ARTICLE
Histopathologically, alopecia areata (AA) is characterized by a dense
accumulation of mainly T lymphocytes around and within affected hair follicles
[1]. This event is accompanied by an overexpression of ICAM-1, and class
I and II MHC molecules on hair follicle keratinocytes and dermal papilla
cells (DPC) [2, 3]. The cascade of immunological events is not lethal
for crucial elements of the hair follicle, which is why AA is usually
reversible [4]. Cytokines may be responsible for the interruption of the
hair cycle, and several lines of evidence suggest that IL-1 may be responsible
for the induction of reversible hair loss such as that occurring in alopecia
areata [5]. If this hypothesis holds true, the hair follicle ought to
be equipped with proteins such as IL-1 receptors to participate in, or
modulate, IL-1-driven immune responses.
The cellular origin of IL-1ß production during AA is unknown.
The crucial role of the dermal papilla in hair cycle regulation and the
lymphocytic infiltration of the papilla in AA has focused our interest
on DPC. Despite their obvious importance, little is known about their
likely immune functions or their capacity to secrete substantial amounts
of cytokines. The aim of this study was, therefore, to investigate the
expression of type I and type II IL-1-receptors as well as IL-1ß
and IL-1-RA in DPC and to elucidate whether cytokines found in acute AA
or cytokines shown to be involved in allergic contact dermatitis (ACD),
are able to modulate the expression of these proteins. In this way, we
wanted to clarify whether DPC are able to exert a modulating effect on
IL-1-mediated immune responses, and whether such findings may help us
understand the therapeutic effect of allergic contact dermatitis (ACD)
in AA.
Materials and methods
Chemicals
Guanidinium thiocyanate, PGE2, penicillin, streptomycin, trypsine, EDTA,
diclofenac and PMA were purchased from Sigma (Deisenhofen, Germany), and
phenol, isopropanol and chloroform from Merck (Darmstadt, Germany). The
kit for reverse transcription (first strand cDNA synthesis kit) was purchased
from Pharmacia (Uppsala, Sweden). The taq polymerase came from
Boehringer Mannheim (Mannheim, Germany). Human recombinant cytokines (INF-gamma,
IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TNF-alpha, GM-CSF and TGF-ß1)
were bought from Laboserv (Gießen, Germany). A neutralizing, mouse,
anti-human IL-1-ß antibody and monoclonal rat (IgG2b) and mouse
(IgG1) anti-human antibodies recognizing specifically type 1 or type 2
IL-1 receptors (no cross-reactivity with receptors for IL-4, IL-2, IL-7,
GMCSF, TNF p80) were obtained from Genzyme (USA). FITC-conjugated rat
and mouse, monoclonal anti-mouse and anti-rat immunoglobulin as well as
mouse IgG1 and rat IgG2b istotype controls were obtained from Biosource
(USA). FITC-conjugated, monoclonal mouse and rat antibodies (IgG1 and
IgG2b subtypes) directed towards Aspergillus niger glucose oxidase
were bought from DAKO (Glostrup, Denmark). Primers for ß-actin,
IL-1ß, icIL-1-RA, sIL-1-RA were designed according to published
sequences [6, 7] and automatically synthesized. These primer pairs were
all intron-spanning in order to avoid amplification of contaminating genomic
DNA (Table I). ELISA assays
for the detection of IL-1ß and IL-1-RA were bought from RD-Biosystems
(USA). Fungizone and Amniomax medium were purchased from Gibco (Heidelberg,
Germany). A HPLC analytical column was bought from Perkin Elmer (USA).
Isolation of human hair follicles and cultivation
of dermal papilla cells
Intact, viable, anagen hair follicles were isolated by microdissection
from scalp biopsies as described [8]. Under a stereo-dissecting microscope,
a scalpel blade was used to cut the skin at the dermo-subcutaneous interface.
The epidermis and upper parts of the corium were removed. Anagen hair
follicles were isolated from the subcutaneous fat by use of a watch-maker's
forceps, by gently gripping the outer root sheath and by subsequent gentle
traction. Dermal papillae were isolated by further microdissection [9]
and placed in Amniomax medium. Only DPC in their third passage were used
for the experiments. Thirty DPC cultures from ten healthy donors (three
DPC culture for each donor) were initiated and studied. These cells have
been shown to possess the capacity to induce hair follicles when grafted
together with embryonal hair buds onto nude mouse skin [10]. DPC were
grown to subconfluency and incubated either for 8 (for RT-PCR), 16 (for
ELISA) or 33 hours (for FACS) with various substances (IL-1ß (20
ng/ml), IL-2 (500 U/ml), IL-4 (100 ng/ml), IL-6 (250 ng/ml), IL-8 (200
ng/ml), IL-10 (20 ng/ml), IL-13 (250 ng/ml), TNFalpha (100 ng/ml), INF-gamma
(50 ng/ml), TGFß1 (20 ng/ml), PGE2 (1 µM), diclofenac (10 mg/ml),
GM-CSF (60 ng/ml) and PMA (1 µM)). For PMA-incubation, a time-kinetic
of IL-1ß and IL-1-RA expression was established. In some experiments
(PMA, IL-1ß and TNFalpha treatment) a neutralizing, human anti-IL-1ß-antibody
(10 µg/ml) was added. Several co-stimulations with different mediators
were performed. Every experiment was performed at least in triplicate.
ELISA analysis
Non-adherent cells were removed by centrifugation at 450 x g for 5 min
and supernatants as well as cell lysates were harvested. Cell lysis was
performed by freeze-thawing cycles, three times with sterile PBS buffer.
Cell membranes were removed from the lysates by centrifuging at 18.000
x g for
15 min. ELISA analysis was performed as indicated by the supplier. Results
for cell lysates were expressed per mg total cellular protein, and for
supernatants as pg/ml. The concentration of proteins was determined as
described by Bradford [11].
Semiquantitative RT-PCR analysis
After the indicated incubations, total RNA from approximately 106
DPC was isolated according to Chomczynski and Sacchi [12]. One µg
total RNA was reverse-transcribed with random hexamer primers and mouse
moloney tumor virus. Each primer pair was tested on total RNA and subsequent
PCR amplification without prior reverse-transcription. No PCR products
were obtained in controls. For each primer pair a three-temperature step,
PCR cycle program was carried out
(2 min annealing, 2 min extension at 72° C and 1 min denaturation
at 94° C, Table I).
Semiquantitative measurement of PCR-products was established as described
[13]. In brief, linear amplification conditions were determined by: (1)
identical amounts of cDNA were subjected to increasing PCR cycles; and
(2) increasing amounts of cDNA experienced a defined PCR cycle number.
PCR cycle numbers used are given in Table
I. Semiquantitative analysis of the PCR-products was achieved
by high-performance liquid chromatography (HPLC) [13]. Before calculation
of IL-1ß, sIL-1-RA and icIL-1-RA mRNA expressions, the probes were
normalized for ß-actin mRNA as a house-keeping gene. This was achieved
by use of one third of the cDNA and amplification of the ß-actin
mRNA to each cytokine mRNA followed by HPLC analysis. Similar experiments
were performed by use of GAPDH as a house-keeping gene with identical
results (not shown). All experiments were performed at least in triplicate.
The mRNA expression of type I and type II IL-1 receptors was not determined
by RT-PCR.
HPLC operating conditions
The samples were injected into the column, equilibrated at 40°
C for 2 min with 70% buffer A (25 mM Tris-HCl, pH 9.0) + 30% buffer B
(25 mM Tris-HCl, pH 9.0 containing 1.0 mol/l NaCl). The eluate was then
brought to 50% B within 1 min, followed by a 15 min linear gradient from
50% B to 65% B using an eluate flow rate of 1 ml/min.
FACS analysis
To detect and quantify IL-1 receptors present on DPC cell surfaces,
we used monoclonal rat and mouse anti-human antibodies (at 10 µg/ml)
directed towards type I and type II IL-1 receptors. The antibodies belonged
to the IgG1 (mouse) and IgG2b (rat) isotype. Controls were performed by
the use of isotype-controls (10 µg/ml). Secondary antibodies were
either FITC-conjugated, monoclonal mouse anti-rat immunoglobulins or FITC-conjugated,
monoclonal rat anti-mouse immunoglobulins (10 µg/ml). FITC-conjugated,
monoclonal mouse and rat antibodies (IgG1 and IgG2b subtypes) directed
towards Aspergillus niger glucose oxidase were also used as control
antibodies (10 µg/ml). After stimulation with various cytokines and
removal by trypsinization the approximately 106 DPC were kept
in PBS and incubated on ice for 60 min with 0.5% BSA. The cells were then
washed twice with PBS and incubated with FITC-conjugated secondary antibody
for a further 60 min. After additional washing procedures with PBS, fluorescence
intensity was determined by FACScan analysis (Becton-Dickinson).
Results
Time-dependent induction of IL-1ß
and IL-1RA transcripts and proteins by PMA
Our approach allowed the detection of IL-1ß and IL-1-RA proteins
expressed in cell lysates of cultured DPC. Incubation with phorbolester
(PMA, 1 µM) led to a time-dependent increase of both proteins. The
cDNAs for ic-IL-1-RA and s-IL-1-RA were found to be constitutively expressed
in DPC, but only transcripts for IL-1ß and ic-IL-1-RA showed an
increase after PMA treatment. PMA-elicited, IL-1ß mRNA expression
declined towards the end of the stimulation
period, whereas steady state mRNA levels of ic-IL-1RA remained constantly
upregulated (Figs. 1 and
2). In cell culture supernatants (even after 36 h treatment)
no IL-ß or IL-1RA proteins were found before and after PMA-treatment
(data not shown).
Effect of several mediators on IL-1ß and
IL-1RA protein expression
PMA, IL-1ß and TNF alpha were inducers for IL-1ß and IL-1-RA
in DPC lysates. Treatment with IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, GM-CSF,
INF-gamma and TGF-ß1 had no effect with regard to constitutive or TNF-alpha-
or IL-1ß-elicited IL-1ß- or IL-1-RA expression. The induction
of IL-1ß and IL-1RA by IL-1ß or TNF-alpha was abrogated by addition
of neutralizing, human anti-IL-1ß-antibodies. Several co-incubations
were performed (e.g., IL-1ß + IL-10; IL-1ß + INF-gamma),
but differences in IL-1ß or IL-1-RA expression were not noted. PGE2
appeared to be a strong inducer of IL-1-RA, whereas IL-1ß expression
was not affected. IL-1ß and IL-1RA were detected in cell lysates
only (Table II). Semiquantitative
RT-PCR was additionally performed and the results in terms of protein
accumulation were reflected by changes in specific trancript levels for
IL-1ß and ic-IL-1-RA (not shown).
Enhanced surface expression of type I and type II
IL-1 receptors after co-incubation with INF-gamma and TNF-alpha
Co-incubation of dermal papilla cells for 33 h with INF-gamma and TNF-alpha
led to a strong upregulation of type I and type II IL-1 receptors (Fig.
3), whereas any other treatment (as described in Materials
and methods) had no effect on constitutive or INF-gamma/TNF-alpha-elicited
surface expression of the studied receptors (data not shown).
Discussion
Our experiments show that DPC in vitro express crucial members
of the IL-1 family. They do not, however, secrete substantial amounts
of IL-1ß or IL-1-RA into cell culture supernatants. Our observation
confirms other reports, where only cell-associated IL-1-RA were detected
[14-16]. With regard to IL-1-RA we found only induction of ic-IL-1-RA
mRNA after treatment with PMA, PGE2, IL-1ß or TNF-alpha. The other tested
cytokines were ineffective, in contrast to other cell types [17-20]. DPC
are able to induce IL-1-RA via an autocrine pathway. Neutralizing
anti-IL-1ß antibodies antagonize TNF-alpha-elicited IL-1-RA expression.
In AA, several cytokines were found to be aberrantly expressed in
situ and one of those is IL-1ß. This is not surprising because
any immune response is in some way modified by cytokines. However, in
AA the immune response preferentially affects the hair bulb, reflecting
the presence of, so far unidentified, antigens. It is possible to assume
that diffusable factors such as cytokines affect crucial structures within
the hair bulb, thus influencing the control of the hair cycle and leading
to hair loss.
All hair follicles alternate between periods of active hair growth and
periods of rest. These cycles involve both epithelial and mesenchymal
structures of the hair follicle, and recent advances have identified various
factors which might be encompassed during this process. Several agents
such as glutathione-S-transferase have been found to be sequentially expressed
after the induction of anagen follicle in a mouse model [21]. Apparently,
this delicate and so far not completely understood process is, in the
case of AA, the target of an immune response that primarily does not interfere
with hair growth. Recent data suggest that IL-1ß may be one of the
factors stopping the hair cycle [5], and our results illustrate that the
dermal papilla can be regarded as being capable of participating in IL-1ß-driven
immune responses. The presence of IL-1-RA and IL-1 receptors within the
dermal papilla may be important to maintain a physiologic homeostasis
during IL-1 induction and release. Because prostanoids are commonly released
during inflammation, it was remarkable to observe that PGE2 is a strong
inducer of IL-1-RA in DPC. Further studies should show whether IL-1-RA
induction by PGE2 plays a role in the treatment of AA with contact allergens.
If this theory holds true, then it is unlikely that other cytokines such
as IL-10, INF-gamma or TGF-ß1, which are released after DCP treatment
[6] and which have no effect on constitutive or IL-1ß-elicited IL-1-RA
expression, are involved in meditating hair regrowth in AA patients during
DCP treatment.
In active AA, we have found aberrant mRNA levels for INF-gamma, IL-1ß
and IL-2 in scalp biopsies [6]. The cells which synthesize these cytokines
are unknown. In vitro, DPC fail to secrete IL-1ß after stimulation
with INF-gamma as has been shown for keratinocytes [22]. It is therefore unlikely
that DPC are the source of IL-1ß during the immune response present
in early AA. Hair matrix keratinocytes or outer root sheath keratinocytes
may be more important candidates.
In conclusion, within the rather sheltered microenvironment of the dermal
papilla, DPC can be regarded as immunologically active cells able to participate
in IL-1-triggered immune responses. Future studies may show whether the
controversial release of PGE2 during allergic contact dermatitis induces
around the hair bulb a microenvironment in which a predominant expression
of IL-1RA and a reduced expression of IL-1ß are responsible for
the induction of hair regrowth in alopecia areata. If so, a preliminary
explanation for the beneficial effect of ACD in AA could be given.
CONCLUSION
Acknowledgements
This work was supported by a grant from the Deutsche Forschungsgemeinschaft
(Ho 1598/1-2), Bonn, Germany.
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