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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.00-0699fje. |
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Department of Internal Medicine, Charité, Humboldt University, Berlin, Germany; and
* Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Hamburg, Germany
2Correspondence: Charité, Campus Virchow Klinikum, Medizinische Klinik/Biomedizinisches Forschungszentrum, Raum 2.0549, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: petra.arck{at}charite.de
SPECIFIC AIMS
Stress has long been suspected as a possible cause of hair loss in various species, even though conclusive experimental evidence is still lacking. A wide range of neuropeptides, neurotransmitters, and neurohormones mediate and modulate systemic stress responses [e.g., corticotropin-releasing hormone, substance P (SP), ACTH, ß-endorphin, prolactin, progesterone, catecholamines]. A growing body of evidence now supports that these factors can indeed alter hair growth, certainly in mice and probably also in humans. This encouraged us to further dissect the putative associations between stress and hair growth in appropriate stress-response models. We specifically investigated whether exposure to stress in a mouse model 1) changes the physiological patterns of proliferation and/or apoptosis in resting (i.e., telogen) hair follicles, using Ki67/TdT-mediated dUTP-digoxigenin nick-end labeling (TUNEL) as markers; 2) alters the number, perifollicular location, and/or activation status of macrophages and/or mast cells in murine back skin; 3) induces changes in hair growth that can be correlated with changes in peri- and/or intrafollicular immunocytes; 4) induces changes in murine skin that can be abrogated by blocking SP effects, using the highly selective NK1-SP receptor antagonist RP 67580.
In addition, we tested whether the administration of SP alone sufficed to reproduce the observed effects of sonic stress on selected parameters of skin immunology and hair biology.
PRINCIPAL FINDINGS
1. Stress increases apoptosis and decreases proliferation in the hair follicle
We first assessed whether stress is associated with changes in intrafollicular proliferation and/or apoptosis in vivo by exposing mice to an established stress source-emitting ultrasonic sound. The TUNEL technique was used to detect apoptotic cell bodies in situ and immunohistochemistry to visualize proliferating cells marked by Ki67 using a previously developed immune fluorescence-labeling technique. Normally, resting (telogen) HFs show only very few if any apoptotic cells and display minimal proliferative activity. Exposure to sonic stress resulted in a significant increase in the number of TUNEL+ cells per hair follicle unit (Table 1
). In contrast, the number of proliferating HF keratinocytes (identified by their Ki67 immunoreactivity) declined significantly under sonic stress (Table 1)
.
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2. Stress-induced intrafollicular apoptosis is reproduced by injection of SP and abrogated by coadministering a SP receptor antagonist
Next, we wished to determine whether these effects of sonic stress could be reproduced by systemic injection of one key modulator implicated in stress responses, the neuropeptide SP. In our experimental setup, the mice received a single intraperitoneal injection of recombinant SP before skin harvesting. A dramatic increase of TUNEL+ HFs was indeed seen after SP injection, simulating the highly significant increase in apoptotic cells triggered by sonic stress.
To further probe whether SP may be a key neuropeptide involved in HF stress responses, we antagonized the SP receptor by injecting a highly selective SP NK1 receptor antagonist (SP-RA). This NK1 antagonist largely abrogated the effect of stress on apoptotic HFs in stressed mice (Table 1)
. Neither SP nor SP-RA injection had a significant effect on the number of Ki67+ proliferating intrafollicular cells (Table 1)
.
3. Stress increases perifollicular macrophage clusters
We had previously shown that the accumulation of perifollicular macrophage infiltrates can be associated with HF degeneration (‘programmed organ deletion’) and that several proinflammatory cytokines, which are typically released by activated macrophages, up-regulate intrafollicular apoptosis. Therefore, we compared the presence of perifollicular clusters of MHC class II+ inflammatory cells (which had been shown to represent largely activated macrophages) in stressed and nonstressed mice. Sonic stress significantly boosted the mean number of MHC class II+ perifollicular cell clusters in the dermis of murine back skin (Table 1)
.
4. Stress stimulates mast cell degranulation
We had previously demonstrated that in mice mast cells are involved in regulating the HF transformation from resting (telogen) to active hair growth (anagen), most likely via the release of secreted products that are liberated or generated secondarily in the course of mast cell degranulation. Interfollicular mast cells can be viewed as hair cycle regulators and are involved in the control of HF regression. Because of this postulated physiological role of mast cells as ‘central switchboards of tissue remodeling’, activated (=degranulated) mast cells were also evaluated in the current sonic stress model. As shown in Table 1
, sonic stress induced a significant increase in the number of activated (i.e., strongly degranulated) mast cells in dermis of mice that had been exposed to stress.
5. Sonic stress down-regulates the number of intrafollicular 
T cells
T cells are recognized as the predominant intraepidermal T cell population in murine skin and have been implicated in the effector arm of stress responses. Stress induced a significant decrease in numbers of T cells after exposure to stress (Table 1)
. Injection of recombinant SP led to an expected significant decrease of T cells in the infundibulum in nonstressed mice, and the effect of stress could be abrogated by SP-RA.
6. SP injection mimics the effects of stress T cells, perifollicular macrophage clusters, and mast cell degranulation
Given the central role of SP in stress responses and the possible involvement of SP in hair growth control, recombinant SP was injected into nonstressed mice in order to probe whether this could mimic at least some of the phenomena produced by sonic stress. SP mimicked the effect of stress exposure activated mast cells in dermis and on MHC II+ cells and T cells in the infundibulum (Table 1)
.
7. SP NK1 receptor antagonist normalizes most stress-induced alterations
SP-RA normalizes most stress-induced alterations, i.e., the effect of sonic stress on MHC II+ cell clusters and T cells in the infundibulum could be abrogated by SP-RA (Table 1)
.
8. Stress induced an increased abortion rate
To demonstrate that a profound systemic stress response had indeed occurred in the sonic stress experimental setup, pregnant mice were used beside nonpregnant control mice, since the effects of stress on triggering an increased abortion rate in mice is well documented. As expected, a significant increase in the number of abortions was observed in mice exposed to stress. An increased abortion rate was also be induced by SP injection, but was abrogated by SP-RA (Table 1)
.
CONCLUSIONS AND SIGNIFICANCE
Previous studies have largely been restricted to correlating the temporal coincidence of stressful life events with clinical evidence of hair loss (e.g., in patients with alopecia areata or telogen effluvium) or have examined the effect of various neuropeptides, neurotransmitters, or neurohormones implicated in stress responses on hair growth. However, none of these studies has provided direct evidence that stress actually alters hair growth in vivo. The current pilot study reports the first experimental evidence available in the literature that stress can indeed negatively affect hair growth in vivo, and our key results strongly support the concept that stress inhibits hair growth. Our demonstration that one defined external stressor increases HF apoptosis and decreases keratinocyte proliferation in the HF offers the first such evidence. This study also identifies SP-dependent signaling events as well as mast cells, macrophages, and T cells as potential pathways by which stress may exert its inhibitory influence on hair growth in the context of what may constitute a brain–hair follicle axis (BHA). Taking into account the available related literature, the following hypothetical scenario can be envisioned, according to which stress may lead to hair growth inhibition in vivo (Fig. 1
).
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Induction of a central stress response may activate the hypothalamic-pituitary-adrenal stress response axis. For many of the corresponding hormones released along this axis—i.e., CRH, ACTH, prolactin, and glucocorticoids—hair growth-inhibitory effects have recently been described in mice. In addition, this central stress response may lead to SP release from sensory nerve fibers in the skin (SP+ peptidergic nerve fibers are found in murine subcutis and prominently around human HFs). Locally released SP may directly inhibit the growth of HF keratinocytes or induce apoptosis and inhibition of their proliferation via inducing the release of hair growth-inhibitory cytokines like tumor necrosis factor 
and interleukin 1 from macrophages and mast cells. Mast cell activation via stress-induced SP, ACTH, and/or CRH release is associated with the secretion of proteases (e.g., tryptase) that can activate so-called protease-activated receptors on peptidergic nerve fibers, thus triggering the release of an additional substance in a positive feedback loop. The induction of dense perifollicular infiltrates of macrophages may increase the risk of inducing ‘programmed organ deletion’ of HFs by destroying the follicle’s regenerative capacity via an attack of activated macrophages on the follicle’s epithelial stem cells.
Additional support for our hypothesis that external stressors can alter signaling along a putative BHA may arise from studies of alternative stress models that demonstrate comparable effects on HF growth and/or on hair growth-modulatory activities of mast cells and macrophages. However, other classical murine stress models (e.g., ice water swimming, restraint, crowded housing conditions, food and water deprivation) must all be expected to be confounded by direct effects of these stressors on skin and hair that, in all likelihood, interfere even more profoundly with hair growth than sonic stress. For example, dietary restrictions alone surely inhibit hair growth due to the recognized, exquisite dependence of hair growth on caloric intake; restraint or crowded housing conditions carry the risk of artifactual hair growth inhibition by mechanical irritation; and ice water swimming might cause cryotrauma to the skin and HFs. Therefore, the murine sonic stress model used here is probably the best available model for testing the effects of stress on hair growth in vivo.
In summary, the current study represents an important step toward bridging the longstanding clinical assumption of (vaguely defined and ill-documented) negative effects of stress on hair growth with the mainstream of current neuroimmunological research by showing that stress may indeed inhibit hair growth, directly or indirectly, via triggering hair growth-inhibitory changes in mast cell/macrophage activities along a putative BHA. The indications of a central role of SP in stress-induced hair growth inhibition provided here encourages one to explore the clinical use of SP receptor antagonists for managing stress-related hair loss disorders. In fact, one wonders in the light of these data and in view of the recent emergence of NK1 antagonists as promising antidepressants whether SP receptor antagonists might serve a dual role, i.e., for alleviating stress-induced hair loss and the depressive mood that comes with it.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0699fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.00-0699fje 
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