CLINICAL AND LABORATORY INVESTIGATIONS
Detection of psoriasin/S100A7 in the sera
of patients with psoriasis
K.S. Anderson*†, J. Wong*, K. Polyak†, D. Aronzon* and C. Enerbäck‡
*Cancer Vaccine Center and †Department of Medical Oncology, Dana-Farber Cancer
Institute, Harvard Medical School, Boston, MA 02115, U.S.A.
‡Department of Clinical Genetics and Department of Dermatology, Sahlgrenska University
Hospital, S-41345 Göteborg, Sweden
Correspondence to Karen S. Anderson.
E-mail: kanderson@partners.org
Conflicts of interest
None declared.
Copyright Journal Compilation © 2009 British Association of Dermatologists
KEYWORDS
autoantibodies • biomarker • psoriasis • S100A7
ABSTRACT
Background Psoriasis is a disease of dysregulated inflammation and epithelial
hyperproliferation in the skin, involving both the innate and adaptive immune system.
Psoriatic keratinocytes express high levels of psoriasin (S100A7), a small calciumbinding protein.
Objectives To determine if patients with active psoriasis have elevated serum levels of
psoriasin and psoriasin-specific autoantibodies.
Methods Blood was collected from 14 patients with psoriasis vulgaris at the start of
narrowband ultraviolet (UV) B therapy and from 11 of these patients every 2 weeks
during the course of the UVB treatment. Patient and control sera were tested for
psoriasin antigen levels by sandwich enzyme-linked immunosorbent assay, and for
psoriasin autoantibody titres using recombinant purified psoriasin and overlapping
peptides.
Results We confirmed strong and specific expression of psoriasin in psoriatic
epidermis by immunohistochemistry. Systemic psoriasin antigen levels tended to be
lower in patients (mean 213 ng mL−1) than in controls (mean 331 ng mL−1, P = 0·308)
and decreased with increasing disease severity. Psoriasin-specific autoantibodies were
detected in a subset of patients with psoriasis and healthy normal donors (mean 0·347
vs. 0·255 units, P = 0·246). The epitopes recognized by the autoantibodies were
mapped to an external loop domain of the molecule but did not show corresponding Tcell immunogenicity.
Conclusions Although psoriasin is overexpressed in psoriatic skin lesions, systemic
levels of psoriasin tended to be lower with increasing disease severity, which may be
due to the presence of psoriasin-specific autoantibodies. Neither psoriasin nor
psoriasin-specific autoantibodies appear to be promising serum biomarkers for clinical
psoriasis.
Accepted for publication 11 August 2008
DIGITAL OBJECT IDENTIFIER (DOI)
10.1111/j.1365-2133.2008.08904.x About DOI
Article Text
Psoriasis is considered to be a genetically determined disease of dysregulated
epidermal hyperproliferation and inflammation, which is initiated and maintained by
multiple mechanisms of the immune system. Recent findings suggest a pathological
collaboration between innate immunity (mediated by antigen-presenting cells and
natural killer T lymphocytes) and acquired immunity (mediated by T lymphocytes)
that results in the inflammatory infiltrate seen in psoriatic plaques.1 Hyperproliferation
of the keratinocytes may be induced by the chemokines and cytokines secreted by
inflammatory cells.
Calcium-binding S100 proteins are involved in many biological processes.2 Several
S100 proteins, specifically S100A7 (psoriasin), S100A8 (calgranulin A) and S100A9
(calgranulin B) have been found to be markedly upregulated in psoriatic skin
lesions.3,4 The expression of all three S100 proteins was induced by the
proinflammatory cytokine interleukin (IL)-22 in psoriatic skin, and the serum level of
IL-22 correlated with psoriasis disease severity.5 We and others have previously
demonstrated a strong correlation between S100A7 and S100A9 expression and
keratinocyte differentiation in normal and psoriatic skin.4,6 Psoriasin expression is also
regulated by interferon (IFN)-γ,7 it has been proposed to be a chemoattractant of
CD4+ lymphocytes in the skin,8 and it upregulates vascular endothelial growth factor.9
These findings suggest a potential role for psoriasin in the dysregulated differentiation,
increased angiogenesis and intense inflammatory reaction characteristic for psoriatic
lesions.
Elevated antigen levels of S100A8/S100A9 molecules have been detected in the sera
of patients with psoriasis and were found to correlate with disease activity.10 As
S100A7, S100A8 and S100A9 are coexpressed in psoriatic skin keratinocytes and are
implicated in the pathogenesis of psoriasis,11,12 we hypothesized that
psoriasin/S100A7 may also be detected in the sera of patients with psoriasis and may
be a potential biomarker of disease severity. However, we found that the level of
serum psoriasin was decreased in patients with psoriasis and decreased with increasing
disease severity. Because psoriasin is partially secreted, we hypothesized that
psoriasin may induce psoriasin-specific autoantibodies that could be used as surrogate
biomarkers in patients with psoriasis. Secreted antigens, including the extracellular
domain of Her2 in breast cancer, can induce autoantibodies that are highly specific
and sensitive biomarkers for breast cancer.13 To test this hypothesis, we developed a
highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of
IgG antipsoriasin autoantibodies, confirmed the specificity of these antibodies by
Western blotting, and mapped the interactive epitopes using overlapping peptides. A
subset of patients with psoriasis and healthy donors had detectable autoantibodies to
psoriasin, but this was independent of disease severity and serum psoriasin levels.
Although the number of patients analysed was fairly small, based on our results
neither psoriasin antigen nor antipsoriasin autoantibodies appear to be promising
serum biomarkers for psoriasis.
Material and methods
Patients and serum samples
Patients with psoriasis vulgaris treated at the psoriasis dermatological clinics at the
Department of Dermatology, Sahlgrenska University Hospital, Göteborg, Sweden,
who had not received systemic (e.g. methotrexate or ciclosporin) or topical (e.g. local
glucocorticoids) treatment for at least 4 weeks prior to sample collection, were
selected for the study. The study was performed in the autumn and the patients had not
received strong summer sun exposure just before the study. Patients with other types
of psoriasis (guttate psoriasis and psoriatic arthritis) were not included. Overall 14
patients were analysed, four women and 10 men. The mean age of the patients was
47 years (range 21–64). Patients received narrowband ultraviolet (UV) B treatment
3 days a week for 8–10 weeks. Patients were treated by whole-body exposure using
Philips TL-01 lamps. Increments were according to standard procedures. Peripheral
blood was collected at the start of and every 2 weeks during the course of UVB
treatment. Serum was immediately frozen on liquid nitrogen and stored at −80 °C.
Peripheral blood mononuclear cells (PBMC) were isolated using ACCUSPIN™
(Sigma-Aldrich, St Louis, MO, U.S.A.) and cellular viability was confirmed by trypan
blue exclusion assay. The study was approved by the ethics committee of Göteborg
University and all patients gave written informed consent. Control serum was obtained
with informed consent from healthy blood donors at the Dana-Farber Cancer Institute.
Immunohistochemistry
Tissue samples for immunohistochemical studies were selected from the files of the
Departments of Dermatology and Pathology at Sahlgrenska University Hospital.
Informed consent was requested from the patients under approval by the ethics
committee of the University of Göteborg. Tissue samples were de-identified prior to
the study. Immunohistochemical analysis was performed essentially as described.14
S100A7 protein purification and peptides
cDNA encoding for full-length human S100A714 was subcloned as an N-terminal Histagged fusion molecule in pQE30 (Qiagen, Valencia, CA, U.S.A.) and expressed in
bacteria. Protein was purified under denaturing conditions in 6 mol L−1 guanidine-HCl
with 100 mmol L−1 NaCl and 10 mmol L−1β-mercaptoethanol. Protein was purified
using a nickel NTA column (Qiagen) using a slow urea step gradient with 0·5 mol L−1
NaCl and 20% glycerol. Protein purity was confirmed by sodium dodecyl sulphate–
polyacrylamide gel electrophoresis (SDS-PAGE), and concentration determined by
bicinchoninic assay (Pierce Biotechnology, Rockford, IL, U.S.A.). Overlapping
peptides (15-mer, overlapping by 10) spanning the full-length molecule were obtained
from Invitrogen (Carlsbad, CA, U.S.A.). S100A7–glutathione-S-transferase (GST)
fusion proteins and GST control protein were expressed from pGEX4T-1 (Amersham,
Piscataway, NJ, U.S.A.) and purified as described.15
Sandwich enzyme-linked immunosorbent assay for
the detection of S100A7 in serum
Monoclonal antibodies 817, 1068 and 396 were generated by standard immunization
with recombinant psoriasin.14 A mixture of two capture antibodies, clones 817 and
1068, each at 2 μg mL−1 in carbonate binding buffer pH 9·6, were coated overnight at
4 °C on to 96-well Nunc-Immuno plates (Nalge Nunc, Rochester, NY, U.S.A.). The
wells were blocked with 1% bovine serum albumin, then 50 μL of human serum was
added in duplicate to each well at room temperature for 1 h. The detection antibody
396 was biotinylated using an EZ-Link biotinylation kit (Pierce Biotechnology), used
at 2 μg mL−1, and detected with streptavidin–horseradish peroxidase (HRP) and
SuperSignal ELISA Substrate (Pierce Biotechnology) using a Victor3 plate reader
(PerkinElmer, Waltham, MA, U.S.A.).
Enzyme-linked immunosorbent assay and Western
blot for the detection of S100A7-specific
autoantibodies
His-tagged psoriasin antigen and peptides were diluted to 5 μg mL−1 in binding buffer
and coated overnight at 4 °C on to 96-well Nunc-Immuno plates (Nalge Nunc) as
described.15 Plates were blocked with 2% nonfat milk and 50 μL of sera (1 : 300
dilution) was added in duplicates followed by incubation with secondary antibody
(goat antihuman IgG-HRP, 1 : 5000; Jackson ImmunoResearch, West Grove, PA,
U.S.A.) and detection with TMB+ Substrate (Dako, Carpinteria, CA, U.S.A.). For
Western blotting, 10 μg of S100A7-GST or GST antigen was separated by SDSPAGE, transferred to nitrocellulose membrane, immunoblotted with patient sera at
1 : 500 and goat antihuman IgG-HRP (Jackson ImmunoResearch) and detected with
ECL reagent per manufacturer's instructions (NEN, Boston, MA, U.S.A.).
In vitro stimulation of peripheral blood mononuclear
cells and interferon-γ enzyme-linked immunospot
assay
PBMC from patients with psoriasis were stimulated in cell culture for 1 week in the
presence of 10 ng mL−1 IL-7 (Endogen Inc., Woburn, MA, U.S.A.) from day 0 and
20 IU mL−1 IL-2 (Chiron Corp., Emeryville, CA, U.S.A.) added on days 1 and 4.
Peptides were added on day 0 at 10 μg mL−1. On day 7, 50 000 PBMC per well were
coincubated with peptide pools corresponding to final concentrations of 4 μg mL−1 of
each individual peptide in triplicate in enzyme-linked immunospot (ELISPOT) plates
(Millipore, Bedford, MA, U.S.A.) for 24 h. IFN-γ secretion was detected following the
manufacturer's instructions (Mabtech AB, Nacka Strand, Sweden) and imaged using
an ImmunoSpot Series Analyzer (Cellular Technology Ltd, Cleveland, OH, U.S.A.).
Statistical analysis
Psoriasis Area and Severity Index (PASI) score was confirmed with a self-reported
patient opinion index of disease severity (a scale of 0–10 with 10 at start of treatment).
Patients were considered responders if their PASI score improved by at least 75%
compared with baseline or their self-opinion score improved to below 4. Three
patients who withdrew before at least three samples were collected were not included
in the analysis. Kappa statistic was used to analyse associations among the outcome of
responder status using the PASI and patient opinion at the last available time point.
The statistical significance of the differences between patients and normals in ELISA
was analysed using two-sided Student's t-test.
Results
Clinical response to ultraviolet B treatment
In order to determine if psoriasin may be used as a serological biomarker of psoriasis,
14 patients with active psoriasis were enrolled in a clinical trial through the
dermatology clinic in Göteborg, Sweden. Individual PASI scores were recorded over
time (Fig. 1). Baseline PASI scores ranged from 2·0 to 25·3 (mean 8·5). Patients
received standard UVB therapy and serum samples were collected at weeks 0, 2, 4, 6
and 10 weeks follow-up. Patients were deemed eligible for the analysis if assessed at
4 weeks or later after therapy was initiated (n = 11) and the last time point was used
for evaluation. Nine of 11 eligible patients (82%) had at least a 75% improvement in
PASI score and all patients had a decrease in PASI score with UVB therapy
(P < 0·003). This is consistent with published data on the rate of response to UVB
therapy.16 Two patients (patients 8 and 14) had a transient increase in PASI score at
week 6 after initiation of treatment, but they subsequently improved. PASI score was
confirmed with a self-reported patient opinion index of disease severity shown in
Table 1. Ten of 11 (91%) patients had a severity index of improvement < 5. Kappa
statistic of 0·62 (n = 11) showed significant agreement between the two scoring
systems.
Fig 1. Decrease in Psoriasis Area and Severity
Index (PASI) score with ultraviolet (UV) B
treatment of psoriasis. Fourteen patients with
moderate to severe psoriasis vulgaris at baseline
underwent UVB treatment and evaluation every
2 weeks until study completion. Individual PASI
scores at 2-week intervals are shown, and decreased
in all patients with treatment.
[Normal View ]
Table 1 Decrease in clinical disease severity with time (A–E) on ultraviolet (UV) B
treatment
PASI score
Patient opinion
Patient
A
B
C
D
E
A
B
C
D
E
1
2
3
4
5
6
7
8
9
10
11
12
8·4
8·3
3·9
25·3
3·3
2
15·6
9
4·2
5·4
4·2
8·2
8·4
5·4
1·8
15·7
3·3
2
2·7
1·3
12·9
0·7
0·5
1·6
0·8
6·4
0
1·5
0·9
10
7
6
6
10
4
5
4
5
5
2
3
2
3
0
4
2
7
3·4
4·5
2·5
5·7
5
1·9
2·7
6·3
0·4
1·35
2·2
0
0
8
3
4·25
3
0
3
0·2
0
7
9
8
6·5
6
6
7
5·5
0·4
10
10
10
10
10
10
10
10
10
10
10
10
0
0
0
13
14
3·6
17·2
2·8
14·4
1·6
11·2
0·4
12·8
0
4·8
10
10
8·5
8
6
7
3
8
0
5
Fourteen patients with moderate to severe psoriasis vulgaris at baseline (A) underwent UVB
treatment and evaluation every 2 weeks (B, C and D) until study completion (E). Clinical
improvement after UVB treatment is shown by Psoriasis Area and Severity Index (PASI)
score and self-reported patient opinion of severity (range 1–10, with 10 at baseline).
Psoriasin is specifically overexpressed in psoriatic
skin lesions
Elevated antigen levels of S100A8/S100A9 molecules have been detected in the sera
of patients with psoriasis, and they correlated with disease activity.10 As
psoriasin/S100A7 expression is also upregulated in psoriatic skin6 and it is a partially
secreted protein,14 we hypothesized that psoriasin would also be elevated in the serum.
To develop a highly sensitive sandwich ELISA for psoriasin, three distinct
monoclonal antibodies were raised in mice by S100A7 injection.14
Immunohistochemical analysis of psoriatic skin lesions using the monoclonal antibody
clone 1068 demonstrated high psoriasin expression in psoriatic keratinocytes and in
the epithelial cells of normal hair follicles (Fig. 2). Psoriasin expression was often
stronger in keratinocytes in the superficial parts of the hyperplastic epithelium and
absent in the basal layers. In the keratinocytes, strong staining was often observed in
the peripheral parts of the individual cells and faint staining could sometimes be
observed in apparent intercellular bridges (desmosomes). Low expression of psoriasin
was observed in keratinocytes of normal skin. In some cases, the epithelium was
completely negative, but it often showed faint staining in superficial parts, which
represent the more differentiated cells (data not shown).
Fig 2. S100A7 (psoriasin) is strongly
overexpressed in psoriatic skin. Immunostaining
using monoclonal antibody 1068 directed against
psoriasin at low (a) and high (b) power. There is
preferential staining in differentiated spinous layer
cells of the central and superficial parts of the
hyperplastic and elongated rete ridges.
[Normal View ]
Serum levels of S100A7 antigen are decreased in the
sera of patients with psoriasis
We developed a sandwich ELISA for the detection of S100A7 protein in the sera of
patients with psoriasis. Detection limits were calibrated using recombinant purified
His-tagged S100A7 and S100A7-GST (Fig. 3 and data not shown). Both His-S100A7
and S100A7-GST had comparable limits of detection. GST alone was not detected and
the data depicted in Figure 3 are representative of more than five replicate assays. The
ELISA had a detection limit of 20 ng mL−1, which is significantly below the
365 ng mL−1 reported mean serum levels of S100A8/S100A9 in normal individuals.10
The range of detection was 20–2000 ng mL−1 and titration curves of recombinant
S100A7 protein were included with every assay. ELISA analysis of serum protein
concentration of psoriasin was performed in duplicate on all 14 patients with psoriasis
at baseline and run concurrently with sera from 14 healthy normal volunteers twice. In
contrast to S100A8/S100A9, the mean serum levels of S100A7 antigen were
decreased in patients with psoriasis (Fig. 4a, mean 213 ng mL−1) compared with
normals (mean 331 ng mL−1) but this was not statistically significant (P = 0·308).
S100A7 serum protein levels were comparable with the reported mean serum levels of
S100A8/S100A9 in normals (365 ng mL−1).10
Fig 3. Development of an enzyme-linked
immunosorbent assay (ELISA) for the detection of
psoriasin antigen. Recombinant His-tagged
psoriasin protein was purified and titrated in a
sandwich ELISA to determine the level of
sensitivity of detection of psoriasin protein. The
assay consisted of two capture monoclonal
antibodies (mAbs) (including mAb 1068 shown in
Figure 2) and an independent biotinylated mAb
detector antibody. The assay shown here was
representative of over five different quantitative
assays. Both His-tagged and glutathione-Stransferase (GST)-tagged psoriasin protein showed
similar limits of detection to 20 ng mL−1 psoriasin,
and GST protein alone was not detected (not
shown).
[Normal View ]
Fig 4. Serum levels of psoriasin/S100A7 are
decreased in patients with psoriasis. (a) Serum
levels of psoriasin were assayed using the psoriasin
enzyme-linked immunosorbent assay in sera from
all 14 patients with psoriasis at baseline, and in sera
from 14 healthy volunteers. Levels of psoriasin
detected in the sera were lower in patients (mean
213 ng mL−1) vs. healthy donors (mean
331 ng mL−1) but this difference was not significant
(P = 0·308). (b) Psoriasin levels do not increase
with increased disease severity as measured by
Psoriasis Area and Severity Index (PASI) score. The
patients with psoriasis shown in (a) were separated
into two groups, those with lower PASI score
(n = 7, mean psoriasin level 239 ng mL−1) and those
with higher PASI score (n = 7, mean psoriasin level
188 ng mL−1): differences in psoriasin level between
the groups were not significant (P = 0·25).
[Normal View ]
The patients assayed in Figure 4(a) were divided into two groups by disease severity
as measured by PASI score. The mean levels of psoriasin detected in the sera of
patients with low PASI score (mean 239 ng mL−1) decreased with increased severity
of the disease (188 ng mL−1, P = 0·504; Fig. 4b). This result is in contrast to the
correlation of S100A8/S100A9 level with disease severity, where mean levels of 1315
ng mL−1 were observed in patients with high PASI score.10 UVB therapy did not
significantly affect S100A7 antigen levels in the 11 patients included in the study (data
not shown, P = 0·56).
Autoantibodies to S100A7 are not elevated in the
serum of patients with psoriasis
We hypothesized that the lack of increased psoriasin protein levels in the serum of
patients with psoriasis could be due to autoantibodies to S100A7 that may result in the
clearance of antigen from the serum and could themselves be potential biomarkers of
disease. To detect these autoantibodies we developed a sandwich ELISA using
purified recombinant His-tagged S100A7 antigen refolded in urea following
denaturation with guanidium chloride. Sera from the 14 patients with active psoriasis
and from 14 healthy controls were assayed in duplicate, repeated twice (Fig. 5a). We
determined that patients with active psoriasis had slightly increased autoantibodies to
S100A7 compared with healthy controls; however, these differences were not
statistically significant (mean 0·347 vs. 0·255 units, P = 0·246). Antipsoriasin
autoantibody levels also did not correlate with disease severity and serum S100A7
protein levels (data not shown). The half-life of IgG in serum is 23 days;17 thus, IgG
autoantibody levels might decrease following 4–6 weeks of therapy. Correlating with
this, autoantibodies to p53 antigen, a biomarker of breast cancer, decrease rapidly and
specifically after induction chemotherapy.15 In the 11 patients analysed S100A7
autoantibody levels did not significantly change after UVB treatment (P = 0·83). The
specificity of the autoantibody identified by ELISA for psoriasin was confirmed by
Western blotting sera from a patient with breast cancer (a disease also associated with
S100A7 overexpression14) that had elevated S100A7 autoantibodies detected by the
ELISA (Fig. 5b).
Fig 5. Detection of S100A7-specific antibodies in
sera. (a) Detection of S100A7-specific antibodies by
enzyme-linked immunosorbent assay (ELISA).
Recombinant His-tagged S100A7 protein was
adhered to 96-well plates, and detected with sera
from healthy donors (left) and patients with
psoriasis (right). No detectable difference in
antibody levels was observed (mean 0·347 vs.
0·255 units, P = 0·246). O.D., optical density. (b)
S100A7 autoantibodies detected by ELISA are
specific to S100A7 protein. Recombinant
glutathione-S-transferase (GST) and S100A7-GST
fusion proteins were purified and separated by
sodium dodecyl sulphate–polyacrylamide gel
electrophoresis and detected by Coomassie staining
(left). The antigens were immunoblotted using
1 : 500 dilution of sera from a patient with breast
cancer that had elevated levels of S100A7
autoantibodies by the ELISA shown in (a). The
S100A7-GST fusion protein is specifically detected
(arrow), while the control GST protein is not (right).
[Normal View ]
Epitope mapping of anti-S100A7 antibodies
S100A7 molecules are small, homodimeric Zn- and Ca-binding molecules. To confirm
the specificity of the anti-S100A7 autoantibodies that we identified using ELISA, 15mer overlapping peptides spanning the full-length psoriasin protein were coated on to
96-well plates and assayed with pretreatment patient sera from patients 6, 9 and 12
(Fig. 6). The immunodominant regions of the S100A7 protein included the N-terminal
portion and peptides 8, 9 and 17. Using the crystal structure of S100A7,18 we
determined that these peptides 8, 9 and 17 are located on the same external region of
the molecule (Fig. 7).
Fig 6. Mapping of the immunogenic regions of
S100A7. Overlapping peptides (15-mers) were
adhered to 96-well plates, and the antibody-positive
psoriasis sera from Figure 5 were added at 1 : 300
dilution. Peptide-specific autoantibodies to the
peptides shown were detected.
[Normal View ]
Fig 7. Structural mapping of the immunodominant
epitopes of S100A7. A ribbon diagram of the
homodimeric S100A7 molecules is shown, with the
immunogenic peptides 8 (blue), 9 (red) and 17
(green) outlined in colour.
[Normal View ]
Patients with psoriasis do not have detectable T cells
to psoriasin peptides
The data from Figures 5(a) and 6 suggested that a subset of patients develops active
immunity to psoriasin. Because antibody responses to proteins can be associated with
measurable T-cell responses and psoriasis is associated with a T-cell inflammatory
process, we examined PBMC from the patients at baseline for the presence of IFN-γsecreting T cells to psoriasin-derived peptides. Using the set of 18 overlapping 15-mer
peptides spanning the entire molecule, we created three pools of six peptides, and
stimulated PBMC for 1 week with the peptides. PBMC were also stimulated with no
peptide control and with phytohaemagglutinin (PHA; positive control). After 1 week
of stimulation, the cells were restimulated on ELISPOT plates with the peptides, and
IFN-γ-secreting cells were quantitated by standard ELISPOT analysis (Fig. 8). T-cell
function was confirmed with PHA stimulation. No psoriasin-specific T cells were
detected in the PBMC of the 14 patients at baseline.
Fig 8. Interferon (IFN)-γ-secreting T cells to
psoriasin peptides are not detected in patients with
psoriasis. Peripheral blood mononuclear cells
(PBMC; 50 000 cells per well) were stimulated in
vitro for 1 week with three separate pools (pools 1,
2 or 3) each containing six overlapping peptides of
psoriasin, or no peptide. IFN-γ secretion of activated
T cells was measured by enzyme-linked
immunospot analysis, and a representative patient
sample is shown. No psoriasin-specific T cells were
detected in the PBMC of the 14 patients at baseline.
The cells could be readily stimulated to secrete IFNγ with the mitogen phytohaemagglutinin (PHA).
[Normal View ]
Discussion
Active psoriatic skin lesions are associated with increased serum levels of S100A8
(calgranulin A), S100A9 (calgranulin B) as well as that of several cytokines including
IFN-γ, IL-12, IL-17, IL-18 and IL-22.19 Based on these prior data, we hypothesized
that, similar to S100A8/S100A9, elevated levels of S100A7 protein would be detected
in the sera of patients with active psoriasis, and that these levels would be correlated
with treatment and disease severity. However, we found slightly decreased levels of
S100A7 in the sera of patients with active psoriasis, and these levels decreased with
increasing disease severity. The absolute levels detected in psoriasis were markedly
lower than in a published report on the S100A8/S100A9 levels in serum.10 Whereas
this may reflect differences in the relative expression or secretion of the different S100
proteins in active psoriasis, we confirmed by immunohistochemistry that S100A7 is
strongly expressed in psoriatic lesions and that it can be detected by the antibody used
in our ELISA system.
This led us to hypothesize that psoriasin/S100A7 antigen may be cleared by the
systemic circulation. This could occur by rapid protein binding or by induction of
autoantibodies that enhance clearance of the complex. Psoriasin/S100A7 forms a
complex with fatty acid-binding protein in keratinocytes20 and both proteins can be
detected by Western blotting in the urine of patients with melanoma.21 Numerous selfantigens have been identified that induce autoantibodies that can be detected in the
sera of patients.13 Certain antigens, such as Ro/SS-A, La/SS-B and others22,23 that are
associated with autoimmune disease, are used as biomarkers of disease severity. Other
cancer autoantibodies are induced by strong overexpression of the antigen, such as
mutant p53 antigen,24 her2/neu,25 MUC126 and NY-ESO-1.27 We reasoned that the
high expression and partial secretion of psoriasin made it a good candidate
autoantigen. We detected psoriasin-specific antibodies in a subset of patients with
psoriasis, but this did not correlate with the presence of disease, disease severity,
antigen levels, or presence of T-cell immunity. As psoriasin is expressed at low levels
in healthy skin, this may stimulate the development of low levels of autoantibodies to
psoriasin in a subset of healthy donors. It remains to be determined what impact, if
any, autoantibodies to psoriasin have on the clinical outcome of psoriasis. Psoriasinspecific antibodies were also recently detected in patients with ovarian cancer.28
It is not surprising that psoriasin induces a specific immune response detectable in the
systemic circulation. Psoriasis was originally considered to be primarily a keratinocyte
proliferation disorder based on the clinical signs of epidermal hyperplasia. The
persistent inflammatory infiltrate in psoriasis led to the proposal that the epidermal
abnormalities were driven by mediators of T-cell activation. This was initially
suggested by the therapeutic effect of T-cell inhibitors, such as ciclosporin.29
Mediators of adaptive immunity, including Th1 cells, antigen-presenting cells and
IFN-γ, are implicated in the pathogenesis of psoriasis,30 as are mediators of innate
immunity, such as tumour necrosis factor-α.31 Despite the intense inflammatory
infiltrate, we did not detect psoriasin-specific IFN-γ-secreting T cells in the peripheral
blood of patients with active psoriatic lesions, suggesting that the T-cell infiltration is
not specific to psoriasin.
Acknowledgments
We thank Imgenex for providing monoclonal antibodies for these studies. This study
was supported by a research grant from the Swedish Psoriasis Foundation and the
Welander Foundation.
References

1
Gaspari AA. Innate and adaptive immunity and the pathophysiology of
psoriasis. J Am Acad Dermatol 2006; 54:S67–80. Links

2
Donato R. Intracellular and extracellular roles of S100 proteins. Microsc Res
Tech 2003; 60:540–51. Links

3
Madsen P, Rasmussen HH, Leffers H et al. Molecular cloning, occurrence,
and expression of a novel partially secreted protein 'psoriasin' that is highly upregulated in psoriatic skin. J Invest Dermatol 1991; 97:701–12. Links

4
Broome AM, Ryan D, Eckert RL. S100 protein subcellular localization during
epidermal differentiation and psoriasis. J Histochem Cytochem 2003; 51:675–
85. Links

5
Wolk K, Witte E, Wallace E et al. IL-22 regulates the expression of genes
responsible for antimicrobial defense, cellular differentiation, and mobility in
keratinocytes: a potential role in psoriasis. Eur J Immunol 2006; 36:1309–23.
Links

6
Martinsson H, Yhr M, Enerbäck C. Expression patterns of S100A7 (psoriasin)
and S100A9 (calgranulin-B) in keratinocyte differentiation. Exp Dermatol
2005; 14:161–8. Links

7
Petersson S, Bylander A, Yhr M et al. S100A7 (psoriasin), highly expressed in
ductal carcinoma in situ (DCIS), is regulated by IFN-gamma in mammary
epithelial cells. BMC Cancer 2007; 7:205. Links

8
Jinquan T, Vorum H, Larsen CG et al. Psoriasin: a novel chemotactic protein.
J Invest Dermatol 1996; 107:5–10. Links

9
Krop I, Marz A, Carlsson H et al. A putative role for psoriasin in breast tumor
progression. Cancer Res 2005; 65:11326–34. Links

10
Benoit S, Toksoy A, Ahlmann M et al. Elevated serum levels of calciumbinding S100 proteins A8 and A9 reflect disease activity and abnormal
differentiation of keratinocytes in psoriasis. Br J Dermatol 2006; 155:62–6.
Links

11
Ryckman C, Vandal K, Rouleau P et al. Proinflammatory activities of S100:
proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and
adhesion. J Immunol 2003; 170:3233–42. Links

12
Nukui T, Ehama R, Sakaguchi M et al. S100A8/A9, a key mediator for
positive feedback growth stimulation of normal human keratinocytes. J Cell
Biochem 2008; 104:453–64. Links

13
Anderson KS, LaBaer J. The sentinel within: exploiting the immune system
for cancer biomarkers. J Proteome Res 2005; 4:1123–33. Links

14
Enerbäck C, Porter DA, Seth P et al. Psoriasin expression in mammary
epithelial cells in vitro and in vivo. Cancer Res 2002; 62:43–7. Links

15
Anderson KS, Ramachandran N, Wong J et al. Application of protein
microarrays for multiplexed detection of antibodies to tumor antigens in breast
cancer. J Proteome Res 2008; 7:1490–9. Links

16
Picot E, Picot-Debeze MC, Meunier L et al. [Narrow-band UVB phototherapy
(Philips TL01 lamps) in psoriasis]. Ann Dermatol Venereol 1992; 119:639–42.
Links

17
Wochner RD, Drews G, Strober W et al. Accelerated breakdown of
immunoglobulin G (IgG) in myotonic dystrophy: a hereditary error of
immunoglobulin catabolism. J Clin Invest 1966; 45:321–9. Links

18
Brodersen DE, Nyborg J, Kjeldgaard M. Zinc-binding site of an S100 protein
revealed. Two crystal structures of Ca2+-bound human psoriasin (S100A7) in
the Zn2+-loaded and Zn2+-free states. Biochemistry 1999; 38:1695–704.
Links

19
Arican O, Aral M, Sasmaz S, Ciragil P. Serum levels of TNF-alpha, IFNgamma, IL-6, IL-8, IL-12, IL-17, and IL-18 in patients with active psoriasis
and correlation with disease severity. Mediators Inflamm 2005; 2005:273–9.
Links

20
Ruse M, Broome AM, Eckert RL. S100A7 (psoriasin) interacts with
epidermal fatty acid binding protein and localizes in focal adhesion-like
structures in cultured keratinocytes. J Invest Dermatol 2003; 121:132–41.
Links

21
Brouard MC, Saurat JH, Ghanem G et al. Urinary excretion of epidermal-type
fatty acid-binding protein and S100A7 protein in patients with cutaneous
melanoma. Melanoma Res 2002; 12:627–31. Links

22
Routsias JG, Tzioufas AG, Moutsopoulos HM. The clinical value of
intracellular autoantigens B-cell epitopes in systemic rheumatic diseases. Clin
Chim Acta 2004; 340:1–25. Links

23
Lernmark A. Autoimmune diseases: are markers ready for prediction? J Clin
Invest 2001; 108:1091–6. Links

24
Soussi T. p53 antibodies in the sera of patients with various types of cancer: a
review. Cancer Res 2000; 60:1777–88. Links

25
Disis ML, Pupa SM, Gralow JR et al. High-titer HER-2/neu protein-specific
antibody can be detected in patients with early-stage breast cancer. J Clin
Oncol 1997; 15:3363–7. Links

26
von Mensdorff-Pouilly S, Petrakou E, Kenemans P et al. Reactivity of natural
and induced human antibodies to MUC1 mucin with MUC1 peptides and nacetylgalactosamine (GalNAc) peptides. Int J Cancer 2000; 86:702–12. Links

27
Chen YT, Scanlan MJ, Sahin U et al. A testicular antigen aberrantly expressed
in human cancers detected by autologous antibody screening. Proc Natl Acad
Sci USA 1997; 94:1914–18. Links

28
Gagnon A, Kim JH, Schorge JO et al. Use of a combination of approaches to
identify and validate relevant tumor-associated antigens and their
corresponding autoantibodies in ovarian cancer patients. Clin Cancer Res
2008; 14:764–71. Links

29
Griffiths CE, Powles AV, Leonard JN et al. Clearance of psoriasis with low
dose cyclosporin. Br Med J (Clin Res Ed) 1986; 293:731–2. Links

30
Sabat R, Sterry W, Philipp S et al. Three decades of psoriasis research: where
has it led us? Clin Dermatol 2007; 25:504–9. Links

31
Bos JD, de Rie MA, Teunissen MB et al. Psoriasis: dysregulation of innate
immunity. Br J Dermatol 2005; 152:1098–107. Links