© 2007 Nature Publishing Group http://www.nature.com/naturemedicine
LETTERS
Increased serine protease activity and cathelicidin
promotes skin inflammation in rosacea
Kenshi Yamasaki1, Anna Di Nardo1, Antonella Bardan1, Masamoto Murakami2, Takaaki Ohtake3, Alvin Coda1,
Robert A Dorschner1, Chrystelle Bonnart4,5, Pascal Descargues4,5, Alain Hovnanian4–6, Vera B Morhenn1 &
Richard L Gallo1
Acne rosacea is an inflammatory skin disease that affects 3%
of the US population over 30 years of age and is characterized by
erythema, papulopustules and telangiectasia1–3. The etiology of
this disorder is unknown, although symptoms are exacerbated by
factors that trigger innate immune responses, such as the release
of cathelicidin antimicrobial peptides4. Here we show that
individuals with rosacea express abnormally high levels
of cathelicidin in their facial skin and that the proteolytically
processed forms of cathelicidin peptides found in rosacea
are different from those present in normal individuals. These
cathelicidin peptides are a result of a post-translational
processing abnormality associated with an increase in stratum
corneum tryptic enzyme (SCTE) in the epidermis. In mice,
injection of the cathelicidin peptides found in rosacea, addition
of SCTE, and increasing protease activity by targeted deletion of
the serine protease inhibitor gene Spink5 each increases
inflammation in mouse skin. The role of cathelicidin in enabling
SCTE-mediated inflammation is verified in mice with a targeted
deletion of Camp, the gene encoding cathelicidin. These findings
confirm the role of cathelicidin in skin inflammatory responses
and suggest an explanation for the pathogenesis of rosacea by
demonstrating that an exacerbated innate immune response can
reproduce elements of this disease.
Cathelicidins have been identified in mammals5, birds6 and fish7, and
are known for their functions to protect the host against infection by
Gram-positive8 and Gram-negative bacteria9,10, and some viruses11.
Several animal models8, and human clinical conditions12, have shown
that the presence of cathelicidin correlates with the ability of the host
to mount an effective defense against infection. In addition, antimicrobial peptides such as cathelicidins and defensins may have a dual
role in immunity because they can act both to kill microbes and
potentially to trigger various host tissue responses. This function has
led to use of the term ‘alarmins’13 in recognition of their capacity to
signal an inflammatory reaction. Among observations of cathelicidin
function are findings that these peptides can promote leukocyte
chemotaxis14, angiogenesis15 and the expression of extracellular matrix
components16. Because many of these effects are similar to the clinical
changes seen in rosacea, we considered that abnormal expression of
cathelicidin peptides may be a factor in its pathogenesis.
To test whether the expression of cathelicidin is altered in rosacea,
skin biopsies were obtained from the naso-malar fold and compared
with skin from a similar location in normal individuals. All specimens
from individuals with rosacea showed abundant cathelicidin by
immunostaining, whereas normal facial skin showed minimal expression (Fig. 1a). Cathelicidin was located diffusely throughout the
epidermis, but was not seen in healthy volunteers. To quantify this
difference, epidermal cathelicidin was measured in tape-stripped
samples of facial skin. Significantly higher cathelicidin expression
was found in rosacea than in normal skin (Fig. 1b, P = 0.015).
Cathelicidin mRNA was also evident in rosacea skin by in situ
hybridization (Fig. 1c), in contrast to normal epidermis where
cathelicidin mRNA is hardly detectable (ref. 17 and data not
shown). Thus, we concluded that the skin of individuals with rosacea,
similar to that of individuals with other inflammatory diseases18,
expressed more cathelicidin than normal facial skin.
Proteolytic processing of the cathelicidin precursor protein into
active peptide is an essential step for function and controls the ability
of cathelicidin to act as an antimicrobial or pro-inflammatory
molecule19,20. Human cathelicidin is secreted as proprotein, named
18-kDa cationic antimicrobial protein (CAP18). CAP18 is biologically
inactive19. Proteolytic cleavage near the C terminus results in release
of the active antimicrobial peptide. Because the function of the
cathelicidin peptide is dictated by the extent of post-translational
processing of CAP18, we analyzed the mass of cathelicidin peptides
from rosacea and normal skin using surface-enhanced laser
desorption-ionization time-of-flight mass spectrometry (SELDITOF-MS) to examine whether the increased expression of cathelicidin
in rosacea signifies a change in CAP18 processing. The mass distributions of cathelicidin peptides were very similar among independent
individuals with rosacea (Fig. 2). Samples obtained from normal facial
skin were also similar to each other, but were markedly different from,
1Division
of Dermatology, University of California, San Diego, and VA San Diego Health Care System, 3350 La Jolla Village Drive, San Diego, California 92161, USA.
of Dermatology, Asahikawa Medical College, Asahikawa 078-8510, Japan. 3Department of Medicine, Asahikawa Medical College, 2-1-1-1 Midorigacka
Hidashi, Asahikawa 078-8510, Japan. 4INSERM, U563, Toulouse F-31000, France. 5Université Paul-Sabatier, Toulouse F-31000, France. 6CHU Toulouse,
Department of Genetics, Place du Dr. Baylac, Toulouse F-31000, France. Correspondence should be addressed to R.L.G. ([email protected]).
2Department
Received 27 March; accepted 13 June; published online 5 August 2007; doi:10.1038/nm1616
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a
Normal
Preimmune IgG
b
c
Antisense
Sense
16
Cathelicidin (fmol/µg protein)
© 2007 Nature Publishing Group http://www.nature.com/naturemedicine
Cathelicidin
Rosacea
14
P = 0.015
12
10
8
6
4
2
0
Rosacea
Normal
and contained fewer cathelicidin fragments than, those from rosacea.
The 37-amino-acid peptide LL-37 was one of the main forms
identified in rosacea, but it was much less abundant in normal skin.
In addition, rosacea skin contained peptides of unique mass that were
absent in normal skin (Fig. 2, arrowheads; deduced sequences are
given in Supplementary Fig. 1 and Supplementary Methods online).
These data demonstrate that both the abundance and the processing
of cathelicidin peptides are altered in rosacea.
Stratum corneum tryptic enzyme (SCTE; also known as kallikrein 5,
KLK5 or hK5), a serine protease of the kallikrein family, is a key
protease that cleaves CAP18 to active peptides in human epidermis21.
On the basis of the abnormal cathelicidin peptide abundance and
distribution pattern observed in rosacea, we next examined whether
the expression of SCTE was altered in rosacea as compared with
normal skin. SCTE was highly expressed in rosacea and colocalized
with cathelicidin in the granular and cornified layers of the epidermis
(Fig. 3a and Supplementary Fig. 2 online). Some rosacea specimens
also expressed SCTE in the basal layer of the epidermis, accompanied
by an increase in cathelicidin expression. By contrast, cathelicidin and
SCTE were much less abundant and localized superficially in normal
skin (Fig. 3a and Supplementary Fig. 3 online). This increase in
immunoreactivity in rosacea correlated with higher protease activity
in the epidermis, as determined by in situ zymography of rosacea
skin and normal skin (Fig. 3b). Total protease activity was measurable
in individuals with rosacea (Fig. 3c), but was typically undetectable in
normal individuals. This activity was a serine protease such as SCTE,
because the serine protease inhibitors aprotinin and AEBSF completely
suppressed the observed proteolytic activity in rosacea skin (Fig. 3c).
Having observed that individuals with rosacea uniformly show an
increase in cathelicidin and an abnormality in enzymatic processing,
we examined whether these findings could explain the clinical presentation of this disease. To test the consequences of abnormal
cathelicidin peptides, human keratinocytes were cultured in the
presence of peptides such as LL-37 and FA-29 that are abundant in
rosacea. These rosacea peptides, but not the shorter peptides DI-27
and KR-20, which are present on normal skin20,22, induced IL-8
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Figure 1 Cathelicidin is abundant in rosacea. (a) Cathelicidin expression
in lesional skin of individuals with rosacea was examined by
immunohistochemistry with antibody to LL-37. Top, anti–LL-37; bottom,
pre-immune IgG. Scale bars, 500 mm. (b) Amount of cathelicidin in skin
was measured by quantitative immuno-dot blot analysis of tape-stripped
samples. The amount of cathelicidin in skin samples was determined by
comparison to synthetic LL-37 as a reference and by normalization to total
protein concentration. The mean of each group is indicated by a broken
line (n ¼ 3). (c) Localization of cathelicidin mRNA in lesional skin of
rosacea individuals was visualized by in situ hybridization with a probe
to LL-37. Brown color indicates positive signal; blue color indicates
methylene blue staining of nuclei. Left, antisense probe; right, sense
probe. Scale bars, 500 mm.
release (Fig. 4a). To test the function of these peptides in vivo, mice
were given subcutaneous injections of LL-37 and FA-29. The concentration of cathelicidin injected (320 mM) was selected to reflect local
physiological concentrations seen in rosacea (maximum 1,500 mM).
Injection of both LL-37 and FA-29 induced erythema and vascular
dilatation in skin after 48 h, and was characterized histologically by a
neutrophilic infiltrate, thrombosis and hemorrhage (Fig. 4b and
Supplementary Fig. 4 online). Conversely, injection of peptide
KR-20 from normal skin did not induce inflammation. The inflammatory reaction to LL-37 was dose dependent and seen at concentrations as low as 3.2 mM (Supplementary Fig. 4); it was also observed
equally in BALB/c and C57BL/6 mouse strains (data not shown).
To test the hypothesis that the innate expression of cathelicidin in skin
can promote inflammation, we tested mice with targeted deletion of the
cathelicidin antimicrobial peptide gene Camp8. Individuals with rosacea
show facial inflammation in response to external stimuli; therefore, the
response of Camp/ mice to irritant stimuli was examined by using an
established mouse skin model of irritation. These experiments supported
Rosacea
6
4
2
0
6
4
2
0
6
4
2
0
3,000
3,500
4,000
4,500 (m/z)
LL-37
Normal
6
4
2
0
6
4
2
0
6
4
2
0
3,000
3,500
4,000
4,500 (m/z)
Figure 2 Altered expression of cathelicidin peptides in rosacea skin. Shown
is the mass of cathelicidin peptides in lesional skin of rosacea (top) and in
normal skin (bottom) as examined by SELDI-TOF-MS. Arrowheads indicate
unique peptide peaks in rosacea skin.
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a
Cathelicidin
SCTE
Merged
b
c
FITC-casein
DAPI
None
Rosacea
Rosacea
Mix
Bestatin
E64
Aprotinin
AEBSF
NEI
Normal
Normal
© 2007 Nature Publishing Group http://www.nature.com/naturemedicine
LEI
EDTA
Pepstatin
0
100
Fluorescence
200
Figure 3 Increased SCTE expression and protease activity in rosacea epidermis. (a) Expression of cathelicidin and SCTE in skin visualized by
immunofluorescence. Top, rosacea skin; bottom, normal skin. Green indicates cathelicidin; red indicates SCTE. Scale bars, 500 mm. (b) Protease activity
in human skin examined by in situ zymography with FITC-conjugated casein substrate (left). Nuclei were also stained with DAPI (right). Scale bars, 500 mm.
(c) Skin surface protease was measured with FITC-conjugated casein substrate. The indicated protease inhibitors were used to identify proteases. Serine
protease inhibitors (aprotinin and AEBSF) and a protease inhibitor mixture (Mix) completely suppressed skin surface protease activity.
the observations made after the injection of excess peptide, because
considerably less inflammation after application of a contact irritant was
seen in Camp/ mice than in wild-type mice (Fig. 4c,d). A similar
decrease in inflammatory infiltrate in Camp/ mice was also seen after
physical abrasion of the skin (data not shown).
To test how the increase in serine protease activity observed in rosacea
contributes to the clinical findings of the disease, we examined mice
deficient in the gene encoding serine peptidase inhibitor Kazal-type 5
(Spink5), which do not express the serine protease inhibitor Lymphoepithelial Kazal-type–related inhibitor (LEKTI) and show increased
SCTE activity23. Skin from Spink5/ mice had altered expression of
cathelicidin peptides similar to that seen in rosacea (Fig. 4e). The main
peak was GLL-34 (m/z 3,877; Fig. 4e, arrowhead), a mouse cathelicidin
peptide similar in activity to human LL-37 (ref. 24). Like LL-37, GLL-34
was not detected in wild-type mouse littermates with normal serine
protease activity. These data support the hypothesis that an increase in
serine protease activity leads to activation of cathelicidin.
To determine whether an increase in SCTE results in greater
inflammation, SCTE was injected subcutaneously in a manner similar
to that used for the cathelicidin peptides themselves. The amount of
SCTE in human skin is as high as 2–13 ng per mg of dry weight of
skin25. From the data of the protease assay, we estimated the amount
of serine protease activity in lesional skin of individuals with rosacea
to be as high as 500 ng of tryptic kallikrein per mg of dry weight of
skin. Therefore, 1 mg of SCTE was injected twice a day for 2 d to
mimic local concentrations seen in rosacea. Injection of active SCTE
induced erythema and inflammatory cell infiltration accompanied by
processing of cathelicidin peptide (Fig. 4f), which were not observed
in control treated skin. This response to SCTE was dependent on the
presence of cathelicidin, because Camp/ mouse showed considerably less cell infiltration after SCTE injection as compared with wildtype mice (Fig. 4g,h). These data show that injection of SCTE
increases cathelicidin processing and induces skin inflammation.
Therefore, the increase in SCTE observed in rosacea would account
for the pathological changes observed this disease.
Our findings demonstrate an association between the clinical signs
of rosacea and abnormal cathelicidin expression and processing.
Overexpression of cathelicidin and the subsequent generation of
abnormally processed peptides by highly increased serine protease
activity results in the accumulation of peptides that can induce
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inflammatory changes in mice characteristic of the disease in humans.
These observations are consistent with an evolving understanding of
the functions of antimicrobial peptides. On the basis of largely in vitro
observations, cathelicidins and other antimicrobial peptides have been
proposed to do more than just kill microbes: they can stimulate
cytokine release20, angiogenesis15, chemotaxis14 and wound repair18,26.
In the case of rosacea, an increase in production of cathelicidin,
combined with greater proteolytic processing, leads to a situation
where accumulation of these peptides can initiate the classical manifestations of this disorder. A change in local antimicrobial peptide
expression may also change the population of commensal microbes on
involved skin. Such a change in the microflora in rosacea1–3 may
contribute further to the manifestations of this disease.
Several clinical observations also support the involvement of abnormal proteolysis in the pathogenesis of rosacea. Protease activity is higher
in facial skin in which rosacea symptoms occur than in other areas of the
body that are spared of the disease (data not shown). Tetracyclines can
indirectly inhibit serine proteases27, thereby explaining their therapeutic
benefit over other antibiotics despite the frequent development of
tetracycline resistance in microflora cultured from the skin of affected
individuals. Indeed, preliminary data show that minocycline decreases
skin protease activity during treatment. Thus, the association between
the administration of a clinically effective drug and a decrease in the
capacity of the skin to proteolytically generate cathelicidin peptides
further supports the notion that protease activity has a role in rosacea.
The balance between cathelicidin and SCTE is disturbed in rosacea;
however, other serine proteases and protease inhibitors may participate
in determining the final steady-state accumulation of cathelicidin
products. Evidence supporting the idea that other serine proteases
have a role in modifying cutaneous inflammation is provided by
transgenic mice overexpressing kallikrein 7 in the skin. These mice
show abnormal formation of the epidermal barrier and inflammation
in the dermis28. It is unclear to what extent an abnormal physical
barrier, as compared with abnormal processing of inflammatory
mediators such as cathelicidin, contributes to this observation. Similarly, mice lacking LEKTI have barrier abnormalities and inflammation
of the skin, and an excess of active cathelicidins21. Although the
inflammatory effects in both models are likely to be partly a consequence of the effects of protease on the stratum corneum, our
findings suggest that the generation of pro-inflammatory forms of
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cathelicidin is also a contributing factor. Thus, surface protease activity
seems not only to influence physical barrier formation but also to
be an essential element in regulating inflammatory signals. This
concept emphasizes the importance of total enzymatic activity, a
process that will be modified by various environmental factors such
as temperature and pH. Because these factors are also well described as
associated with precipitating disease in rosacea29, these clinical associations further support the hypothesis that rosacea is a manifestation of
an abnormality in antimicrobial peptide expression and processing.
In conclusion, individuals with rosacea have an increase in both
serine protease activity and cathelicidin peptides in their facial
skin, resulting in the generation of pro-inflammatory forms of the
a
antimicrobial peptide. This abnormality in enzymatic activity and
peptide expression can lead to the development of many aspects of the
human disease in mice. Our findings suggest a new direction for
understanding the pathophysiology of rosacea. Influencing the balance
of antimicrobial peptides, and their post-secretory processing, provides an opportunity for designing more effective therapy and reveals
the potential for the involvement of proteolysis and cathelicidin
expression in other inflammatory disorders.
METHODS
Immunohistochemistry. All sample acquisition, including skin biopsies and
tape-stripped samples, was approved by the Committee on Investigations
LL-37
b
KR-20
c
4
IL-8 (ng/ml)
3
2
1
Camp –/–
0
LL-37
d
Cells/HPF
FA-29
1,400
1,200
1,000
800
600
400
200
0
DI-27
KR-20
e
30
Spink5 –/–
20
10
0
30
Spink5 +/+
20
Camp –/– Camp +/+
10
(m/z)
0
2,000
4,000
6,000
f
SCTE
Camp +/+
none
8,000
10,000
g
2
1
Camp –/–
2
Boiled
SCTE
Camp +/+
1
3,000
4,000
5,000
800
h
(m/z)
6,000
Cells/HPFs
© 2007 Nature Publishing Group http://www.nature.com/naturemedicine
LETTERS
600
400
Figure 4 Cathelicidin peptides augment cytokine induction in human keratinocytes and skin inflammation.
(a) Human epidermal keratinocytes were stimulated by cathelicidin peptides, and IL-8 in culture media was measured.
200
Data are the mean ± s.d. (n ¼ 3 experiments). (b) Skin surface and histology images of lesions caused by injection of
0
cathelicidin peptides. Left, LL-37–injected skin; right, KR-20–injected skin. Scale bars, 5 mm (skin surface), 500 mm
Camp –/– Camp +/+
(histology). (c,d) Skin irritation caused by epicutaneous DNFB application to Camp / and wild-type littermate
+/+
(Camp ) mice. Shown are histology (c) and mean ± s.d. of leukocyte counts per HPF from 3 randomly selected regions (d). Wild-type mice showed
significantly more cell infiltration than Camp / mice (P o 0.05). Scale bars, 500 mm. (e) Cathelicidin processing in Spink5 –/– mice analyzed by
SELDI-TOF-MS. Skin from Spink5 –/– mice had processed cathelicidin peptides of o7 kDa (top), whereas the main cathelicidin in wild-type (Spink5 +/+)
mice was a non-processed form of 48 kDa (bottom). Arrowhead indicates GLL-34, a representative mouse cathelicidin peptide. (f) SCTE or boiled SCTE was
injected subcutaneously, and histology (left) and cathelicidin processing (right) were examined. SCTE-treated skin showed inflammation and processed
cathelicidin peptide (m/z 4,244, sequence FKKISRLAGLLRKGGEKIGEKLKKIGQKIKNFFQKLV). Scale bars, 500 mm. (g,h) SCTE was injected subcutaneously
into Camp / and wild-type mice. Skins were processed by hematoxylin-eosin staining (g), and the mean ± s.d. (n ¼ 4) of infiltrated cells per HPF was
plotted (h). Camp / mice showed significantly less cell infiltration (P o 0.05).
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Involving Human Subjects of the University of California, San Diego. We
obtained informed consent for all procedures. After the injection of local
anesthesia, 3-mm punch biopsies were taken from the untreated lesional skin of
individuals with rosacea and from skin of normal healthy volunteers. The tissue
was freshly frozen in Tissue-Tek OCT compound (Electron Microscopy
Sciences). We cut 5-mm sections and fixed them in methanol for 30 min at
4 1C. Sections were blocked with 5% donkey serum in PBS for 30 min, incubated
for 1 h with polyclonal chicken IgY antibody to the LL-37 peptide of CAP18,
and then incubated for 30 min with horseradish peroxidase (HRP)-conjugated
goat antibody to chicken IgY. Immunostaining was visualized by using a
Vectastain ABC kit (Vecta Laboratories). We obtained images with an Olympus
BX41 microscope (Scientific Instrument Company). In total, 11 rosacea
samples and 10 normal samples were examined to confirm similar results.
ethanolamine in PBS (pH 8.0). After three washes with 0.5% Triton-X in PBS,
protein chips were assembled in the Bioprocessor reservoir, 50 ml of eluted
sample was applied, and the chips were incubated for 2 h at room temperature.
The protein chips were washed twice with RIPA buffer, once with PBS
containing 0.5% Triton-X and three times with PBS, and then soaked in
10 mM HEPES buffer and air-dried. We applied 0.5 ml of energy absorbance
molecule (50% saturated a-cyano-4-hydroxy cinnamic acid in 50% acetonitrile
plus 0.5% trifluoric acid) twice, and all spots were allowed to dry completely.
Samples were analyzed on a SELDI mass analyzer PBS II with a linear TOF
mass spectrometer (Ciphergen Biosystems) using time-lag focusing. The
specificity of antibodies to LL-37 and mouse CRAMP in this system has been
confirmed by several synthetic cathelicidin peptides20,22. Synthetic LL-37 and
KR-20 peptides were used as references to calibrate the exact mass sizes.
Cathelicidin protein analysis. To obtain skin surface cathelicidin peptides,
facial skin was tape-stripped 20 times from the same lesion with a 23-mm
diameter tape (D-Squame; a gift of CuDerm Corp.). The tapes were immersed
in 1 ml of 1 M HCl containing 1% trifluoroacetic acid and vortexed. We
lyophilized the protein extracts completely and dissolved the pellet in 100 ml of
distilled water. Concentrations of total protein were measured by a BCA protein
assay (Pierce Biotechnology). For quantification of cathelicidin, 5 ml of each
sample, or serially diluted synthetic LL-37 peptide as a standard, was dotted
onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat
dry milk in PBS for 1 h, incubated overnight with rabbit antibody to LL-37 at
4 1C, and then incubated for 1 h with HRP-conjugated goat antibody to
rabbit IgG (DAKO). The immunoreactions were visualized by Western Lighting
chemiluminescence reagent plus (Perkin-Elmer Life Science), and the density of
each dot was measured with NIH Image and compared with standard controls.
Fluorescence immunohistochemistry. We fixed 6-mm frozen sections with
paraformaldehyde, blocked them with 5% goat serum, and incubated them with
polyclonal rabbit antibody to LL-37 or monoclonal mouse antibody to SCTE.
FITC-conjugated goat antibody to rabbit IgG and AlexaFluor568-conjugated
goat antibody to mouse IgG (Molecular Probes), respectively, were used as
secondary antibodies. We mounted sections in ProLong Anti-Fade reagent
(Molecular Probes). Images were obtained by a Zeiss LSM510 laser scanning
confocal microscope coupled with an Axiovert 100 inverted stage microscope.
In situ hybridization. To localize cathelicidin mRNA expression in the skin, we
carried out in situ hybridization as described30. Digoxigenin (DIG)-labeled
riboprobes were prepared by using a DIG RNA labeling kit (SP6/T7; Roche
Applied Science) in accordance with the instructions provided. Freshly frozen
sections were cut at 8 mm, fixed with 4% paraformaldehyde for 10 min at room
temperature (18–23 1C), and immersed in 0.1% active diethyl pyrocarbonate in
PBS at 4 1C for 10 min. The sections were then washed with PBS at room
temperature for 10 min, treated with 1 M triethanolamine solution (pH 8.0)
containing 0.25% acetic anhydride for 15 min at 37 1C, washed with PBS at
least three times, treated with 100% ethanol for 5 min, and then dried.
Prehybridization was performed with 50% formamide in 2 SSC (3 M sodium
chloride and 0.03 M sodium citrate) for 30 min at 45 1C. After removal of
excess solution, sections were hybridized for 16 h at 45 1C with sense or
antisense DIG-labeled cRNA probes for the LL-37 peptide sequence (137 bp,
1 mg/ml) in hybridization solution (1 mg/ml of yeast tRNA, 20 mM Tris-HCl
buffer (pH 8.0), 2.5 mM EDTA, 1 Denhart’s solution, 0.3 M NaCl, 50%
deionized formamide and 50% dextran sulfate). Stringent washing was done
for 60 min at 45 1C with 50% formamide in 2 SSC, and for 10 min at 37 1C
with 2 SSC. RNase treatment was performed with 40 mg/ml of RNase-A
(Roche Applied Science) for 30 min at 37 1C. The sections were then washed
with 2 SSC for 30 min at 37 1C, and reacted with alkaline phosphate–
conjugated Fab fragment antibody to DIG (1:500 in PBS; Roche Applied
Science) for 5 h at room temperature. Alkaline phosphate was visualized by
incubation with 5-bromo-4-chloro-3-indolyl phosphate (X-phosphate) and
nitroblue tetrazolium with addition of levamisole solution (DAKO) overnight
at room temperature. We used methyl green as a nuclear counterstain.
SELDI-TOF-MS. Skin biopsies were frozen in OCT compound and stored at
80 1C. Twenty 10-mm slices were collected in 1.5-ml polypropylene tubes and
dissolved in 100 ml of RIPA buffer (50 mM HEPES, 150 mM NaCl, 0.05% SDS,
0.25% deoxycholate and 0.5% NP-40; pH 7.4) containing protease inhibitors
(Roche Applied Science). Samples were sonicated for 3 min and centrifuged for
10 min at 12,000g. We transferred the supernatant to new tubes and kept it at
20 1C until SELDI-TOF analysis.
Protein chips (RS-100 protein chip array; Ciphergen Biosystems) were
coated with 4 ml of rabbit antibody to LL-37 for human samples and rabbit
antibody to mouse cathelin-related antimicrobial peptide (CRAMP)26 for
mouse samples for 2 h at room temperature, and then blocked with 0.5 M
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In situ zymography. The frozen sections 6 (mm) were rinsed with 1% Tween-20
in distilled water and incubated with 100 ml of BODIPY-FL-casein substrate
(10 mg/ml in 10 mM Tris-HCl, pH 7.8; Molecular Probes) at 37 1C for 3 h.
After removal of excess of substrate solution, nuclei were stained with
4¢,6-diamidino-2-phenylindole (DAPI) and rinsed with 1% Tween-20 in
distilled water. We mounted sections in ProLong Anti-Fade reagent (Molecular
Probes). Images were obtained with an Olympus BX41 microscope (Scientific
Instrument Company).
Peptide synthesis. Cathelicidin peptides were commercially prepared by
Synpep. We used the following peptide amino acid sequences: LLGDFFRKSKE
KIGKEFKRIVQRIKDFLRNLVPRTES (LL-37), FALLGDFFRKSKEKIGKEFK
RIVQRIKDF (FA-29), DISCDKDNKRFALLGDFFRKSKEKIGK (DI-27), and
KRIVQRIKDFLRNLVPRTES (KR-20). All synthetic peptides were purified to
495% by HPLC and their identity was confirmed by mass spectrometry.
Measurement of IL-8 release. Normal human keratinocytes (Cascade Biologics) were grown in EpiLife medium (Cascade Biologics) supplemented with
0.06 mM Ca2, 1% EpiLife defined growth supplement, and 1% penicillinstreptomycin (Invitrogen Life Technologies). Cells were grown at 37 1C in a
humidified atmosphere of 5% CO2 and 95% air. We cultured human keratinocytes to confluence and treated them with the cathelicidin peptides (3.2 mM)
for 6 h. Supernatants were collected and placed in a sterile 96-well plate for
ELISA. Production of IL-8 was determined by ELISA (R&D systems) in
accordance with the manufacturer’s instructions. In brief, 96-well plates were
coated with capture antibody and incubated overnight at 4 1C. Wells were
washed three times, blocked for 1 h at room temperature, and washed another
three times. Standards and samples at a dilution of 1/20 were then added to the
wells. After 2 h at room temperature, the wells were washed five times, and the
detection antibody was added. After 1 h at room temperature, the wells were
washed seven times, substrate solution was added to each well, and the plate was
incubated for 30 min at room temperature in the dark. We added stop solution
to each well and measured the absorbance at 450 nm with correction at 570 nm.
Skin inflammation models and identification of cathelicidin peptides. All
mouse procedures were approved by the Veterans Affairs (VA) San Diego
Healthcare System subcommittee on animal studies. Neonatal mouse skin from
Spink5-deficient mice and wild-type littermates was excised by using an 8-mm
punch biopsy. Samples were homogenized and centrifuged for 10 min at 12,000g
and the supernatant was collected. We analyzed processing of mouse CRAMP by
SELDI-TOF-MS with rabbit antibody to mouse CRAMP, as described above.
BALB/c and C57BL/6 mice, shaved 24 h before treatments, were injected
subcutaneously on the back with 40 ml of peptide (320 mM) twice a day.
Subcutaneous injections were targeted superficially to raise an intact epidermal
bleb, thereby identifying that administration was at the level of the lower
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LETTERS
epidermis or dermis. Forty-eight hours after the initial injection (four injections in total), we assessed skin inflammation by the severity of erythema and
edema. Skin was then biopsied for hematoxylin-eosin staining to examine the
histopathological changes. One representative image of skin surface and
histology from three independents experiments is shown (Fig. 4b).
To determine the role of loss of cathelicidin in inflammation, CRAMPdeficient (Camp/) mice8 and wild-type littermates were used. To induce
cathelicidin, mice were shaved and skin was injured by light abrasion 20 times
with sandpaper (aluminum oxide sandpaper, medium 100 grit, 3M). After
24 h, we induced chemical inflammation epicutaneously by application of 10 ml
of 2% 2,4-dinitrofluorobenzene (DNFB, Sigma-Aldrich) diluted in acetone
onto abraded back skin. Skin was excised 5 d after the application of DNFB,
fixed in 10% formaldehyde solution, and processed for hematoxylin-eosin
staining. Numbers of infiltrated cells in dermis were counted under a microscope. The mean ± s.d. of the counted cells in high power fields (HPFs) from
three randomly selected regions is plotted (Fig. 4h). All mouse experiments
were repeated at least three times to confirm the reproducibility.
For administration of kallikrein, 100 ml of 10 ng/ml of SCTE (hKLK5; R&D
Systems) was injected subcutaneously twice a day. We injected boiled SCTE
(10 min at 100 1C) and vehicle as controls. Forty-eight h after the last injection
(four injections in total), the skin was biopsied with a 6-mm punch and cut in
half. One-half was fixed in 10% formaldehyde solution and subjected for
hematoxylin-eosin staining. The other half was extracted with 200 ml of RIPA
buffer containing protease inhibitors and subjected to SELDI-TOF-MS analysis.
Numbers of infiltrated cells were counted from three HPFs, and the mean ± s.d.
of four mice is plotted (Fig. 4f).
Skin protease activity. Facial skin was tape-stripped 20 times from the same
lesion with two tapes (D-Squame). The tapes were immersed in 1 ml of
1 M acetic acid and incubated at 4 1C overnight. The protein extracts were
lyophilized completely, and the pellet was dissolved in 40 ml of PBS (pH 7.4).
Protease activity of facial skin surface was monitored by using an EnzCheck
protease assay kit (Molecular Probes) in accordance with the manufacturer’s
instructions. In brief, 10 ml of the aqueous solution collected from the skin surface
was mixed with 190 ml of BODIPY-FL-casein substrate in 10 mM Tris-HCl (pH
7.8), and incubated at 37 1C for 24 h. We monitored protease activity as an
increase in fluorescence with SpectraMax GEMINI EM (Molecular Devices
Corporation). In some experiments, protease inhibitors were added, including
a protease inhibitor mixture (Complete EDTA-free, 1 tablet per 50 ml; Roche),
200 mg/ml of bestatin, 20 mg/ml of E64, 20 mg/ml of aprotinin (Sigma-Aldrich),
200 mM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF), 200 mM human
neutrophil elastase inhibitor (methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl
ketone), and 200 mM human leukocyte elastase inhibitor (methoxysuccinylAla-Ala-Pro-Val-chloromethylketone; Calbiochem).
Statistical analysis. Student’s t-test was used for statistical analyses of cathelicidin protein expression in human skin and cell infiltration in mouse skin
inflammation models. A value of P o 0.05 was considered significant.
Note: Supplementary information is available on the Nature Medicine website.
ACKNOWLEDGMENTS
We thank B. Cottrell for instructions in SELDI-TOF-MS analysis. This work was
supported by the National Institutes of Health (R01-AI052453, R01-AR45676),
The National Rosacea Society and a VA Merit Award (R.L.G.); and the
Association for Preventive Medicine of Japan (K.Y.).
AUTHOR CONTRIBUTIONS
K.Y. conducted SELDI-TOF-MS experiments, protease assays including in situ
zymography and in vivo studies, and wrote the manuscript. A.D.N. conducted
the in vivo skin irritation model. A.B., M.M. and T.O. performed
immunohistochemistry, dot blot and in situ hybridization. A.C. performed
immunofluorescence. R.A.D. prepared and purified peptides. C.B., P.D. and A.H.
contributed to the experiments with Spink5-deficient mice. A.B., V.B.M. and
R.L.G. organized human sample collection. R.L.G. conceived, designed and
supervised all aspects of this work.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
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