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Research Communications |
activation in response to 
-IFN
Departments of
a Dermatology and
b Pathology, University of Edinburgh, Edinburgh EH3 9YW, Scotland, U.K.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-IFN) and are believed
to be necessary to maintain the psoriatic phenotype. In normal KC,
-IFN is a potent inhibitor of proliferation, but proliferation of KC
persists in psoriatic plaques despite the presence of -IFN.
Immunostaining of interferon regulatory factor-1 (IRF-1) revealed that
IRF-1 was localized to the basal cells of the epidermis in normal and
in nonlesional psoriatic skin, but was suprabasal or completely absent
in lesional psoriatic skin. This finding led to the hypothesis that
abnormal signaling in the -IFN pathway may occur in psoriatic KC. To
test this hypothesis, we measured activation of IRF-1 and signal
transducer and activator of transcription (STAT)-1
transcription
factors in KC after stimulation with -IFN. Primary cultures of KC
from normal and nonlesional psoriatic skin were stimulated with -IFN
and subsequent transcription factor activation was measured by
electrophoretic mobility shift assay. Psoriatic KC showed a reduced
induction of IRF-1 and STAT-1 activation after stimulation with
-IFN, compared with normal KC. Reduced activation of IRF-1 and
STAT-1 in response to -IFN indicates a fundamental defect in the
growth and differentiation control of psoriatic KC in the absence of
the influence of other cell types.—Jackson, M., Howie, S. E. M., Weller, R., Sabin, E., Hunter, J. A. A., McKenzie,
R. C. Psoriatic keratinocytes show reduced IRF-1 and STAT 1-
activation in response to -IFN.
Key Words: psoriasis • -interferon • fibronectin • KC • GAS sites
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, interleukin-6, interleukin-8, and
gamma interferon (-IFN) (3)
, that, in addition to the
epidermal-derived cytokines, create an intense inflammatory reaction.
The reciprocal activation of CD8+ T cells and KC in this
cytokine-rich milieu is a crucial component of the immunopathology of
this disease phenotype. The factors involved in triggering and maintaining the chronic plaque of psoriasis remain undetermined, since no adequate animal or in vitro model exists with which to test them directly. Current therapies available for psoriasis reduce the symptoms of the disease, which often recurs once treatment ceases. An understanding of mechanisms underlying psoriasis immunopathogenesis is therefore of great importance for the development of more effective and specific treatments.
Epidermal proliferation persists in psoriatic plaques despite the
presence of -IFN (4)
, a potent antiproliferative
cytokine and inducer of squamous differentiation (5)
,
represent an intriguing paradox. This fact, in addition to experimental
data indicating that psoriatic KC are less sensitive to the
antiproliferative effect of -IFN (6
, 7
),
suggests that there may be a defect in the -IFN signaling pathway.
The -IFN receptor signaling complex consists of two major subunits,
a 90 kDa chain (the primary ligand binding domain) and a 60 kDa ß
chain necessary for signal transduction (8)
. Neither the
nor ß chain of the -IFN receptor (R) have intrinsic tyrosine
kinase activity. Therefore, -IFN receptor (-IFN R) signaling
relies on the activity of JAK-1 and JAK-2 tyrosine kinases (Janus
kinases), which associate with the -IFN R- and -ß chains,
respectively (9)
. After oligomerization of the receptor
chains, the associated JAK-1 and JAK-2 tyrosine kinases reciprocally
activate each other by phosphorylation.
An immediate event after ligand binding and receptor complex
oligomerization is the recruitment of signal transducer and activator
of transcription (STAT-1) to phosphorylated tyrosine-440 on the
-IFN receptor chain (10)
. This recruitment results
in the phosphorylation of STAT-1 by the JAK kinases and the
formation of STAT-1 homodimers, which then translocate to the
nucleus and bind to the gamma-activated sequence (or GAS sites) in the
promoters of -IFN inducible genes (11)
.
A critical gene regulated by STAT-1 in the -IFN pathway is
interferon regulatory factor-1 (IRF-1). IRF-1 is a transcription factor
that binds to an interferon-stimulated response element (ISRE) in the
promotor of -IFN inducible genes such as intercellular adhesion
molecule-1 (12)
, major histocompatibility complex II
(13)
, and inducible nitric oxide synthase
(14)
. The binding of STAT-1 to the GAS sites in the
IRF-1 promoter is an essential event since STAT-1 knockout mice are
unable to induce IRF-1 in response to -IFN (15)
. IRF-1
can, however, be induced directly by other cytokines such as
interleukin-6 and leukemia inhibitory factor (16)
.
The outcome of the -IFN response is also regulated by IRF-2, a
transcription factor with homology to IRF-1. IRF-2 binds to the same
DNA motif and represses the activity of IRF-1 (17)
.
Although both IRF-1 and IRF-2 are constitutively expressed in a variety
of cell types, IRF-2 usually predominates over IRF-1 in resting cells
(exhibiting 10-fold more binding activity) due to the greater stability
of IRF-2 protein (18)
. After stimulation with -IFN, the
transcription of IRF-1 is induced and its protein levels increase to
above the IRF-2 basal level.
IRF-1 has growth inhibitory effects (16) and has been implicated as a tumor suppressor gene (19) because of its role in DNA damage-induced apoptosis (20) . Conversely, IRF-2 has been implicated as having oncogenic activity since overexpression of IRF-2 in fibroblasts caused the loss of growth control in vitro and tumor formation when injected into nude mice (21) . Therefore, the ratio of growth inhibitory IRF-1 to growth-promoting IRF-2 plays a major role in the proliferative capacity of the cell.
Since psoriasis has a genetic component (22)
, the
nonlesional skin of a psoriatic patient is a valid model with which to
investigate differences between psoriatic and normal KC. Nonlesional KC
from psoriatic patients have a higher proliferative index, which is
maintained on transplantation to nude mice (23)
. In
addition, nonlesional skin of a psoriatic individual may form a plaque
after mechanical injury to the epidermis (the Koebner phenomenon).
Therefore, to dissect out the response of psoriatic KC to -IFN from
the intense inflammatory reaction within a psoriatic lesion, we used
nonlesional skin from psoriatic individuals to generate primary KC
cultures in vitro. The use of such established cultures
allows investigation of intrinsic differences in the IRF-1 and
STAT-1 response to -IFN in the KC from psoriatic or an unaffected
individual's skin. Here we provide evidence that both IRF-1 and
STAT-1 activity are reduced in psoriatic KC in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immunohistochemical staining of skin sections
Elliptical biopsies were obtained from lesional and nonlesional
skin of each patient with chronic plaque psoriasis and from healthy
volunteers. Biopsies were fixed in 10% buffered formalin, pH 7.4,
before embedding in low-temperature paraffin wax and cutting into 3
µm sections. Sections were deparaffinized through xylene and graded
ethanol to distilled water. Endogenous peroxidase was blocked by
immersion in 1% hydrogen peroxide (Sigma, Poole, Dorset, U.K.),
followed by permeabilization in boiling 7.14 mM sodium citrate buffer
pH 6 (Sigma) in a microwave oven for 2 x 2 min. Sections were
loaded onto a Sequenza (Shandon) and blocked in 20% normal swine serum
(NSS)/Tris-buffered saline (TBS) pH 7.6. Sections were stained with a
1:300 dilution of polyclonal anti-IRF-1 (Santa Cruz, Autogen, Bioclear,
Wilts, U.K.) or a 1:400 polyclonal anti-s100 (Dako U.K. Ltd, Ely,
Cambridgeshire, U.K.) in NSS/TBS. Anti-s100 stains melanocytes and
Langerhans' cells in the skin, which served as a positive control for
immunostaining. Staining was detected with a 1:400 dilution of
biotinylated swine anti-rabbit F(ab')2 immunoglobulins
(Dako), followed by incubation in avidin/biotin horseradish peroxidase
complex (Dako). All incubations were for 30 min at room temperature.
Staining was visualized by a 5 min incubation in diaminobenzidine in
0.05 M Tris/HCl buffer pH 7.4 containing 0.01% hydrogen peroxide.
Substitution of the primary antibodies with 20% NSS/TBS showed no
detectable staining.
Culture of primary human keratinocytes
Subcutaneous fat was removed from the skin, which was chopped
into 1 cm squares before digestion in 0.25% trypsin/EDTA (Gibco Life
Technologies, Paisley, U.K.) in salt solution (8 g NaCl, 0.4 g
KCl, 1 g glucose/l pH 7) (24)
with the addition of
100 IU/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml
Fungizone (Gibco). After digestion with trypsin, the epidermis was
scraped off with a scalpel blade, followed by disaggregation of
epidermal cells by gentle pipetting. Epidermal cell suspensions were
centrifuged at 800 x g for 5 min at 4°C and
resuspended in complete keratinocyte serum-free medium (KSFM) (Gibco)
supplemented with epidermal growth factor, bovine pituitary extract as
supplied by the manufacturer, penicillin, streptomycin, and Fungizone
(as above).
Primary KC cultures were initiated by plating out epidermal cell
suspensions on a feeder layer of irradiated Swiss 3T3 cells. Six-well
tissue culture dishes (Corning Costar, High Wycombe, Bucks., U.K.) were
coated with 5 µg/cm2 fibronectin (FBN) (kindly provided
by the Scottish National Blood Transfusion Service, Edinburgh, U.K.) in
phosphate-buffered saline (PBS) for 20 min at room temperature before
aspirating the excess FBN/PBS. Swiss 3T3 were -irradiated (5000
RADS), plated out at 2 x 105 per well in FBN-coated
plates, and incubated at 37°C/5% CO2 in a humidified
atmosphere for 3 h to adhere. Epidermal cell suspensions were then
plated out on top of feeders at 3 x 105 cells per
well.
Cultures received fresh KSFM every 2 or 3 days; KC colonies that came to confluence between 10 and 32 days were passaged to expand numbers in tissue culture flasks without an FBN coating or a 3T3 feeder layer. To compare normal and psoriatic KC at the same passage, KC were frozen after passage 1 and thawed to use at passage 2 in experiments.
Stimulation of primary KC with -IFN
KC were plated out at 1 x 105 cells per well
of a 6-well dish and -IFN was added between 70 and 90% confluence.
Initially, 1000 IU/ml -IFN (R&D Systems, Abingdon, Oxon, U.K.) was
added to KC cultures in complete KSFM and cells were harvested at
various times after stimulation. Later experiments used 30 IU/ml.
Nuclear extracts
At the time of harvesting, KC cultures were placed on ice
and washed with ice-cold PBS. Cells from each well were then scraped
off into 1 ml ice-cold PBS, centrifuged at 10,000 x g
for 30 s, and lysed in 1 ml buffer A [20 mM HEPES pH 7.9, 10 mM
KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM PMSF, 0.1 mM NaVO4,
0.2% IGEPAL (Sigma, Poole, Dorset, U.K.), 10 % glycerol]
(25)
. Lysates were centrifuged for 2 min at 10,000 x
g and the supernatant was discarded before re-centrifugation
for 2 min and removal of as much supernatant as possible. The remaining
pellet was gently resuspended in 25 µl of buffer B [20 mM HEPES pH
7.9, 350 mM NaCl, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 20 %
glycerol] and incubated for 30 min on ice before centrifugation at
10,000 x g for 10 min. The resulting supernatant was
aliquotted and snap frozen in liquid nitrogen before storing at
-70°C. Protein estimations on nuclear extracts were performed by
Bradford assay (BioRad Laboratories, Ltd, Herts, U.K.).
Electrophoretic mobility shift assays
Oligonucleotides (oligos) containing the binding sites for
IRF (ISRE) were AAGTGAAAGTGAAAGTGA and TCACTTTCACTTTCACTT
(26)
, and for STAT-1 were GACATTTCCCGTAAATCAT and
ATGATTTACGGGAAATGTC (27)
(Oswell, Southhampton, U.K.).
Oligos were annealed before labeling by heating to 98°C for 10 min
and cooling slowly to room temperature. Double-stranded oligos (50 ng)
were end-labeled with 32P-adenosine triphosphate (ICN
Biologicals, Thame, Oxon, U.K.) using T4 polynucleotide kinase
(Boehringer-Mannheim, Lewes, E. Sussex, U.K.) and purified using
Sephadex G-25 spin columns (Pharmacia, St. Albans, Hert, U.K.). Binding
reactions were carried out by incubating 1 µg nuclear extract in 20
mM HEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.05% IGEPAL, 10 %
glycerol, 1 mg/ml nuclease-free BSA (Boehringer), 100 µg/ml poly
d(I-C) (Boehringer), and 0.25 ng labeled oligos in a volume of 20 µl.
Competition experiments were carried out by adding unlabeled oligo
(either STAT-1 or IRF oligo) to the binding reactions before addition
of the labeled probe. Supershift experiments to confirm the identity of
the bound protein were performed by the addition of 1 µg of specific
antibodies to either STAT-1 IRF-1 or IRF-2 (Santa Cruz, Autogen
Bioclear U.K. Ltd, Wilts, U.K.) to the binding reactions. Binding
reactions were incubated at room temperature for 20 min before
separating DNA–protein complexes without tracking dye on a 6%
polyacrylamide, 0.25X TBE gel at 200 volts for 3–4 h. Gels were fixed
in 10% acetic acid before vacuum drying and exposure to
autoradiographic film (Kodak Xomat XAR) and a PhosphorImager screen to
quantify the binding with a BioRad PhosphorImager and Molecular analyst
software (BioRad).
Statistics
Data was tested for statistically significant differences by the
Mann-Whitney test. Values of P<0.05 were considered
significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of IRF-1 activity is reduced in psoriatic KC
The immunohistochemical staining demonstrates that the IRF-1
expression pattern is altered in psoriatic skin, suggesting that this
may be an important molecule in psoriatic immunopathology. Since IRF-1
is a major component of the -IFN signaling pathway, it was important
to determine whether the DNA binding activity of IRF-1 is altered in
nonlesional-psoriatic KC. IRF-1 DNA binding activity was detected in
unstimulated KC (Fig. 2
A) and increased when KC cultures were stimulated with
1000 IU/ml -IFN (shown by the arrow in Fig. 2A
). The
induction of binding was observed 1 h after stimulation,
increasing to a maximum after 5–8 h in both normal and psoriatic KC.
The higher mobility complex in Fig. 2A
was shown to be IRF-1
by supershifting the DNA–protein complex with IRF-1 specific
antibodies (shown by the arrow in the right-hand panel, lane A). This
complex was not supershifted by IRF-2 antibodies (lane B in the
right-hand panel Fig. 2A
). The lower (IRF-1) complex was
competed away with the addition of 200 ng cold IRF-1 oligos, but not
with the STAT-1 oligos (lanes D–G, Fig. 2A
). Experiments to
determine the kinetics of IRF-1 activity with 1000 IU/ml -IFN were
repeated in different normal and psoriatic KC. The quantification of
these experiments is shown in Fig. 2B
. The induction of
IRF-1 activity is greatly reduced in psoriatic KC compared with normal
KC. Further experiments stimulating normal and psoriatic KC with 30
IU/ml -IFN showed reduced activity of IRF-1 in psoriatic KC 5 h
after the addition of -IFN (P<0.05, Fig. 3
).
|
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Induction of STAT-1 activity is reduced in psoriatic KC
Since IRF-1 had reduced DNA binding activity in psoriatic skin, it
is possible that alterations to -IFN signaling may occur earlier in
the pathway. STAT-1 induction was examined in KC derived from five
unaffected individuals and from the nonlesional skin of five patients
with chronic plaque psoriasis. One representative experiment is shown
in Fig. 4
A. Neither normal human KC nor psoriatic KC exhibited
STAT-1 binding when unstimulated. STAT-1 activity was induced 5
min after the addition of 1000 IU/ml -IFN to cultures (Fig. 4A
). The kinetics of STAT-1 induction were similar for
both normal and psoriatic KC cell lines, with maximal binding activity
between 15 and 30 min after the addition of -IFN. Quantification of
STAT-1 induction was carried out by phosphorimaging and is shown in
Fig. 4B
. Although STAT-1 activity was induced in
psoriatic KC after the addition of 1000 IU/ml -IFN, levels of
STAT-1 binding were significantly reduced compared with normal KC
(at 10, 15, 30, and 60 min (P<0.04). The identity of the
bound protein was shown to be STAT-1 by the addition of specific
anti-STAT-1 antibodies (Santa Cruz), which caused a supershift in
the complex (shown by the arrow in lane S1 Fig. 4A
). The
STAT-1 complex was competed away with cold STAT-1 oligos, but not
by IRF-1 oligos (data not shown), further confirming that the protein
bound to the oligos was STAT-1. In dose response experiments, no
differential STAT-1 induction was observed between normal and
psoriatic KC. Both psoriatic and normal KC lines showed significant
induction of STAT-1 at a dose of 50 IU/ml; maximal induction of
STAT-1 binding was achieved at 500 IU/ml -IFN (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-IFN in psoriasis is
unclear since psoriatic KC continue to proliferate despite high levels
of -IFN. One hypothesis is that the -IFN signaling pathway may be
defective in psoriatic KC. We provide evidence to support the notion of
altered -IFN signaling in psoriatic KC.
Immunohistochemical staining revealed changes in the pattern of IRF-1
expression in lesional psoriatic skin compared with nonlesional or
normal skin. IRF-1 expression in normal skin was restricted to the
basal layer KC, whereas lesional psoriatic KC showed no basal staining
but had either light or no suprabasal expression. This evidence
suggests that IRF-1 may play a role in the maintenance of the psoriatic
phenotype. The pattern of IRF-1 expression seen here correlates with
the expression pattern of -IFN R chain in one study
(28)
, suggesting that the areas of functional -IFN
signaling in lesional psoriatic skin may be different from those seen
in normal skin. These preliminary observations led to the question of
whether nonlesional psoriatic KC have altered IRF-1 DNA binding
activity compared with normal KC. Using electrophoretic mobility shift
assay, we have demonstrated that the DNA binding activity of IRF-1 is
reduced in psoriatic KC compared with normal KC. Since IRF-1 has
tumor-suppressing activities, reduced activity by this molecule may
contribute to the lack of growth control in psoriatic lesions. -IFN
induces proliferation in a myeloid cell line that has inhibited IRF-1
expression (29)
. It could be speculated that reduced
activity of IRF-1 in psoriatic KC may lead to growth-promoting activity
by -IFN in vivo.
Since the induction of IRF-1 activity is reduced in nonlesional
psoriatic KC, it follows that the induction of STAT-1 DNA binding
may also be reduced. Indeed we have shown a reduced induction of
STAT-1 DNA binding in psoriatic KC after 15 min incubation with
-IFN. STAT-1 activation has been demonstrated to have growth
inhibitory effects by mediating expression of p21/WAF1
(30)
, caspase 1, and apoptosis (31)
. Reduced
activity of STAT-1 by psoriatic KC in response to -IFN suggests
that this transcription factor may contribute to the lack of growth
control in psoriatic epidermis. STAT-1 is present in the cytosol of
unstimulated cells and its activity is regulated by phosphorylation.
Therefore one explanation for reduced STAT-1 activity could be
reduced phosphorylation of STAT-1 by the JAK kinases. There is some
evidence that nitric oxide inhibits JAK2 activity in vitro
(32)
. Since the synthesis of nitric oxide synthase is
increased in psoriatic epidermis (33)
, this may be a
plausible explanation. Alternatively, reduced STAT-1 activity may be
a result of decreased surface expression of -IFN receptors in
psoriatic KC.
In many cell types, expression of the -IFN and ß receptor
chains differs significantly. The chain is generally constitutively
expressed, whereas the ß chain is at a very low constitutive level
but is regulated by external stimuli (reviewed in ref 8
). Also, there
is no correlation between the level of chain expression and the
magnitude of the -IFN-induced response in cells (34)
.
The only studies investigating the -IFN R expression in psoriatic KC
documented the chain and were also conflicting. Scheynius et al.
(35)
detected expression of -IFN chain receptors
restricted to the lower layers of the epidermis in lesional psoriatic
skin, but expressed throughout the thickness of normal and uninvolved
psoriatic skin. Conversely, van den Oord (28)
found
-IFN R chain expressed suprabasally in involved psoriatic skin
but restricted to the basal layer in normal skin. The distribution of
the signaling -IFN R ß chain in psoriatic skin may be crucial in
determining whether functional signaling complexes are present.
Potentially a down-regulation of the ß chain could account for a
reduced level of signaling complexes on the KC cell surface. Autocrine
down-regulation of -IFN R on psoriatic KC by endogenous -IFN
production might provide an explanation for reduced -IFN signaling.
Indeed, analysis of the conditioned culture fluids of passage 3 normal
and psoriatic KC cultures revealed that 0/3 normal cultures but 4/4
different cultures from psoriatic donors produced easily measurable
levels of -IFN (R. C. McKenzie and E. Sabin, unpublished data).
The actions of -IFN are very complex since it can promote
inflammation by the induction of adhesion molecule expression (ICAM-1
or HLA DR). In addition, -IFN can have an antiproliferative effect
on many cells including epidermal KC. A reduced sensitivity of
psoriatic KC to the growth inhibitory effects of -IFN has been
demonstrated in vitro (6
, 7
).
Psoriasiform lesions and epidermal proliferation can, however, be
induced in the epidermis of psoriasis patients and normal individuals
upon local administration of -IFN (36
,
37
), thus supporting a proinflammatory role for -IFN in
psoriatic skin. However, this may reflect the effects of
pharmacological rather than physiological doses of -IFN. There is
evidence that -IFN given systemically can improve some inflammatory
dermatoses, whereas lesional injection increases the disease severity
(26)
. The presence of locally administered or produced
-IFN cannot alone account for the proliferative status of the
epidermis. Induction of -IFN was demonstrated in the KC of allergic
contact dermatitis patients whose skin had been nickel-challenged,
although epidermal hyperproliferation was not evident
(38)
.
In conclusion, this study has demonstrated that the induction of IRF-1
and STAT-1 activity is reduced in nonlesional psoriatic KC. Both
IRF-1 and STAT-1 have growth inhibitory effects, and thus are
implicated in the maintenance of epidermal growth control. Reduced
activity of components in the -IFN signaling pathway may play a role
in the reduced sensitivity of psoriatic KC to -IFN, both in
vitro and in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations; FBN, fibronectin; GAS,
gamma-activated sequence; IFN, interferon; -IFN R, gamma IFN
receptor; IRF-1, interferon regulatory factor-1; ISRE, interferon
stimulated response element; ICAM-1, intercellular adhesion molecule-1;
JAK, Janus kinases; KC, keratinocyte(s); KSFM, keratinocyte serum-free
medium; NSS, normal swine serum; oligos, oligonucleotides; PBS,
phosphate-buffered saline; STAT-1, signal transducer and activator of
transcription; TBS, Tris-buffered saline.
Received for publication August 28, 1998.
Revision received October 26, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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= psoriatic KC; 
= normal KC.
