in lipids, we first washed the skin of
activity of healthy skin in vivo, we determined whether an antibody to healthy donors (scalp and forearm) with sodium phosphate buffer
psoriasin affected in vivo E. coli–killing on skin surfaces. Because a followed by acetone washing (which extracts skin lipids). We then
commercially available antibody did not neutralize psoriasin antimi- quantified psoriasin in aqueous and acetone washing fluids by ELISA.
crobial activity in vitro, we generated a psoriasin-specific neutralizing In accordance with the immunoreactivity, we detected large amounts
mAb (HL15-4; immunoglobulin G1k (IgG1k)), which inhibited of psoriasin in lipid extracts of sebaceous gland–rich scalp skin. In
Figure 4 E. coli is effectively killed on human
skin and the E. coli–killing activity is inhibited a b 20
P < 0.01
in vivo by a neutralizing antibody to psoriasin.
(a) E. coli (ATCC 35218) was applied at various P < 0.01
concentrations (horizontal axis) under occlusion
(fold increase)
E. coli growth
on standardized test fields of the forearm of a
healthy volunteer. After an incubation time of 10
2 h, the bacteria were recovered by repeated
rinsing of the test areas, then serial dilutions NS
were plated and the remaining CFU were
quantified the next day. Control, bacteria without
skin contact (inoculum). Numbers above bars 0
Buffer OM HL
indicate the killing rate (%). Data are the mean
7 s.d. of one representative experiment of four,
each done in triplicate. (b) For investigation of
the effect of the neutralizing mAb HL15-4 in vivo, standardized test areas on the forearm skin of healthy volunteers were preincubated with 10 ml of the
HL15-4 mAb to psoriasin (HL; 2 mg/ml), an equivalent concentration of mAb OM30-15 (OM; IgG1k irrelevant control antibody raised against ovomucoid)
or the diluent (Buffer; 10 mM sodium phosphate buffer) before the application of E. coli (ATCC 35218; 2 Â 102 CFU). Data are the mean 7 s.d. of
independent experiments, each done in triplicate, in eight different healthy volunteers. In all people tested, the in vivo neutralization of psoriasin resulted
in a highly significant increase in E. coli growth compared with both controls (Wilcoxon signed-rank test).
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46.2 amounts at the skin surface suggest that psoriasin might be inducible.
29.1
To address this hypothesis, we tested whether E. coli compounds were
able to induce psoriasin expression in keratinocytes. Using real-time
quantitative PCR, we found psoriasin gene induction after treatment
3.8
25.0
of keratinocytes for 16 h with various E. coli culture supernatants
15.5 (Fig. 6a). Psoriasin mRNA expression was induced after 2–4 h of
9.0
incubation (data not shown), and we confirmed secretion of the
protein by ELISA (Fig. 6b). To investigate whether inflammatory
5.7 7.4 mediators also induce psoriasin in vitro, we tested various proin-
5.2
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
flammatory cytokines for induction of psoriasin in primary keratino-
22.9 6.25 8.8 cytes. Interleukin 1b and, to a lesser degree, tumor necrosis factor
25.0 induced psoriasin transcription (Fig. 6c). We confirmed the secretion
6.0
of psoriasin protein with ELISA (Fig. 6d).
To determine whether induction of psoriasin occurs in vivo, we
>20 ng / cm2
used culture supernatants of bacteria that strongly induced psoriasin
in vitro (Fig. 6b) and applied them for 6 h under occlusion to the
5.3
10 – 20 ng / cm2 forearm skin of healthy donors. ELISA showed increased psoriasin
5 – 9.9 ng / cm2 release on skin areas treated with bacterial culture compounds
5.1 compared with that of buffer-treated controls (Fig. 6e).
< 5 ng / cm2
29.0
DISCUSSION
Figure 5 Psoriasin is secreted in vivo on the body surface. Standardized To understand why human skin is highly resistant to E. coli coloniza-
areas of various body locations on healthy volunteers (n ¼ 8) were rinsed tion and infection, we analyzed human skin for the presence of E. coli–
with 10 mM sodium phosphate buffer, pH 7.4, to determine the concentra- killing factors and identified psoriasin (S100A7)21 as a principal and
tion of psoriasin present on the skin by ELISA, in duplicate. Data represent preferentially E. coli–killing antimicrobial component of healthy
the median concentration of psoriasin/cm2 in all people for each region. human skin. The abundance of psoriasin on human skin together
with its high antimicrobial activity against E. coli suggest that psoriasin
may be an important factor in controlling E. coli growth on the skin
contrast, we found only small amounts of psoriasin in acetone surface. To confirm this hypothesis, we specifically inhibited psoriasin
washing fluids of the forearm skin (Supplementary Fig. 5 online). using a mAb. These in vivo experiments on the skin of various healthy
people identified psoriasin as a principal E. coli–killing factor.
Bacteria and proinflammatory cytokines induce psoriasin Psoriasin is upregulated in epithelial cells undergoing abnormal
The focal expression of psoriasin in skin, especially in areas rich in differentiation21 and tumorigenesis24,25 and has been isolated from
bacteria, and the highly variable donor- and site-dependent psoriasin vernix caseosa26, suggesting that it could be involved in utero and
a b e
c d
Figure 6 Bacteria and proinflammatory cytokines induce psoriasin. (a,b) Primary keratinocytes were stimulated for 16 h with culture supernatants of
various E. coli strains (horizontal axes). After stimulation, psoriasin gene expression was analyzed by quantitative real-time PCR (a) and psoriasin release
was determined by ELISA (b). (c,d) Primary keratinocytes were stimulated for 16 h with 10 ng/ml of various cytokines (horizontal axes). After stimulation,
psoriasin gene expression was analyzed by quantitative real-time PCR (c) and psoriasin release was determined by ELISA (d). IL, interleukin; TNF, tumor
necrosis factor; IFN, interferon. Data are means 7 s.e.m. of one representative experiment of four, each done in triplicate. (e) Standardized areas on the
forearm of a healthy volunteer were rinsed with 10 mM sodium phosphate buffer, pH 7.4, to determine the basal secretion of psoriasin in each test field by
ELISA. To confirm that this procedure was effective in eluting psoriasin from the surface, each area was rinsed again and the remaining psoriasin present
in the rinsing fluid was measured by ELISA. The test fields were then left untreated or were treated for 6 h with TSB medium or with E. coli culture
supernatant (ATCC 35218 or ATCC 11775) under occlusive conditions. After incubation, secreted psoriasin present in the rinsing fluid was determined by
ELISA. Data are from one representative experiment of four, each done in duplicate.
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6. ARTICLES
possibly protects the newborn from E. coli infection during birth. periplasm of many Gram-negative bacteria34. It has been suggested
Sequencing of the human S100 gene cluster has identified five copies that stationary-phase bacteria have a particular need to defend
of S100A7-like genes (S100A7a–S100A7e)12 in the human genome, themselves against oxidative damage by endogenously generated
thus showing evidence of gene duplication during primate evolu- hydrogen peroxide35, which makes stationary-phase cells particularly
tionary history. We found that only the S100A7c variant was released vulnerable to endogenous oxidants, an effect that has been connected
in vivo and in vitro, whereas various other not-yet-characterized to their progressive loss of viability36. Zn2+ and Cu2+ compartmenta-
psoriasin variants and post-translationally modified and mutated lization could therefore be a critical step in bacterial defense against
psoriasin forms were also found in skin extracts. oxidative stress; psoriasin might use this to be antimicrobial. It is
Although the physiological function of S100 proteins is not fully possible that in patients with hyperzincemia37, who suffer from
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
understood, members of this protein family are believed to mediate a recurrent infections, increased Zn2+ may also affect host-derived
variety of functions in eukaryotic cells27,28. A few studies have zinc-dependent innate defense effector molecules, such as psoriasin,
indicated putative involvement of S100 proteins in innate host resulting in inactivation of bactericidal activity.
defense; calprotectin shows Zn2+-sensitive, C. albicans–selective bio- In summary, our results indicate that psoriasin is probably key in
static activity18,19. Furthermore, a short C-terminal peptide fragment the local innate defense of healthy skin against the gut bacterium
of S100A12 has Gram-negative bacteria–killing properties29. Many E. coli. One of the main biological functions of epithelial S100
S100 proteins are expressed in epithelial tissues28, an observation that proteins could be to target selective microbial growth as well as to
may suggest involvement of these proteins in epithelial defense. In kill various potentially pathogenic microbes on body surfaces by
support of this hypothesis, we found psoriasin mRNA expressed in a sequestering essential transition metal ions.
variety of epithelial tissues.
In skin, psoriasin was focally expressed and released from kerati- METHODS
nocytes, particular in areas known to have a high bacterial coloniza- Isolation of psoriasin from human skin and keratinocyte cultures. All
tion rate11. In addition to keratinocytes, sebocytes, the lipid-secreting human materials were collected in compliance with the laws and guidelines
cells of sebaceous glands, also showed expression of psoriasin, suggest- of the ethics committee of the medical faculty of Christian-Albrechts University
(Kiel, Germany; AZ, A130/03). Healthy volunteers were informed in detail and
ing that it is also secreted together with lipids. Therefore, lipids may
gave written consent for all in vivo experiments. Pooled heel stratum corneum
reflect an additional reservoir of lipophilic antimicrobial proteins,
(80–120 g) was extracted with acidic ethanolic citrate buffer as described38.
such as psoriasin, at lipid-rich body surfaces. This is consistent with After ‘diafiltration’ (Amicon filters; cut-off, 3 kDa) against 10 mM Tris–citrate
our acetone washing experiments, in which we detected large amounts buffer, pH 8.0, extracts were then applied to a heparin-sepharose cartridge (10-
of psoriasin in lipid-rich scalp skin but not in extracts derived from  5-mm; Pharmacia) previously equilibrated with the diafiltration buffer. After
the forearm. washing, bound proteins were eluted with 2 ml of 2 M NaCl in 0.1 M Tris–
We found only small amounts of psoriasin in skin-washing fluid citrate buffer, and the heparin-bound material was then diafiltered against 0.1%
from the dry skin of the extremities, where immunohistochemical (volume/volume (v/v)) trifluoroacetic acid in HPLC-grade water.
analyses showed psoriasin expression to be ‘patchy’. Our results Heparin-bound material was purified by preparative wide-pore C8 RP-
suggest that psoriasin is induced by the presence of local, colonizing HPLC with a column (300- Â 7-mm, C8 Nucleosil; 250- Â 12.6-mm, Macherey
and Nagel) that was previously equilibrated with 0.1% (v/v) trifluoroacetic acid
bacteria, because increased psoriasin expression was always present in
in HPLC-grade water containing 20% (v/v) acetonitrile. Proteins were eluted
places where bacterial colonization mainly occurs. This hypothesis is
with a gradient of increasing concentrations of acetonitrile containing 0.1%
supported by our findings that various E. coli strains secreted factors (v/v) trifluoroacetic acid (flow rate, 2 ml/min). Aliquots (10–30 ml) of each
that induced psoriasin secretion in vitro and in vivo. In contrast to fraction were lyophilized, dissolved in 5 ml of 0.1% (v/v) aqueous acetic acid
previous studies21, we found psoriasin induction by interleukin 1b or and tested for antimicrobial activity against E. coli (ATCC 11303) and S. aureus
tumor necrosis factor in primary keratinocytes. However, the focal (ATCC 6538) by a radial diffusion plate assay13.
psoriasin induction is unlikely to be caused by these cytokines, because Fractions containing strong antimicrobial activity against E. coli but low or
they are present only in inflamed skin. no activity against S. aureus were further purified by MonoS cation exchange
The antimicrobial spectrum of psoriasin showed a preference for E. HPLC followed by C2-C18 RP-HPLC as described for the purification of HBD-3
coli, unlike that known for b-defensins5,6 and the cathelicidin LL-37 (ref. 6) and RNase 7 (ref. 3). The concentrations of proteins present in HPLC
fractions were estimated by ultraviolet absorbance integration at 215 nm or
(ref. 30), suggesting a different mode of action of psoriasin. Ultra-
280 nm with ubiquitin (Sigma) for calibration.
structural analyses suggested that psoriasin possibly kills E. coli by a
Electrophoretic mobility was assessed by SDS-PAGE in the presence of 8 M
mechanism different from that of many cationic antimicrobial pep- urea and tricine in nonreducing conditions as described for chemokines38.
tides15. As seen for the S100 protein calprotectin, which shows Peptides were visualized by silver staining38.
candida-killing activity that can be inhibited by Zn2+ (refs. 18,19), Proteins were sequenced with a pulsed-liquid-phase 776 automated protein
we found that the antimicrobial activity of psoriasin was also Zn2+ sequencer (Perkin Elmer Applied Biosystems). Electrospray-ionization mass
sensitive. Structural investigations have shown that psoriasin binds spectrometry analyses were done in the positive ionization mode with a
Zn2+ (ref. 31), and the absence of antibacterial activity in Zn2+-loaded quadrupole orthogonal accelerating time-of-flight mass spectrometer (QTOF-
psoriasin suggests that psoriasin probably kills E. coli by Zn2+ II hybrid mass spectrometer; Micromass).
sequestration. This hypothesis was supported by experiments with
the zinc chelator TPEN15, in which we noted E. coli–killing activity, as Antimicrobial assays. The antimicrobial activity of purified psoriasin was
has been reported for other, less selective bivalent metal chelators32. estimated with a microdilution assay system13. Test organisms were incubated
for 3 h at 37 1C with purified Zn2+-free, natural skin–derived, 11,366-Da
Although the exact mechanism of psoriasin E. coli–killing activity
psoriasin (freshly lyophilized from HPLC purifications and stored as a pool in
still remains to be elucidated, Zn2+ deprivation ultimately would affect 10 mM sodium phosphate buffer at –80 1C) in 10 mM sodium phosphate
Zn2+-dependent enzymes; Zn2+ as well as Cu2+ are essential transition buffer, pH 7.4, containing 1% (v/v) trypticase soy broth (TSB). The antibiotic
metal ions for functional E. coli CuZn superoxide dismutase33. This activity of psoriasin was analyzed by plating of serial dilutions of the incubation
enzyme has high sensitivity to chelators of divalent cations34 and is mixtures and determination of the number of CFU the next day. The limit
almost exclusively synthesized in the aerobic stationary phase in the of detection (1 colony per plate) was equal to 1 Â 102 CFU/ml. The following
62 VOLUME 6 NUMBER 1 JANUARY 2005 NATURE IMMUNOLOGY

7. ARTICLES
microorganisms were used: E. coli (ATCC 35218), S. aureus (ATCC 6538), analysis (Supplementary Fig. 4 online). For ELISA, 96-well immunoplates
P aeruginosa (ATCC 15442) and S. epidermidis (ATCC 14990; inoculum, (MaxiSorp; Nunc) were coated at 4 1C for 20 h with 50 ml of 0.05 M carbonate
1 Â 104 CFU/ml). buffer, pH 9.6, containing 50 mg/ml of the HL15-4 mAb to psoriasin.
For testing the influence of salt or bivalent cations on antimicrobial activity, Subsequently, wells were blocked twice with 200 ml of 1% BSA in PBS for
psoriasin was pretreated for 1 h at 25 1C with various concentrations of NaCl, 5 min at 25 1C. After three washing steps with 200 ml PBS plus 0.1% Tween 20,
bivalent cation chlorides and sulfates or TPEN (Sigma) in water. Pretreated 100 ml per sample and serial dilutions of natural skin–derived psoriasin were
psoriasin was then directly analyzed for remaining bactericidal activity against incubated for 30 min at 25 1C. Plates were washed thrice with PBS plus 0.1%
E. coli (ATCC 35218) in the microdilution antimicrobial test system (inoculum, Tween 20 and wells were incubated for 30 min at 25 1C with 50 ml of the HL44-
1 Â 104 to 1 Â 106 CFU/ml). 8 mAb to psoriasin diluted 1:50 in PBS plus 0.1% Tween 20. Plates were washed
again three times with PBS plus 0.1% Tween and were incubated with 50 ml of
© 2005 Nature Publishing Group http://www.nature.com/natureimmunology
Culture and stimulation of keratinocytes. Primary keratinocytes were pre- biotinylated goat anti-mouse IgG2a (Southern Biotechnology Associates)
pared from tissue after surgery following established methods2 and were grown diluted 1:5,000 in PBS plus 0.1% Tween 20. After three washings with 200 ml
in EpiLife medium (Sigma). For stimulation and RNA isolation, cells were PBS plus 0.1% Tween 20, plates were filled with 50 ml/well of streptavidin-
grown in 6-well culture plates (9.6 cm2/well; Falcon) and were used after the peroxidase (1:10,000 dilution in PBS plus 0.1% Tween 20; Roche Diagnostics).
second passage at a confluence of 70–80%. Cells were stimulated for the The plates were then incubated for 30 min at 25 1C, washed six times as
indicated time (Fig. 6a–d) with different recombinant cytokines (Pepro-Tech) described above and incubated for 15 min at 25 1C in the dark with ABTS (2,2-
and E. coli culture supernatants. For the production of E. coli culture super- azino-bis-3-ethylbenzthiazoline-6-sulfonic acid; Roche Diagnostics) as the
natants, bacteria were grown in TSB in shaking conditions at 37 1C until they development agent. Absorbance was measured at 405 nm with a multichannel
reached an optical density of 1.0. Then, 1 ml of this culture was added to 9 ml photometer (Sunrise; Tecan). Standard curves were generated with natural
TSB and the resultant culture was incubated for 24 h in 75-cm2 flasks (Sarstedt) skin–derived psoriasin; the detection limit of the ELISA was a concentration of
without shaking. Subsequently, bacteria were heat-killed in a water bath at 1 ng/ml of psoriasin.
65 1C for 60 min and then were centrifuged at 5,000g for 15 min. The
resulting supernatants were diluted 1:3 in EpiLife medium and were used
Transmission electron microscopy of bacteria. Approximately 1 Â 108 CFU of
for stimulation experiments.
E. coli (ATCC 35218) were treated for various lengths of time (30–180 min) at
Real-time RT-PCR. A fluorescence thermocycler (LightCycler; Roche Molecu- 37 1C with psoriasin (250 mg/ml) in 100 ml of 10 mM sodium phosphate buffer,
lar Biochemicals) was used for real-time RT-PCR experiments following the pH 7.4, containing 1% (v/v) TSB. The bacteria were then centrifuged at 5,000g
instructions of the manufacturer. With this technique, the fluorescence-labeled for 5 min, immersed for 2 h in cold (4 1C) 5% phosphate-buffered glutar-
amplification product is measured continuously. Total RNA obtained from aldehyde, pH 7.8, repeatedly rinsed in cold phosphate buffer, ‘post-fixed’ for 2 h
epithelial cells was isolated by the TRIzol method (Invitrogen) and 2 mg of RNA in 4% phosphate-buffered osmic acid, dehydrated in acetone and, finally,
was reverse-transcribed with standard methods (Invitrogen). The cDNA embedded in Araldit (Araldit Cy212; Sigma), as described6. Bacteria were
equivalent to 10 ng of RNA served as template for PCR in a volume of 10 ml examined with an EM 910 electron microscope (Zeiss).
(4 mM MgCl2, 0.5 mM of each primer and 1Â Lightcycler-FastStart DNA
Master SYBR Green I mix). Samples were amplified in capillaries after initial Immunoblot. For immunoblot analyses, 100 mg stratum corneum extract,
denaturation at 95 1C for 10 min for 45 cycles (10 s of denaturation at 95 1C, serial dilutions of skin-derived psoriasin or diluent (as a control) were
5 s of annealing at initial 68 1C (‘touchdown’ of À1 1C/cycle to 62 1C) and 10 s separated by 16.5% tricine SDS-PAGE containing 8 M urea. Proteins were
of extension at 72 1C). SYBR Green I fluorescence was measured at 72 1C at the transferred to a nitrocellulose membrane and blocked for 1 h in blocking buffer,
end of each cycle. Melting curves were generated after each run to confirm then were incubated for 18 h at 4 1C in buffer containing a 1:2,000 dilution of
amplification of specific transcripts (cooling of the samples at 65 1C for 15 s, mAb to psoriasin (clone HL15-4 or clone HL44-8). The washed membrane was
heating at 0.2 1C/s to 95 1C with continuous measurement of the fluorescence). then incubated in a 1:20,000 dilution of goat anti-mouse IgG–horseradish
Intron-spanning primers specific for psoriasin were derived from psoriasin peroxidase conjugate (Dianova), followed by incubation with chemilumi-
mRNA (forward, 5¢-AGACGTGATGACAAGATTGAC-3¢; reverse, 5¢-TGTCCT nescent peroxidase substrate (Sigma). Bands were visualized with a Diana III
TTTTCTCAAAGACGTC-3¢). Amplification with these primers resulted in a cooled charge-coupled device camera imaging system (Raytest). Densitometry
127–base pair fragment. The specificity of the psoriasin products were verified was quantified with AIDA evaluation software (Raytest).
by sequencing. The housekeeping gene Gapd (glyceraldehyde phosphodehy-
drogenase) was amplified with each cDNA in a separate PCR (GA1, 5¢-
In vivo experiments to determine the E. coli–killing activity of human skin.
CCAGCCGAGCCACATCGCTC-3¢; GA2, 5¢-ATGAGCCCCAGCCTTCTCCAT
For investigating the natural E. coli–killing activity of human skin, up to 1 Â
-3¢; 360 base pairs, intron-spanning-product) to allow calculation of the
107 CFU of E. coli (ATCC 35218) were applied in a volume of 10 ml sodium
relative transcripts.
phosphate buffer (10 mM, pH 7.4) plus 1% TSB on standardized test areas on
Immunohistochemistry. For localization of psoriasin protein expression in the the volar forearms of healthy volunteers. After an incubation time of 2 h under
skin by immunohistochemistry, 5-mm vertical paraffin sections of human skin occlusion with thin aluminium plates (Finn Chambers on Scanpor, diameter,
were deparaffinized and rehydrated, blocked with normal rabbit serum and 8 mm; Epitest) the bacteria were recovered by repeated rinsing of each test area
incubated for 60 min with a 1:1,500 dilution of a mouse mAb (IMG-409, clone with 100 ml sodium phosphate buffer (10 mM, pH 7.4). For quantification of
47C1068; Biocarta). As a specific control, sections with intense positive staining the remaining bacteria, serial dilutions were plated on TSB agar and CFU were
derived from psoriasis lesions were incubated with preabsorbed antibodies with counted after 20 h of incubation at 37 1C. To ensure that all bacteria were
various concentrations of skin-derived psoriasin. The natural psoriasin blocked successfully recovered from the forearms after the rinsing procedure, the test
the psoriasin mAb staining in a dose-dependent way. The sections were fields as well as the aluminium plates, which were used for occlusion, were
incubated with a biotinylated IgG antibody to mouse (anti-mouse; Vector), pressed onto agar plates and the number of colonies was determined after 20 h
followed by incubation with the Vectastain ABC-AP system (Vector), develop- of incubation at 37 1C. As only single E. coli colonies were identified in this way,
ment with Vector Red and counterstaining with hematoxylin. we were confident that most colonies were recovered during the rinsing
procedure of our experimental setting.
Psoriasin ELISA. As a direct test for the presence of psoriasin in skin-washing In some experiments, test areas on the forearm skin were pretreated with
fluids as well as in the supernatants of primary keratinocytes, a sensitive various test solutions in a volume of 10 ml: aqueous ZnSO4 and CaCl2, with
psoriasin ELISA was established with two mAbs (clone HL15-4 (IgG1k) and water as a control, or antibodies with the diluent (10 mM sodium phosphate
clone HL44-8 (IgG2ak)). These antibodies were generated by immunization of buffer) as control. After incubation for 5 min, E. coli (ATCC 35218; 1 Â 102 to
mice with natural 11,366-Da psoriasin using standard methods as de- 1 Â 103 CFU in 10 ml sodium phosphate buffer) was applied onto the test areas
scribed39,40. Specificity of the psoriasin antibodies was verified by immunoblot and incubated under occlusion for 2 h. Bacteria were quantified as described
NATURE IMMUNOLOGY VOLUME 6 NUMBER 1 JANUARY 2005 63