ARTICLE
Stringently controlled variations in the local, cytokine-mediated signaling
milieu of hair follicles (HF) have been implicated as major elements of
hair cycle control in health and disease [1, 2]. Although soluble factors
that induce hair growth (anagen) or initiate HF regression (catagen) in
man have not yet been identified definitely, studies in rodents have revealed
an increasing list of candidate cytokines or growth factors that likely
play an important role during these hair cycle transformations [1].
Several lines of clinical and experimental evidence point towards interleukin
(IL-1) as an important inducer of hair loss in various hair growth disorders.
IL-1alpha and IL-1ß are potent inhibitors of human and rodent hair growth
ex vivo [3, 4], and mice overexpressing IL-1alpha are smaller in size
and show patchy hair loss reminiscent of alopecia areata (AA) [5], a hair
disorder characterized by premature catagen development. Untreated AA
is associated with lesional overexpression of IL-1ß [6]. Ultrastructural
studies have defined the initial changes within the dermal hair papilla
and outer root sheath of HF affected by AA as a lack of structural organization
within the dermal papilla, a marked polymorphism of dermal papilla fibroblasts
and an abnormal, diamond-like shape of the dermal papilla [7]. Remarkably,
all of these morphological changes observed in vivo can be mimicked
by IL-1ß stimulation of isolated human hair follicles in vitro
[8]. In addition, the severity of the inflammatory response in AA, which
probably represents a polygenic disease, may be determined by an interplay
of pro- and anti-inflammatory cytokines. Recently, an increased frequency
of the allele 2 of the interleukin-1 receptor antagonist gene was detected
in AA patients, particularly in those showing extensive hair loss [9].
Because the IL-1 receptor antagonist is the natural antagonist of IL-1
[10], it has been proposed that patients who cannot secrete sufficient
amounts of the IL-1 receptor antagonist, due to a gene polymorphism, may
have a more progressive disease [9].
Hence, it is conceivable that IL-1 is involved
in the control of HF regression (catagen). The question is whether the
cutaneous gene expression profiles of important elements of the IL-1 signaling
system fit with this hypothesis.
To address this question, we have exploited the high degree of synchrony
during depilation-induced HF cycling [11] in mice, and have determined
by semiquantitative RT-PCR, whether the steady state mRNA levels of the
gene expression of IL-1alpha, IL-1ß, IL-1-(RA) receptor antagonist,
IL-1 receptor (R)-I and IL-1-R-II parallel distinct phases of the murine
hair cycle. Several lines of evidence indicate that IL-1-driven tissue
responses are crucially dependent on the balance between the ligands (IL-1alpha,
IL-1ß), the signal transducing receptor (IL-1-R-I) and the presence
of antagonists such as the IL-1-RA and the IL-1-R-II [10]. The fine tuning
of the IL-1 signaling elements appears to be of importance and therefore,
we have investigated the in situ mRNA expression profiles of these
proteins.
Materials and methods
For this purpose, full thickness dorsal skin was obtained from untreated,
6-9-week-old syngenic female C57BL/6 mice in the telogen stage of the
hair cycle, or from mice in various stages of the depilation-induced hair
cycle as described [11-13]. It is important to note that, while depilation
is associated with a mild wound healing response during early anagen (day
1-3 after depilation), catagen develops spontaneously, once the HF have
matured into late-stage anagen VI HF (day 17-20 after depilation) [13].
Total RNA was isolated from full-thickness back skin samples (Trizol reagent,
Gibco, Germany), and was reverse transcribed (First strand cDNA synthesis
Kit, Pharmacia, Freiburg, Germany). At least three mice were studied per
hair cycle stage. Subsequently, semiquantitative PCR was performed as
described [14, 15] by using the following amplimers:
m-ß-actin: 5' GTG GGC CGC TCT AGG CAC CAA 3' and 5' CTC TTT
GAT GTC ACG CAC GAT TTC 3'; PCR-Product: 540 bp, 35 cycles, 60° C
annealing temperature
m-IL-1alpha: 5' AAG ATG TCC AAC TTC ACC TTC AAG GAG AGC CG 3' and 5'
AGG TCG GTC TCA CTA CCT GTG ATG AGT TTT GG 3'; PCR-Product: 491 bp, 36
PCR cycles, 60° C annealing temperature
m-IL-1ß: 5' GCA ACT GTT CCT GAA CTC A 3' and 5' CTC GGA GCC
TGT AGT GCA G 3'; PCR product: 390 bp, 36 PCR cycles,
60° C annealing temperature
m-IL-1-R-I: 5' CCT GCT CTG GTT TTC TTC CT 3' and 5' CGG CAG TTT
CTC CTT AGT GT 3'; PCR product: 587 bp, 35 PCR cycles,
60° C annealing temperature
m-IL-1-R-II: 5' TGC AAA GTG TTT CTG GGA AC 3' and 5' ATA TTG CCC
CCA CAA CCA AG 3'; PCR product: 333 bp, 35 PCR cycles,
60° C annealing temperature
m-IL-1-RA: 5' GAC CCT GCA AGA TGC AAG CC 3' and 5' GAG CGG ATG
AAG GTA AAG CG 3'; PCR product: 250 bp, 30 PCR cycles, 52° C annealing
temperature
Amplification was performed by using taq polymerase (Pharmacia, Freiburg,
Germany) and an automated thermal cycler (MWG Biotech, Germany). All PCR
products were verified by cloning (TA cloning kit, Invitrogen, San Diego,
USA) and sequencing (by Eurogentech, Belgium).
Results and comment
Our results (Fig. 1)
indicate that the highly synchronized, induced murine hair cycle is associated
with profound fluctuations in the steady state mRNA levels of members
of the IL-1 signaling system. Both IL-1alpha and IL-1ß showed an expression
peak shortly after depilation, which may result from the trauma of depilation.
These expression levels declined during the course of anagen development.
Most interestingly, IL-1alpha and IL-1ß transcript levels increased
dramatically with the onset of spontaneous catagen (around day 18) and
peaked during telogen (day 25).
These fluctuations in the IL-1alpha and IL-1ß transcript levels were
paralleled by substantial changes in expression of the corresponding signal
transducing type I IL-1 receptor. The steady state mRNA levels for the
IL-1-R-II also changed in a hair cycle-dependent manner, but to a lesser
extent than for the IL-1-R-I (Fig.
1). The expression levels for the IL-1-RA did not display significant
steady state-fluctuations throughout the depilation-induced murine hair
cycle (data not shown).
Between days 17 and 20 after anagen induction
by depilation, most anagen hair follicles in this mouse model spontaneously
complete the anagen VI-catagen-telogen transformation of the hair cycle
[11, 13]. Therefore, our findings are consistent with the concept that
IL-1alpha, IL-1ß, IL-1-RI and IL-1-RII are involved in the control of
catagen development. It is reasonable to ask whether the catagen-associated
increase in expression of the IL-1 signaling family reflects only the
consequences of a catagen transformation induced by other signals (e.g.,
upregulation and release of IL-1 by activated macrophages or mast cells,
both of which have been implicated in the control of HF regression [2,
16, 17]) or whether IL-1 overexpression within, or in the vicinity, of
the anagen VI hair follicle is a crucial signaling event necessary for
the anagen-catagen switch to occur. In view of the significant growth
inhibitory effects of IL-1 on human and murine HF in vitro, and
in consideration of the alopecia areata-like hair loss in IL-1alpha transgenic
mice, it is likely that elements of the IL-1 signaling system play a functionally
important role in the control of catagen development in vivo. However,
this remains to be conclusively demonstrated in appropriate in vivo
models, e.g., by testing whether IL-1 injection into anagen VI
skin induces catagen, in a manner that is comparable to catagen induction
by topical application of dexamethasone [13].
Abbreviations:
AA, alopecia areata; HF, hair follicle; IL-1, interleukin-1; IL-1-RI,
interleukin-1-receptor type-I; IL-1-RII, interleukin-1-receptor type-II;
IL-1-RA, interleukin-1-receptor antagonist.
CONCLUSION
Acknowledgements
This work was supported by grants (Ho 1598/1-3; Pa 345/3-3) from Deutsche
Forschungsgemeinschaft (DFG) to R.H. and R.P. The excellent technical
assistance of R. Pliet and E. Hagen is gratefully acknowledged.
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