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*
Institute of Immunology, University Hospital Benjamin Franklin, Free University Berlin, Berlin, Germany;
Departments of Dermatology and
Clinical Immunology, Charité, Humboldt University, Berlin, Germany; and
§
Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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protein
on the cell surface, suggesting that human KC can employ IL-15 for
juxtacrine signaling. While IL-15 exerted no significant effect on KC
proliferation and IL-6 or IL-8 secretion, IL-15 inhibited both
anti-Fas and methylcellulose-induced KC apoptosis in vitro. This is
in line with the recognized potent anti-apoptotic effects of IL-15.
IL-2, whose receptor shares two components with the IL-15R, failed to
inhibit KC apoptosis. Together with the role of IL-15 in sustaining
chronic immune reactions, this invited the question of whether a
reduction of KC apoptosis by IL-15 may be involved in the pathogenesis
of psoriasis, a chronic hyperproliferative inflammatory skin disease
characterized by abnormally low KC apoptosis in the epidermis.
Remarkably, compared with nonlesional psoriatic skin and skin of
healthy volunteers, lesional psoriatic epidermis showed high IL-15
protein expression in the epidermis and enhanced binding activity for
IL-15. Therefore, antagonizing the inhibitory effects of IL-15 on KC
apoptosis deserves exploration as a novel therapeutic strategy in
psoriasis management. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(2, 3, 4) as well as IL-1, IL-6, and TNF- (1, 5, 6). The expression of several of these cytokines is up-regulated
in inflammatory skin diseases, where these cytokines are thought to
mediate skin inflammatory immune responses and leukocyte chemotaxis
(e.g., keratinocyte cytokines, such as TNF-, IL-8, and GM-CSF, are
known to be responsible for the leukocyte infiltrate characteristic of
the mature lesion of psoriasis (7, 8). Taken together,
KC-derived cytokines are likely to play a critical role in various
inflammatory skin diseases. One parameter in the maintenance of epidermal homeostasis is that loss of KC adhesion leads to terminal differentiation to corneocytes or apoptosis. KC may undergo apoptosis by loss of cell-cell contact (9), after cross-linking of the Fas (CD95) molecule (10, 11), or by UV radiation (12, 13). KC apoptosis may be inhibited by different growth factors, such as epidermal growth factor (14, 15), KC growth factor (16), or nerve growth factor (17) in vitro. However, the molecular controls of KC apoptosis in vivo are still poorly understood. Therefore, in chronic inflammatory, hyperproliferative skin diseases such as psoriasis, where an abnormally low rate of apoptosis contributes to the development of epidermal hyperplasia (18, 19), it is important to gain a deeper understanding of the roles of specific cytokines and their cognate receptors in down-modulating the normal rate of KC apoptosis. In fact, it has been recently shown that psoriatic KC are abnormally resistant to apoptosis (9, 20). In addition, some of the proinflammatory cytokines that drive development of a psoriatic plaque may suppress KC apoptosis. Interestingly, in addition to its well-established immunosuppressive properties, recent data indicate that methotrexate, one of the best established and most effective antipsoriatic agents, induces KC apoptosis (21).
In this context it is interesting to explore the role of IL-15 in KC apoptosis and the pathogenesis of psoriasis. IL-15 not only is implicated in the maintenance of several chronic inflammatory immune responses (21, 22, 23), but also is a very potent inhibitor of lymphocyte and hepatocyte apoptosis, both in vivo and in vitro (24). Since IL-15 mRNA is also expressed in KC and dendritic cells in the skin (25) and is a potent growth factor and chemoattractant for leukocytes (26, 27), it may play a role in KC biology and pathology. The aim of the present study was to further explore this as yet obscure functional role. Normal, primary foreskin KC were used to study the effects of IL-15 on native, untransformed cells. Moreover, the spontaneously transformed, immortalized HaCaT cells (28, 29) were used, not the least because this KC cell line displays a keratin expression pattern typically seen in psoriatic epidermis and has therefore been employed by some investigators for analyzing an easily handled epithelial cell population that shares some, although certainly not all, features of psoriatic epidermal KC.
In addition, the expression of IL-15 cytokine and IL-15R in situ was analyzed in skin biopsies from psoriatic patients and compared with that in clinically uninvolved or normal skin and in skin biopsies from chronic eczema and lichen simplex, which are also characterized by acanthotic epidermis. For receptor demarcation by immunohistology, a recently generated IL-15-IgG2b fusion protein was employed.
The data reported here suggest a role for IL-15, which is expressed in
a juxtacrine manner on the KC cell surface, in inhibiting KC apoptosis,
probably via the IL-15R -chain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human epidermal cell suspensions were obtained from normal donors undergoing foreskin surgery. KC were propagated in serum-free medium (KGM, BioWhittaker, Heidelberg, Germany) with the following supplements: 0.1 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 7.5 mg/ml bovine pituitary extract, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, and 0.15 mM calcium. KCs were passaged by dissociating the monolayer with 0.025% (w/v) trypsin/0.01% (w/v) EDTA. For the experiments performed, cells were derived from the third to the fifth passage and grown to subconfluence as a monolayer. The human KC cell line HaCaT, provided by N. Fusenig (29), was cultured in DMEM with 5% (v/v) FCS and antibiotics at 37°C with 5% CO2.
Induction of apoptosis by anti-Fas
Semiconfluent keratinocyte cultures were incubated for 24 h
in the presence or the absence of human recombinant IFN-
at a
concentration of 103 U/ml to promote Fas
expression as previously described (30). The cells were
washed twice with PBS and once with culture medium and were treated
with 1 µg/ml anti-Fas Ab (clone SM1/1; Bender Med Systems,
Boehringer Ingelheim, Heidelberg, Germany) for 2 h at 37°C.
After washing twice with PBS, the cells were treated with a
cross-linking goat anti-mouse IgG Ab (Sigma, Deisenhofen, Germany)
in the presence or the absence of human rIL-15 (10 and 50 ng/ml;
Genzyme, Cambridge, MA) or rIL-2 (10 and 50 ng/ml PeproTech, Rocky
Hill, NJ) and cultured for 24, 48, or 72 h at 37°C. As control,
cells were incubated with an irrelevant, isotype-matched IgG2a Ab
(anti-HIV p24) (31) or with the cross-linking Ab
alone. Detached cells were recovered by collecting the supernatant, and
adherent cells were harvested by trypsination. All cells were then used
for viability tests and apoptosis detection.
Induction of apoptosis in methylcellulose (MC)
Induction of apoptosis in semisolid medium was performed as previously described (9, 32). Briefly, sterile 1.68% MC (MG 4000, Serva, Heidelberg, Germany) was prepared by autoclaving 3.37 g MC and a magnetic stir bar in a 250-ml glass bottle. Keratinocyte basal medium (KBM) (100 ml) was heated to 60°C, added to the MC powder, and stirred at room temperature for 20 min. An additional 100 ml of serum-free KBM was added, and the mixture was stirred at 4°C for 1 h. Aliquots of the MC solution were centrifuged at 10,000 rpm for 90 min at 4°C. Freshly trypsinized HaCaT cells or human keratinocytes were resuspended in 1.68% MC solution at 106 cells/ml with or without the addition of human rIL-15 (20 ng/ml; Genzyme) or rIL-2 (20 ng/ml; PeproTech) in a 50-ml conical tube and incubated at 37°C for 24, 48, or 72 h in a humidified 5% CO2 atmosphere. Cells were recovered from suspension by repeated dilution of the MC by KBM followed by centrifugation.
Detection of apoptosis
KC were harvested at the indicated time points, and cytospins of
104 cells were prepared, air-dried, fixed, and
stained for apoptotic cell death (TUNEL assay) (24) with
an apoptosis-detection kit (ApopTaq, Oncor, Gaithersburg, MD) following
the manufacturer’s instructions. Cell nuclei were counterstained with
Hoechst 33342 dye (Sigma), and apoptotic and nonapoptotic cells were
counted in 10 microscopic fields (
700 cells). Cells were referred to
as undergoing apoptosis based on two microscopic criteria: 1) positive
TUNEL staining, and 2) an apoptotic phenotype of the cells, i.e.,
shrinkage and fragmentation of the nucleus, as observed in the Hoechst
33342 counterstaining. The percentage of apoptosis was calculated.
Shown is the inhibition of apoptosis (percentage) by the added IL-15 or
IL-2 compared with the apoptosis induced without IL-15 or IL-2 in the
culture medium (without cytokines added, no inhibition). As proof,
apoptosis was also detected by FACS staining of propidium iodide
inclusion as previously described (33).
RT-PCR
RNA was extracted from cells by using the RNA Clean reagent
(AGS, Heidelberg, Germany) according to the manufacturer’s
instruction. A 5-µg aliquot of total cellular RNA was reverse
transcribed using random hexanucleotides as primers and the Superscript
II preamplification kit (Life Technologies, Karlsruhe, Germany). cDNA
was amplified in 50-µl PCR reaction mixture containing 250 µM of
each dNTP, 200 nM of primers, 5 µl of 10-fold PCR buffer
(Perkin-Elmer/Cetus, Emeryville, CA), and 2.5 U Taq DNA polymerase
(AmpliTaq, Perkin-Elmer/Cetus). The primers used were
(27): human IL-15R: sense, 5'-GCC AGC GCC ACC CTC CAC
AGT AA-3'; antisense, 5'-GCC AGC GGG GGA GTT TGC CTT GAC-3'; human
IL-15: sense, 5'-GGC TTT GAG TAA TGA GAA TTT CGA-3'; antisense, 5'-ATC
AGT TGC AAT CAA GAA GTG TTG-3'; human IL-2R: sense,
5'-AAGCTCTGCCACTCGGAACACAAC-3'; antisense, 5'-TGATCAGCAGGAAAACACAGC-3';
human IL-2Rß: sense, 5'-GAATTC CCTGGAGAGATGGCCACGGTCCCA-3';
antisense, 5'-GAATTCGAGGTT TGGAAATGGATGGACCAAGT-3'; human
IL-2R:sense, 5'-AGCCCCAGC CTACCAACCTCACT-3'; antisense,
5'-TTAAAGCGGCTCCGAACACGAA-3'; and human ß-actin: sense,
5'-GTGGGGCGCCCCAGG CAC CA-3'; antisense,
5'-CTCCTTAATGTCACGCACGATTTC-3'. All primers used were purchased
from TIB Molbiol (Berlin, Germany). Samples were amplified in a DNA
thermocycler (Perkin-Elmer/Cetus) for 35 cycles. Each cycle consisted
of denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and
extension at 72°C for 2 min. Aliquots of PCR products were then
electrophoresed on 1.5% agarose gel and visualized by ethidium bromide
staining. To evaluate mRNA expression semiquantitatively, in addition
to the PCR product from 35 cycles, 15 µl of the PCR product from the
25 cycles and the 30 cycles was run simultaneously. ß-Actin message
expression was used to normalize the cDNA amount to be used. A mock PCR
(without cDNA) was included to exclude contamination in all
experiments.
Immunohistology
Skin biopsies of psoriatic plaques and of uninvolved skin were obtained from 12 patients. Moreover, three biopsies each from patients with chronic eczema and lichen simplex were used to analyze additional chronic hyperproliferative, inflammatory skin diseases that also displayed acanthotic epidermis. Control biopsies from 10 normal, healthy donors were obtained from surgical skin explants from patients. Biopsies were immediately snap-frozen in liquid nitrogen and stored at -80°C. Ten-micron cryostat sections were air-dried, fixed in acetone (-20°C, 10 min), and stained with mouse anti-human IL-15 (34) or biotinylated IL-15-IgG2b fusion protein (FP) following standard avidin-biotin complex technique as previously described (35). Controls were obtained by omission of the primary Ab or by using biotinylated murine IgG2b (clone 49.2; PharMingen, Hamburg, Germany). To verify the specificity of the IL-15-IgG2b binding to the IL-15R, additional staining was performed on Con A-activated human peripheral blood T cells after stimulation with IL-2 (10 ng/ml for 24 h) (27). IL-15 binding to these cells was blocked by preincubation of the FP with anti-IL-15 Ab (M111, Genzyme, Alzenau, Germany). Additional control staining was performed by preincubation of psoriatic skin sections with a 100-fold excess of IL-15 for 30 min. This completely blocked binding of the biotinylated IL-15-IgG2b FP to the cognate IL-15R in the skin sections.
FACS staining
Expression of the IL-15R or surface IL-15 cytokine on primary
KC or HaCaT was analyzed on unstimulated or on cells treated with
IFN- (103 U/ml) for 24 h. LPS-activated
(24-h incubation) PBMC, which express surface IL-15 as well as the
IL-15R (36, 37), were stained as a positive control.
The IL-15R was detected with mouse anti-huIL-15R (M116,
provided by Immunex, Seattle, WA), and IL-15 surface expression was
detected with a mouse anti huIL-15 Ab (M111, Genzyme), both followed by
FITC-labeled goat anti-mouse IgG (Southern Biotechnology
Associates, Birmingham, AL). Isotype-matched Abs were used as controls,
and the fluorescence was analyzed with a FACScan (Becton Dickinson,
Heidelberg, Germany).
Statistical analysis
Results are presented as the mean ± SD, unless stated otherwise. Mann-Whitney U test was used to determine the significance of difference between treatment groups or between patients and healthy donors (p < 0.05 was considered significant). The in vitro experiments were repeated at least three times, with highly comparable results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain on their cell
surface
IL-15 and IL-15R expression was analyzed on primary foreskin KC
from different donors and on the HaCaT cell line. mRNA transcription
was analyzed in KC treated with IL-15 (10 ng/ml) or IFN-
(103 U/ml) or left untreated for 24 h.
Incubation with IL-15 was performed, since this reportedly
down-regulates its own high affinity receptor chain (IL-15R) on T
cells (38). IFN- was studied in this assay, because it
is overexpressed in psoriatic lesions (39), stimulates KC
expression of activation markers (HLA-DR) (40) and
cytokine receptors (IL-1R) (41), and enhances the
expression of IL-15 in epithelial cells of human fetal retina
(42).
By RT-PCR, unstimulated primary KC showed expression of IL-15 cytokine
and receptor mRNA (Fig. 1
). Steady state
levels of the receptor transcripts were slightly down-regulated by
incubation with IL-15. In contrast, IFN- seems to up-regulate mRNA
levels for both IL-15 cytokine and receptor (Fig. 1). HaCaT cells
showed substantially higher baseline mRNA levels for IL-15 and
IL-15R. However, neither IL-15 nor IFN- stimulation altered
significantly IL-15 cytokine or receptor steady state levels (Fig. 1).
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and surface IL-15
cytokine expression as determined by FACS analysis. Cell surface
expression was studied, since it has been shown that IL-15 secretion in
the supernatant is very tightly controlled and that most cells that
transcribe IL-15 mRNA do not secrete detectable amounts of this
cytokine (43). Therefore, biological effects may be
mediated by membrane-bound IL-15 (in fact, we had previously detected
membrane-bound IL-15 on monocytes (37)). As shown in Fig. 2, primary KC and HaCaT cells both
express IL-15 cytokine as well as IL-15R protein on their cell
surface. Stimulation for 24 h with IFN- did not up-regulate the
surface expression of IL-15 cytokine or its receptor at the protein
level (not shown). Since monocytes express IL-15 and IL-15R on their
surface (37) and are a major source of IL-15
(36), we stained LPS-activated monocytes and analyzed them
by FACS as positive control for IL-15 and IL-15R surface expression.
As shown in Fig. 2, E and F, expression was
higher on monocytes compared with KC (Fig. 2, A–D).
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KC express IL-2Rß and IL-2R mRNA
The IL-15R consists of three components: the high affinity IL-15R
-chain, the IL-2Rß, and the common -chain (IL-2R)
(22). We have recently shown that inhibition of apoptosis
in fibroblasts is mediated by the IL-15R -chain (44).
However, IL-15 can activate cells (e.g., NK cells) by binding and
signaling through the IL-2Rß and -chain (45).
Therefore, we used PCR to detect the IL-2R on primary KC and HaCaT
cells. As shown in Fig. 3, both primary
KC and HaCaT cells express mRNA for IL-2Rß and -chain, but fail to
express the high affinity IL-2R -chain. This shows that KC express
all components of the IL-15R, namely, the high affinity IL-15R
-chain as well as the IL-2Rß and -chain.
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We and others have previously demonstrated that IL-15 is a very
potent inhibitor of apoptosis of various cell types in vitro and/or in
vivo, including on epithelial cell population (24). In a
fibroblast cell line, we were able to demonstrate that the IL-15R
-chain probably mediates these anti-apoptotic effects by
blocking the recruitment of adaptor proteins, thereby inhibiting
apoptosis induction at a very early stage of the apoptotic signaling
cascade (44). Since the above experiments had shown that
IL-15 and IL-15R proteins are expressed by KC itself, we tested
whether IL-15 also inhibits human KC apoptosis in vitro.
Primary KC and HaCaT cells were incubated in semisolid MC medium, which
induces apoptosis by inhibiting cell-cell contact (9).
After 24 h 10% of the KC were TUNEL positive, i.e., had
undergone apoptosis; after 48 h 30% and after 72 h in
culture >60% of the KC undergo programmed cell death, comparable to
the previously published values (Fig. 4A) (9).
Inhibition of apoptosis was calculated as the percent inhibition to be
able to compare the effects of IL-15 on MC- or anti-Fas-induced
apoptosis (MC or anti-Fas without IL-15 was set equal to 0%
inhibition).
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A). Therefore, inhibition of
apoptosis by IL-15 (not apoptosis itself) is highest after 24-h
culture, while the total percentage of apoptosis increases over time,
as stated above. Thus, apoptosis itself is not absent after 72 h,
but the effect of the added IL-15 is absent (added at time zero, i.e.,
at the time point apoptosis is induced). The failure of IL-15 to
inhibit apoptosis is probably due to degradation of the cytokine. In
addition, IL-15 could not rescue those cells that are primed to undergo
apoptosis; rather, it seems to inhibit the induction of apoptosis at an
early time point (i.e., it rescues cells before apoptosis could be
induced).
To address whether these apoptosis inhibitory effects of IL-15
reflected general down-modulatory properties of IL-15 on human KC
apoptosis, we used a second, receptor-mediated method to induce
apoptosis via an mAb to Fas that is expressed by KC (30, 46) (this was confirmed in the present study by FACS
analysis; not shown) and whose cross-linking induces apoptosis by
oligomerization of this membrane-bound receptor. KC were treated for
24 h with IFN- to maximize Fas-mediated apoptosis as previously
described (30). Then, KC were incubated with an
anti-Fas Ab, which thereafter was cross-linked by a second Ab. This
technique induced apoptosis in 10% KC after 24 h and 60% KC
after 72 h of culture. Simultaneous addition of IL-15 in two
different concentrations to the cell culture significantly suppressed
Fas-induced apoptosis (Fig. 4B). The effect was most potent
in primary KC (after 48 h), where addition of IL-15 (10 ng/ml)
suppressed apoptosis by 75% compared with that in controls
(anti-Fas alone). Addition of a 5-fold higher dose did not further
increase this anti-apoptosis effect. In HaCaT cells, apoptosis was
maximally inhibited after 72 h (60%) by 10 ng/ml IL-15. In HaCaT
cells, higher concentrations of IL-15 (50 ng/ml) exhibited stronger
anti-apoptotic effects. After 24 h 60% apoptosis inhibition
by IL-15 was seen in HaCaT cells, but it was not significantly modified
at 48 or 72 h (Fig. 4B). This inhibition again seems to
represent the rescue from apoptosis at an early time point, before or
shortly after apoptosis is induced by Fas cross-linking.
Although the TUNEL method is a reliable and specific method for the
detection of apoptotic KC cell death in vitro (9),
terminally differentiating KC in vivo may also show a positive TUNEL
reaction (18, 47). Therefore, a second method was applied,
which detects apoptotic cell death by flow cytometric propidium iodide
staining (33). This method confirmed the results obtained
with the TUNEL method (data not shown). In addition to TUNEL and
propidium iodide staining, morphological signs of anti-Fas-induced
apoptosis were analyzed by light microscopy and were photodocumented.
In primary KC grown in a confluent monolayer (Fig. 5A) addition of the
anti-Fas and the cross-linking Abs induced marked morphological
alterations consistent with the morphological features of apoptosis,
such as cell condensation and shrinkage, appearance of apoptotic
bodies, and loss of adhesion (Fig. 5A). Incubation with an
isotype-matched, irrelevant control Ab did not generate these
phenomena. Coincubation with anti-Fas and IL-15 (50 ng/ml) markedly
inhibited apoptosis-associated cell detachment and shrinkage, as shown
in Fig. 5A. The same effects were seen in HaCaT cells (Fig. 5B).
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To further clarify the anti-apoptotic effects of IL-15 on KC
we used IL-2 in the same apoptosis assay. IL-15 can bind to KC via the
IL-15R -chain as well as via the IL-2Rß and -chain, whereas
IL-2 binds and signals exclusively via the IL-2Rß and -chain. In
addition, IL-2 can signal via the high affinity IL-2R -chain, which
is not expressed on KC (see Fig. 3), but fails to transmit a signal via
the IL-15R -chain (22). As shown in Fig. 6 induction of apoptosis by
methylcellulose (Fig. 6A) and by anti-Fas Ab (Fig. 6B) in HaCaT is not suppressed by IL-2. In fact, the
anti-Fas pathway in contrast is, rather, increased by 50% (Fig. 6B). Similar results were obtained using primary KC (not
shown). Since both cytokines bind to the IL-2Rß and -chain, and
since IL-2 fails to inhibit KC apoptosis, this suggests that inhibition
of apoptosis by IL-15 in KC is mediated by the IL-15R -chain, as
previously shown for fibroblasts (44).
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In vitro IL-15 is not a growth factor for KC, since incubation of HaCaT and primary KC with IL-15 could not induce significant proliferation, as assessed by [3H]thymidine incorporation (not shown). Also, incubation of both, primary KC and HaCaT with IL-15 or IL-2 failed to induce significant IL-6 and IL-8 release into the supernatant (not shown). Taken together, IL-15 itself is not a growth factor for KC and cannot induce KC to secrete this two proinflammatory cytokines.
IL-15 cytokine and IL-15 binding sites are overexpressed in lesional psoriatic epidermis
Since these experiments had revealed that IL-15 inhibits human KC apoptosis in vitro, it was interesting to begin to explore whether IL-15 might play a role in hyperproliferative inflammatory skin diseases such as psoriasis or other dermatoses such as chronic eczema and lichen simplex. IL-15 mRNA and protein are produced by human KC (25), but nothing was known about cutaneous expression of the IL-15 cytokine and its receptor. To analyze the distribution of IL-15 cytokine and IL-15 binding sites, immu-nohistologic staining was performed on skin sections from normal, healthy donors as well as on sections derived from lesional and nonlesional skin of patients with psoriasis vulgaris as well as from patients with chronic eczema or lichen simplex. Staining of psoriatic plaques for IL-15 with an mAb revealed high expression throughout the entire epidermis (from the stratum basale, which contains epidermal stem cells, to the stratum granulosum, which is characterized by KC that undergo programmed cell death by terminal differentiation and cornification) (19) as well as in the dermis directly adjacent to the epidermal basal membrane. Skin sections from chronic eczema/lichen simplex with acanthotic epidermis showed IL-15 expression mainly restricted to the basal layer of the epidermis and the subepidermal compartment, com-parable to healthy skin. In addition, single cells in the dermis stained positively for IL-15, probably representing hemopoietic cells of the inflammatory infiltrate that is characteristic of these dermatoses (48).
By immunohistology, in normal skin from healthy donors only
subepidermal expression of IL-15 was seen, and no IL-15
immunoreactivity could be detected in the epidermal KC (Fig. 7A). However, a few
IL-15-positive cells were detected in the epidermal and subepidermal
layers, probably representing macrophages or dendritic cells. As
positive control we used skin biopsies from hypertrophic scars, since
they overexpress IL-15 (49), as confirmed here (Fig. 7A). Omission of the anti-IL-15 Ab as a negative control
showed the specificity of the staining (Fig. 7A).
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B). As an additional control, it was
demonstrated that human cells did not bind the murine IgG2b tail of the
FP, as determined by staining with biotinylated murine IgG2b. The
specificity of the staining was further demonstrated by preincubation
of psoriatic skin with a 100-fold excess of IL-15 cytokine for 30 min,
which completely blocked binding of the IL-15-IgG2b fusion protein
(Fig. 7B).
Nonlesional skin from psoriatic patients and normal skin from healthy
donors were found to constitutively express binding sites for IL-15 on
epidermal KC at very low levels (Fig. 7B). In contrast,
staining of skin sections from lesional psoriatic skin revealed a
striking overexpression of IL-15 binding sites on epidermal KC (Fig. 7B). Acanthotic epidermis in chronic eczema/lichen simplex
did not show this strong epidermal overexpression of IL-15 binding
sites. The staining in the two disease settings was restricted to the
basal layer of the epidermal KC and to the subepidermal dermis. Single
IL-15-binding cells could be detected in the dermis, probably
representing cells of the inflammatory infiltrate associated with
chronic eczema/lichen simplex.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-2Rß, and -chain. In addition, we provide evidence that human KC
express the cytokine as well as the cognate high affinity receptor on
the cell surface, which suggests that juxtacrine signaling of KC is
mediated via surface-bound IL-15. Furthermore, we show that in vitro
IL-15, but not IL-2, strongly inhibits MC- and anti-Fas-induced KC
apoptosis. Since epidermal hyperplasia in chronic inflammatory
hyperproliferative skin diseases such as psoriasis may at least in part
be due to abnormally reduced or delayed apoptosis (9), it
was intriguing to note that immunohistology revealed an altered
distribution pattern of IL-15 cytokine, with IL-15 also detected on
epidermal KC above the basal membrane, rather than being restricted to
the dermis, compared with uninvolved or normal human skin. Additional
staining of acanthotic epidermis from chronic eczema/lichen simplex
showed expression of IL-15 and its specific binding sites to be
restricted to the basal epidermal layer and to inflammatory cells in
the upper dermis, while no overexpression could be detected in the
epidermis as seen in psoriasis.
Loss of cell-cell contact promotes KC differentiation and apoptosis in
normal epidermis (50, 51). Inhibition of this
physiological control process of KC turnover by IL-15 supports a role
for IL-15 in the promotion of epidermal hyperplasia in psoriasis. In
addition, KC express the Fas receptor (30), which induces
apoptosis after receptor cross-linking (10). As
demonstrated here, Fas-induced apoptosis is potently inhibited by
IL-15. If confirmed, e.g., in skin organ culture assays of psoriatic vs
healthy human skin during administration of IL-15R antagonists,
IL-15R-mediated signaling may well become a promising target in the
management of chronic inflammatory, hyperproliferative skin diseases.
Pharmacological blockade or down-regulation of IL-15 release, IL-15R
overexpression, and/or IL-15R-mediated signal transduction pathways
might represent useful targets for adjuvant therapy in the management
of skin diseases such as psoriasis.
Interestingly, psoriatic KC are now recognized to display abnormal
resistance to apoptosis (9, 20). Also, the induction of
apoptosis in KC represents one mode of action of the well-established
and highly effective anti-psoriatic agent methotrexate
(21). This encourages us the pursuit of adjuvant treatment
modalities in psoriasis that counteract KC strategies to rescue them
from apoptosis, such as overexpression of anti-apoptotic IL-15 and
IL-15R.
The constitutive expression of IL-15R, IL-2Rß, and -chain as
well as of IL-15 on the protein level demonstrated here on normal KC
and on HaCaT cells in vitro suggests a physiological role of IL-15 in
KC biology. Staining of skin sections for IL-15 binding sites also
revealed low level expression of IL-15 binding sites in the epidermis
in normal skin from healthy donors. Furthermore, the transcription of
mRNA for IL-15 by KC in vitro demonstrated here confirms previously
published results (25, 42).
The secretion of IL-15 is tightly controlled, and expression of mRNA does not necessarily correlate with cytokine secretion into the supernatant (43). However, membrane-bound IL-15 on the KC surface may serve as a juxtacrine signal for directly adjacent cells; directly neighboring KC that express IL-15R may be stimulated by IL-15 expression on the cell surface of KC in their immediate vicinity.
Since the IL-15R -chain shows a very high affinity for IL-15
(Ka = 1011
M-1) (53),
functional significance (e.g., cytokine production by macrophages) was
reached at extremely low concentrations (picomolar to attomolar range)
(23). All three chains of the IL-15R are expressed by KC,
and IL-15 as well as IL-2 may signal through the ß- and -chains of
the IL-2R. However, suppression of apoptosis in the in vitro model
employed here seems to be mediated by the IL-15R, since IL-2 did not
reduce the rate of KC apoptosis.
Taken together, expression of membrane-bound IL-15 may affect epidermal homeostasis primarily by enhancing the survival of those KC that express cognate receptors. The suppression of KC apoptosis by IL-15 in vitro demonstrated here suggests one scenario for how this cytokine may inhibit the normal pattern of KC cell death and terminal differentiation of KC in the upper layers of the epidermis, thus leading to epidermal hyperplasia (acanthosis) in psoriasis, but not in other chronic inflammatory diseases such as lichen simplex cronica and chronic eczema. As one of the most common hyperproliferative skin diseases, psoriasis vulgaris is characterized by KC hyperproliferation as well as profound changes in KC maturation and turnover rate (54, 55). In addition, an abnormal resistance of psoriatic KC to apoptosis (9, 20) may contribute to the epidermal hyperplasia. However, little is known about the underlying mechanisms of this defect in the apoptosis control machinery of psoriatic KC. Psoriatic KC have abundant amounts of the cell survival protein Bcl-xL (20). Whether this overexpression correlates with enhanced survival and how Bcl-xL is up-regulated in the cells are as yet unclear. We are currently testing whether IL-15 is involved in the regulation of Bcl-xL in KC, since IL-15 has been shown to up-regulate Bcl-2 in T cells (56).
Our findings provide the first indicator that delayed apoptosis of
psoriatic KC (9) may be mediated at least in part by
KC-derived IL-15. Epithelial cell production of IL-15 was not only seen
in normal KC, but also in HaCaT cells, which are often advocated as an
in vitro model for studying hyperproliferative skin diseases
(57, 58, 59). The observed dramatic up-regulation of IL-15
binding to KC and the expression of IL-15R mRNA in psoriatic plaques
are in line with the concept that IL-15 may inhibit apoptosis in
psoriatic epidermis, which overexpresses IL-15 cytokine and the IL-15R.
This overexpression suggests a correlation of the IL-15 cytokine and
receptor expression and the dramatic overaccumulation of KC in
psoriatic epidermis. However, acanthotic epidermis, as such, is not
necessarily correlated to IL-15/IL-15R overexpression, since in chronic
eczema and lichen simplex neither IL-15 nor IL-15 binding sites were
found to be overexpressed above the basal layer of the epidermis.
The putative role of IL-15 in the pathogenesis of psoriasis may go beyond that of an inhibitor of KC apoptosis. Given that psoriasis increasingly surfaces as a T cell-mediated immune disease (8, 60), it is interesting to note that IL-15 is a potent chemoattractant and inhibitor of lymphocyte apoptosis as well as a T cell growth factor (24, 26, 27). Injection of IL-15 in mouse skin results in a local inflammatory immune response and the accumulation of T cells in the dermis and epidermis (R. Rückert, unpublished observation). In view of recent indications that a subset of NK cells is involved in initiating the onset of a psoriatic epidermal lesion (61), it is particularly interesting to note that IL-15 is now accepted as a major NK cell growth factor (62). Therefore, KC-derived IL-15 may also account to some extent for T cell accumulation, proliferation, and survival in psoriatic epidermal plaques. Since IL-15 also activates neutrophils, enhances their phagocytic activity, and inhibits their apoptosis in vitro (63, 64), KC-derived IL-15 may play a role in the abnormal influx and activation of neutrophils in psoriatic epidermis (65, 66, 67). This local effect of IL-15 on leukocytes is further supported by the IL-15/IL-15R positively stained cells in the dermal compartment in chronic eczema and lichen simplex chronica, which probably represent infiltrating inflammatory cells, a characteristic feature of both dermatoses (48).
In summary, the current report provides new insights into the role of IL-15 in KC physiology and pathology, namely in the control of KC apoptosis, and may lead to a better understanding of the pathophysiology of common hyperproliferative, inflammatory skin diseases such as psoriasis. Epidermal hyperplasia may be mediated at least in part by juxtacrine IL-15 signaling, which inhibits KC apoptosis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Silvia Bulfone-Paus, Institute of Immunology, University Hospital Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany.
3 Abbreviations used in this paper: KC, keratinocytes; MC, methylcellulose; IL-15-IgG2b FP, fusion protein consisting of human IL-15 and murine IgG2b; KBM, keratinocyte basal medium.
Received for publication March 2, 2000. Accepted for publication May 22, 2000.
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