Substance P as an Immunomodulatory Neuropeptide in a Mouse Model for Autoimmune Hair Loss (Alopecia Areata)

See related commentary on pg 1289

ORIGINAL ARTICLE

Substance P as an Immunomodulatory Neuropeptide in a Mouse Model for Autoimmune Hair Loss (Alopecia Areata) Frank Siebenhaar1,2, Andrey A. Sharov1, Eva M.J. Peters3, Tatyana Y. Sharova1, Wolfgang Syska1,2, Andrei N. Mardaryev1, Pia Freyschmidt-Paul4, John P. Sundberg5, Marcus Maurer2 and Vladimir A. Botchkarev1,6 Alopecia areata (AA) is an autoimmune disorder of the hair follicle characterized by inflammatory cell infiltrates around actively growing (anagen) hair follicles. Substance P (SP) plays a critical role in the cutaneous neuroimmune network and influences immune cell functions through the neurokinin-1 receptor (NK-1R). To better understand the role of SP as an immunomodulatory neuropeptide in AA, we studied its expression and effects on immune cells in a C3H/HeJ mouse model for AA. During early stages of AA development, the number of SP–immunoreactive nerve fibers in skin is increased, compared to non-affected mice. However, during advanced stages of AA, the number of SP-immunoreactive nerves and SP protein levels in skin are decreased, whereas the expression of the SP-degrading enzyme neutral endopeptidase (NEP) is increased, compared to control skin. In AA, NK-1R is expressed on CD8 þ lymphocytes and macrophages accumulating around affected hair follicles. Additional SP supply to the skin of AA-affected mice leads to a significant increase of mast cell degranulation and to accelerated hair follicle regression (catagen), accompanied by an increase of CD8 þ cellsexpressing granzyme B. These data suggest that SP, NEP, and NK-1R serve as important regulators in the molecular signaling network modulating inflammatory response in autoimmune hair loss. Journal of Investigative Dermatology (2007) 127, 1489–1497. doi:10.1038/sj.jid.5700704; published online 1 February 2007

INTRODUCTION Alopecia areata (AA) is an autoimmune disorder of the hair follicle (HF) characterized by intra- and perifollicular inflammatory cell infiltrates consisting of CD4 þ and CD8 þ T lymphocytes, macrophages, and Langerhans cells that target HF keratinocytes, melanocytes, and dermal papilla fibroblasts (Shapiro and Madani, 1999; McDonagh and Messenger, 2001; McElwee et al., 2003; Paus et al., 2003, 2005; Hordinsky and Ericson, 2004). Aberrant expression of human leukocyte antigen class I and II antigens occurs in keratinocytes of the HF bulb leading to the autoimmune 1

Departments of Dermatology, Pathology and Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, USA; 2Department of Dermatology and Allergy, University Medicine-Charite´, Humboldt University of Berlin, Berlin, Germany; 3Department of Internal Medicine, Psychoneuroimmunology, University Medicine-Charite´, Humboldt University of Berlin, Berlin, Germany; 4Department of Dermatology, Philipp University Marburg, Marburg, Germany; 5The Jackson Laboratory, Bar-Harbor, Maine, USA and 6Laboratory of Skin Development, Regeneration and Carcinogenesis, Medical Biosciences, School of Life Science, University of Bradford, Bradford, UK Correspondence: Dr Vladimir A. Botchkarev, Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, Massachusetts 02118, USA. E-mail: [email protected] Abbreviations: AA, alopecia areata; HF, hair follicle; SP, substance P; NEP, neutral endopeptidase; NK-1R, neurokinin-1 receptor

Received 6 November 2005; revised 23 August 2006; accepted 5 October 2006; published online 1 February 2007

& 2007 The Society for Investigative Dermatology

attack by CD8 þ T lymphocytes and followed by the development of inflammatory cell infiltration in and around the HFs (Gilhar et al., 1998, 1999; Hoffmann, 1999). Cytokines secreted by inflammatory cells (tumor necrosis factor-a, IL-1b, IFNg) stimulate premature HF involution (catagen) followed by hair loss (Hoffmann, 1999; Hordinsky and Ericson, 2004; Paus et al., 2005). In about 20% of aged C3H/HeJ mice, AA develops spontaneously and affects the HF in its cyclic portion (above the bulb region to just below the level of the sebaceous gland) (Sundberg et al., 1995a, b; McElwee et al., 2003; Sundberg and King, 2003). The C3H/HeJ mouse model for AA shares many features of the human condition, with minor variations in the location of AA lesions and in the expression patterns of major histocompatibility complex I class antigens (Freyschmidt-Paul et al., 1999a; McElwee and Hoffmann, 2002). In non-affected C3H/HeJ mice, AA may be induced by grafting skin from affected animals (McElwee et al., 1998). It was shown in human skin/Scid model that AA can be induced by the transfer of T cells isolated from affected skin that recognize a hair follicle autoantigen, suggesting that AA is a T-cell-mediated disease (Gilhar et al., 1998; Gilhar and Kalish, 2006). The murine HF is richly innervated by sensory nerve fibers expressing the neuropeptide substance P (SP) that are located in close vicinity to the HF bulge region, a specialized HF compartment containing stem cell populations (Peters et al., 2001). Cutaneous SP expression and the number of www.jidonline.org 1489

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RESULTS Number of SP-immunoreactive nerve fibers in skin affected by AA fluctuates with progression of the disease

To determine whether SP expression is changed in AAaffected skin, the number of SP-immunoreactive nerve fibers 1490 Journal of Investigative Dermatology (2007), Volume 127

assessed by immunohistochemistry was compared between anagen VI skin samples collected from mice at early and advanced stages of AA development, as well as from nonaffected C3H/HeJ mice (Figure 1). Consistent with our previous data (Peters et al., 2001), SP-immunoreactive nerve fibers were seen in close vicinity to and occasionally inside the epidermis of unaffected control skin, as well as in the interfollicular dermis (Figure 1a and d). In C3H/HeJ mice at early stages of AA development, the number of intra/ subepidermal and dermal SP-immunoreactive nerve fibers was significantly increased (Po0.05), compared to nonaffected mice (Figure 1b, e, and g). By contrast, a significant decrease (Po0.01) in the number of SP-immunoreactive nerve fibers was seen in the skin of mice at advanced stages of induced AA (Figure 1c, f, and g), as well as in mice with spontaneous AA (data not shown). Also, in skin lesions of mice with advanced AA, SP protein levels determined by ELISA were significantly reduced compared to control mice not affected by AA (73.1 þ

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SP-immunoreactive nerve fibers are significantly increased during the early active growth phase (anagen II–III) of the murine hair cycle (Paus et al., 1994; Peters et al., 2001). SP was shown to act as a growth factor for cultured keratinocytes (Tanaka et al., 1988), and treatment of mice with SP induces anagen in resting HFs and promotes anagen progression in denervated, organ cultured murine skin (Paus et al., 1994; Peters et al., 2001). By contrast, in fully developed growing mouse HFs (anagen VI), SP treatment induces a deleterious perifollicular neurogenic inflammation with subsequent premature regression (catagen) of affected HFs (Arck et al., 2003). A similar perifollicular neurogenic inflammatory response can be observed after exposure of mice to stress (Arck et al., 2003; Arck et al., 2005), and this response can be suppressed by additional treatment with neurokinin-1 receptor (NK-1R) antagonists and nerve growth factor neutralizing antibodies (Arck et al., 2001, 2003; Peters et al., 2005). Interestingly in this context, the link between AA and stress is described in humans (Gupta et al., 1997; Misery and Rousset, 2001). Human HFs affected by AA are innervated by SP-immunoreactive nerve fibers, which may modulate the perifollicular immune response (Hordinsky and Ericson, 1996, 2004). Numerous data suggest that SP via binding to the NK-1R plays an important immunomodulatory role in the skin by influencing the function of immunologic effector cells involved in AA pathogenesis (CD4 þ and CD8 þ lymphocytes, macrophages, and mast cells,) (reviewed by Scholzen et al., 1998; Katayama et al., 2001; Legat et al., 2002; Steinman, 2004). During the inflammatory response, SP elicits local vasodilatation and recruitment of immune cells into inflammation sites and can stimulate cytokine secretion from these cells (reviewed by (Legat et al., 2002; O’Connor et al., 2004)). A similar response is observed in stressed and SP-treated healthy murine skin (Arck et al., 2001, 2003; Peters et al., 2005). However, roles for SP in the pathogenesis of AA, as well as systematic analysis of neuroimmune interactions in AA lesions have not been performed so far. To better understand the immunomodulatory role of SP in AA, we studied the expression and the effects of SP on immune cells in C3H/HeJ mice affected by AA. We analyzed the levels of SP protein and the number of SP-immunoreactive nerve fibers in AA-affected skin, as well as the expression of the SP-degrading enzyme neutral endopeptidase (NEP) as compared to non-affected skin. We also analyzed NK-1R expression on CD8 þ cells and macrophages accumulating around HFs in skin affected by AA. Finally, we performed prolonged treatment of AA-affected mice to analyze SP effects on AA skin and immune cells. With this approach, we aim to demonstrate that SP is an important player in the molecular signaling network regulating the inflammatory response in autoimmune hair loss.

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Figure 1. Immunohistochemistry of SP in skin affected by AA. Full-thickness samples of anagen VI skin taken from C3H/HeJ mice at early and advanced stages of AA development and from non-affected control mice were embedded and processed for SP immunohistochemistry. (a and d) Presence of SP þ nerve fibers in the (a) epidermis and (d) dermis of control skin (arrows). (b and e) Number of (b) epidermal and (e) dermal nerve fibers is increased in skin at early stage of AA (arrows). (c and f) Lack of SP þ nerve fibers in the (c) epidermis and (f) dermis of mice at advanced stage of AA. Cell nuclei are counterstained with Hoechst 33342. (g) Immunohistomorphometry of the cutaneous SP þ nerve fibers in the control and AA-affected mice. Statistical analysis is performed using Student’s t-test (means7SEM, *Po0.05, **Po0.01). Bar ¼ 20 mm. Abbreviations: DER – dermis, EP – epidermis.

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10.6 pg/ml vs 215.9 þ 28.7 pg/ml, respectively, Po0.01). Thus, these data suggested that intraneuronal expression of SP significantly fluctuates in skin affected by AA, increasing during the early stages and decreasing during advanced stages of the disease. NEP expression is increased in skin affected by AA

To test whether levels of NEP, an SP-degrading enzyme that limits SP activity (Grady et al., 1997; Olerud et al., 1999), are changed in AA-affected skin, we assessed the expression of NEP in AA-affected and non-affected skin by RT-PCR and immunohistology (Figure 2). By RT-PCR, the expression of NEP messenger RNA was markedly increased in anagen VI skin affected by AA, compared to control anagen VI skin (Figure 2a). Increased levels of NEP transcripts were seen in both spontaneous and induced AA at advanced stages of the disease (Figure 2a). By immunohistochemistry, only weak expression of NEP was seen in the control skin and NEP expression was virtually absent from the HF outer root sheath

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Figure 2. Increased expression of NEP in skin affected by AA. The expression of NEP was assessed by (a) semiquantitative RT-PCR and (b and c) immunohistochemistry in skin affected by AA (spontaneous and induced by grafting), as well as in non-affected skin from control mice. (a) Total RNA was isolated from full-thickness skin samples of C3H/HeJ mice and RT-PCR was performed with primers specific for NEP and b-actin. Expression of the NEP messenger RNA was markedly increased in spontaneously developing and experimentally induced AA lesions, compared to control skin (representative data out of three independent experiments). (b and c) Weak expression of NEP was seen in the HF outer root sheath in (b, arrows) control skin. Increase of the NEP expression in the (c, large arrows) HF outer root sheath and appearance of expression in the (c, small arrows) inner root sheath. Cell nuclei in (b and c) are visualized by Hoechst 33342. Bar ¼ 20 mm.

of anagen VI hair follicles (Figure 2b). By contrast, skin of mice with spontaneous or induced AA at advanced stages exhibited pronounced NEP expression, which was found to be substantially increased in the HF outer root sheath (Figure 2c). In these samples, NEP expression was also increased in the interfollicular epidermis (data not shown) and was additionally seen in the proximal inner root sheath of the HFs affected by AA (Figure 2c). These data suggest that NEP as an SP-degrading enzyme may contribute to the reduction of SP levels and activity in skin at advanced stages of AA development. SP treatment promotes hair follicle regression, stimulates mast cell degranulation, and differentially affects the number of immune cells in AA

To explore whether SP release is involved in the maintenance and severity of AA lesions, agarose beads soaked either with SP or with normal mouse serum (control) were implanted into the skin of mice affected by AA. This or similar models were used previously in hair research for studying the long-term effects of biologically active peptides that are released slowly into the skin from implanted beads (Botchkarev et al., 1999a, b, 2001; Paus et al., 1994; Peters et al., 2001). Given that SP after intracutaneous administration is rapidly degraded by NEP (Scholzen et al., 1998), the goal of using this model was also to avoid repeated injections of SP into the skin which may cause additional inflammation in mice affected by AA. In skin samples examined 4 weeks after SP treatment, the percentage of catagen HFs was significantly increased, compared to control skin (Figure 3a), suggesting that excess of SP supply to the skin in AA promoted HF regression, as it has previously been observed after capsaicin treatment or under stress (Maurer et al., 1997; Arck et al., 2003). Also, SP administration was accompanied by a significant increase (Po0.01) in the percentage of mast cells exhibiting signs of extensive degranulation, whereas the percentage of nondegranulated mast cells was significantly (Po0.01) reduced, compared to controls (Figure 3b and d). This observation indicates massive neurogenic inflammation, as it has previously been observed in the close vicinity of prematurely regressing hair follicles after capsaicin treatment or under stress (Maurer et al., 1997; Arck et al., 2003). In addition, CD8 þ lymphocytes in AA-affected skin were found to be significantly increased (Po0.05) after SP treatment both inside and outside of HFs, compared to vehicle-treated skin (Figure 3e–g). By contrast, the number of CD4 þ lymphocytes and macrophages was not affected by SP treatment (Figure 3e, data not shown). This suggests that together with mast cell degranulation, SP treatment also affects the cellular composition of perifollicular inflammatory infiltrates in skin affected by AA by increasing the proportion of CD8 cells. NK-1R is expressed on CD8 þ lymphocytes in skin affected by AA

To define the cellular targets for SP in skin affected by AA, double-immunofluorescence staining was performed to www.jidonline.org 1491

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Figure 3. SP treatment in skin affected by AA leads to catagen acceleration, and is accompanied by increase in the number of degranulating mast cells and CD8 þ lymphocytes. C3H/HeJ mice affected by AA were injected intracutaneously with agarose beads soaked either with SP or vehicle. Skin was harvested 4 weeks after treatment and cryosections were processed for immunohistochemistry and alkaline phosphatase staining for hair cycle staging. Statistical analyses were performed by Student’s t-test (means7SEM, *Po0.05, **Po0.01). (a) After SP treatment, the percentage of catagen HFs was significantly increased as compared to vehicle-treated skin. (b) Graph demonstrating the increase in number of mast cells with extensive degranulation after SP treatment, compared to control. (c and d) After SP treatment, skin mast cells showed markedly increased (d) degranulation, whereas non-degranulating mast cells are seen in (c) control skin. (e) Graph demonstrating selective increase in number of CD8 þ cells infiltrating HF epithelium after SP treatment, whereas number of CD4 þ cells and macrophages remains unchanged, compared to control. (f and g) Numerous CD8 þ cells are seen inside of the HF epithelium after SP treatment (arrows). Bar ¼ 20 mm.

co-visualize the expression of NK-1R and markers specific for each type of immune cell seen in the inflammatory infiltrates around affected HFs (mast cells, CD4 þ and CD8 þ lymphocytes, and macrophages). Surprisingly, in skin affected by AA, mast cells were found to lack NK-1R expression by the detection method employed here (data not shown). However, intense NK-1R expression was seen in CD8 þ lymphocytes and MOMA-2 þ macrophages infiltrating the HF epithelium (Figure 4a and c). In addition, weak NK-1R expression was seen on keratinocytes of the HF outer and inner root sheaths, but not on CD4 þ lymphocytes (Figure 4b). These data suggest that NK-1R is differentially expressed by distinct types of immune cells in skin affected by AA and CD8 þ lymphocytes may serve as one of the targets for the immunoregulatory effects of SP. SP stimulates the expression of granzyme B in CD8 þ cells

To define whether SP treatment results in changes of the expression of cytotoxicity associated molecules in skin affected by AA, the expression of granzyme B, an important marker of activated effector CD8 þ cells (Barry and Bleackley, 2002; Waterhouse et al., 2004), was assessed in AA-affected and treated by SP or by vehicle control. In skin affected by AA, granzyme B-positive cells were seen in the interfollicular dermis and only a few of them showed coexpression of CD8 antigen (Figure 4d and e). However, after SP treatment the number of granzyme B/CD8 double-positive cells was significantly (Po0.01) increased, as compared to vehicle-treated skin (Figure 4d–f). This suggests that SP 1492 Journal of Investigative Dermatology (2007), Volume 127

treatment leads to the increase of granzyme B expression in CD8 þ cells and may therefore result in the increase of their cytotoxic activity in skin affected by AA, especially within the HF epithelium. Thus, in addition to the effects on skin mast cells and HF keratinocytes, immunomodulatory activity of SP in AA lesions may also be realized via stimulation of granzyme B expression in CD8 cells. DISCUSSION It was shown previously that neuropeptides contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and diabetes mellitus (reviewed by Frieri, 2003). Here, we demonstrate that the neuropeptide SP is also involved in the regulation of neuroimmune interactions in the AA lesions that are characterized by autoimmune inflammation around actively growing HFs involving T lymphocytes, macrophages, mast cells, and dendritic cells (Shapiro and Madani, 1999; McDonagh and Messenger, 2001; McElwee et al., 2003; Hordinsky and Ericson, 2004; Paus et al., 2005, 2006). Using the C3H/HeJ mouse model for AA (Sundberg et al., 1995a; McElwee et al., 2003), we show that the number of SP-immunoreactive nerve fibers in skin affected by AA changes significantly compared to non-affected control skin and shows a strong dependence on the disease progression, increasing during the early stages of AA development and decreasing during advanced stages accompanied by hair loss (Figure 1). In addition, we found that expression of the SPdegrading enzyme NEP increased in AA-affected skin

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Figure 4. Expression of NK-1R and effects of SP on the expression of granzyme B by CD8 lymphocytes in skin affected by AA. Skin of C3H/HeJ mice affected by AA was harvested and cryosections were processed for double immunovisualization of NK-1R and CD8 þ , CD4 þ , or MOMA-2. The expression of granzyme B, an important marker of activated effector CD8 þ cells, was assessed in SP-treated and control AA skin. (a) Expression of NK-1R on CD8 þ lymphocytes infiltrating the HF epithelium (large arrows). Some CD8 þ cells do not express NK-1R (arrowheads) and NK-1R þ /CD8-negative cells are seen in perifollicular dermis (small arrows). (b) Lack of NK-1R expression on CD4 þ lymphocytes (arrowheads). NK-1R-expressing cells are also seen in the dermis (arrows). (c) Expression of NK-1R on subset of MOMA-2 þ macrophages (large arrows). Single CD8 þ and MOMA-2 þ cells are shown by small arrows and arrowheads, respectively. (d) Graph demonstrating the increase in number of CD8 þ cells expressing granzyme B after SP treatment, compared to control. Student’s t-test, means7SEM, **Po0.01. (e) Single granzyme B þ cells (large arrow) and numerous CD8 þ cells (small arrows) are seen in the interfollicular dermis of control skin. (f) After SP treatment, the number of granzyme B/CD8 double-positive cells (yellow fluorescence, large arrows) was significantly increased. CD8 þ cells that lack expression of granzyme B are indicated by small arrows. Cell nuclei are counterstained with Hoechst 33342. Bar ¼ 20 mm. Abbreviations: DER – dermis; HF – hair follicle.

(Figure 2) indicating for active SP degradation. These findings correlate with increased mast cell degranulation following the experimental elevation of SP levels by implanting SPsoaked agarose beads into the skin affected by AA (Figure 3). Along the same lines SP treatment was found to increase AA symptoms: catagen HFs are significantly increased and CD8 cell numbers and their expression of the important cytotoxic mediator granzyme B are markedly elevated (Figures 3 and 4). SP is a small decapeptide that is rapidly released from nerve fibers during inflammation and is degraded after release by NEP (reviewed by Scholzen et al., 1998). Inflammatory diseases of the skin are frequently associated with fluctuations in the number of peptidergic nerve fibers and with their depletion owing to the massive release of neuropeptides into the sites of inflammation followed by temporary degeneration of axons (Ostlere et al., 1995). Thus, an increase in the number of SP þ nerve fibers during early stages of AA development followed by their decrease in advanced AA lesions may serve as an indicator for SP release from nerve fibers during the progression of autoimmune inflammation and proves SP involvement in the pathogenesis of AA. Our data support previous observations that revealed the presence of SP þ nerve fibers around human HFs affected by AA (Hordinsky et al., 1995; Hordinsky and Ericson, 1996, 2004; Toyoda et al., 2001). However, statistically significant alterations in the number of SP þ nerve fibers in human skin

affected by AA versus normal skin have not ever been clearly demonstrated. Moreover, radioimmunoassay results show decreased levels of SP in scalp biopsies of AA patients (Rossi et al., 1997). Although additional studies are required to further delineate the mechanisms of SP involvement in AA in humans, data presented here suggest that, similar to mice, SP levels in human skin affected by AA may also fluctuate significantly and show a strong correlation with the disease stage and/or activity. Also, our data demonstrating the increased expression of the SP-degrading enzyme NEP in AA-affected skin (Figure 2) are consistent with results that revealed similar changes in skin lesions of AA patients (Toyoda et al., 2001). NEP, which is expressed in the HF outer root sheath during anagen (Figure 2), is critically important for the regulation of tissue responsiveness to SP (Grady et al., 1997; Olerud et al., 1999). As NEP is involved in the termination of proinflammatory effects of SP in a large number of tissues (Scholzen et al., 2001; O’Connor et al., 2004; Scholzen and Luger, 2004), the increased expression of NEP may be considered an important protective and compensatory response mechanism in skin affected by AA after massive release of SP from sensory nerves. The C3H/HeJ mouse model for AA used here offers a unique opportunity to dissect neuroimmune interactions during AA in a defined and timed manner and to help in elucidating the role and targets for SP during the autoimmune www.jidonline.org 1493

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response. Our data on skin response to SP administration and on NK-1R expression suggest that SP may target at least four distinct cell populations in skin affected by AA: hair follicle keratinocytes, mast cells, CD8 lymphocytes, and macrophages (Figures 3 and 4). The increase in hair follicle regression after SP treatment observed here (Figure 3) is consistent with previously published results that show premature termination of active hair growth (anagen) in normal mice after capsaicin or SP treatment (Maurer et al., 1997; Arck et al., 2003). However, mechanisms of these effects remains to be further clarified: in particular, it is unclear whether SP directly affects HF keratinocytes and induces HF regression in AA, or whether this effect is partially or fully mast cell dependent. Because mast cell degranulation can also induce HF entering into catagen (Maurer et al., 1997), mast cells may indeed contribute to the accelerated catagen development seen in AA-affected skin after SP treatment. Although we were not able to detect NK-1R expression on mast cells in AA-affected skin with the detection method applied here, several observations suggest that SP may induce mast cell degranulation through a receptor-independent mechanism (Foreman, 1987; Saban et al., 2002; Kleij et al., 2003) or indirectly, via release of cytokines (Ansel et al., 1993) and other modulators of mast cell activity from inflammatory cells (reviewed by Paus et al., 2006). Mast cells, therefore, may be considered important players involved in neuroimmune interactions modulating the development of AA. The marked increase of mast cell degranulation after SP treatment in AA skin (Figure 3) may be considered one of the features of neurogenic inflammation. It was shown previously that neurogenic inflammation in skin accompanied by extensive mast cell degranulation can be observed after exposure of mice to stress (Arck et al., 2003), and this response can be suppressed by administration of NK-1R antagonists and/or nerve growth factor-neutralizing antibodies (Arck et al., 2001, 2003; Peters et al., 2005). Psycho-emotional stress is considered one of the important factors that trigger AA development in humans (Gupta et al., 1997; Misery and Rousset, 2001). It remains to be explored, however, whether AA development in mice may be affected by stress exposure and whether or not this effect is mast cell dependent. In addition to hair follicle keratinocytes and mast cells, CD8 lymphocytes may be considered a target for SP in AAaffected skin: CD8 cells infiltrating HFs express NK-1R, their number and expression of granzyme B are significantly increased after SP administration (Figures 3 and 4). These observations are in good agreement with data demonstrating the stimulatory effects of SP on cell adhesion and lymphocyte recruitment into inflamed tissue (Lindsey et al., 2000; Kang et al., 2004). Furthermore, SP treatment exacerbates the severity of other experimental autoimmune inflammatory diseases such as arthritis, which is T-cell dependent (Colpaert et al., 1983). It has been shown previously that SP can induce a large variety of responses in lymphocytes and that it stimulates the release of proinflammatory cytokines from these cells (O’Connor et al., 2004). Our data link, the 1494 Journal of Investigative Dermatology (2007), Volume 127

proinflammatory effects of SP to the granzyme B expression in CD8 þ cells (Figure 4). This observation is consistent with reports demonstrating the increase of cytotoxic activity of natural killer cells after SP administration (Lang et al., 2003) and suggests a new mechanism in the controlling CD8 effector functions by SP. Taken together, our data suggest that by interacting with distinct cellular targets in AA-affected skin, SP plays an important role in the pathogenesis of AA. Similar to other inflammatory conditions, SP modulates neurogenic inflammation and autoimmune response in AA at least in part by stimulating mast cell degranulation and CD8 þ cell accumulation in inflammatory lesions and by increasing their cytotoxicity. However, additional efforts employing pharmacological and genetic approaches are required to fully understand the role of SP and other neuropeptides in the pathobiology of autoimmune responses to HF antigens, which, eventually, may result in the development of new approaches for the treatment of AA and other autoimmune disorders. MATERIALS AND METHODS Mice and experimental treatment protocols All animal experiments were conducted in accordance with current federal, state, and institutional guidelines. C3H/HeJ mice affected by AA (Sundberg et al., 1995a, b) and non-affected control mice were obtained from the Jackson Laboratory (Bar-Harbor, ME). To induce AA, skin from C3H/HeJ mice that had developed spontaneous AA lesions was grafted onto the back of non-affected C3H/HeJ mice, as described previously (McElwee et al., 1998). Age-matched mice at spontaneous anagen VI (non-affected by AA, n ¼ 5), as well as mice with spontaneous AA lesions (n ¼ 10) and mice with early and advanced stages of induced AA (3–4 and 8–12 weeks after grafting, respectively, n ¼ 25) were used for experiments. Eight weeks after grafting, C3H/HeJ mice that had developed AA lesions (n ¼ 15) were injected intracutaneously either with agarose beads (Biorad Laboratories Inc., Hercules, CA), soaked with SP (1 mg/ml, Sigma, St Louis, MO), or with vehicle (normal mouse serum, Sigma) (Botchkarev et al., 2001). All mice were sacrificed after 4 weeks of observation and skin samples were collected from the control (n ¼ 5) and experimental (n ¼ 10) groups of mice, as well as from untreated animals affected by AA (n ¼ 10), according to previously described protocols (Muller-Rover et al., 2001). As inflammatory cell infiltrates and the number of immune cells attacking the HFs in AA reach their maximal extent around late anagen HFs and decrease strongly after HFs enter catagen (Sundberg et al., 1995a; McElwee et al., 2003), only skin areas that contained late anagen–early catagen HFs were selected for histomorphometry of immune cells (CD4 þ and CD8 þ lymphocytes, macrophages, and mast cells) after SP treatment.

ELISA and semiquantitative RT-PCR For protein extraction, full-thickness skin samples of C3H/HeJ mice, dissected at the level of the subcutis just below the panniculus carnosus of AA-affected and non-affected mice were pulverized in liquid nitrogen. Skin was treated with lysis buffer (0.5 ml/100 mg; 50 mmol/l Tris/HCl, pH 8.0, 150 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 5 mmol/l iodacetamid,

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10 mg/ml aprotinin, 0.2% SDS, 1% Nonidet, and 1% Triton X-100), all obtained from Sigma and lysed by sonication. After 1-hour shaking at 41C, the mixture was sonicated again. The solution was centrifuged for 30 minutes at 14,000 r.p.m. at 41C, and the supernatants were frozen and stored at 801C. For quantification of SP protein, a commercially available ELISA-kit was used (R&D Systems Inc., Minneapolis, MN) according to the manufacturer’s instructions (Botchkarev et al., 1998, 1999b). Semiquantitative RT-PCR was performed to assess the expression of the NEP and b-actin transcripts. Total RNA was isolated from fullthickness skin samples of AA-affected and non-affected C3H/HeJ mice using Trizol LS reagent (Invitrogen, San Diego, CA). Complementary DNA was synthesized by reverse transcription of total RNA, using a complementary DNA synthesis kit (Invitrogen). The following primer sets were used: NEP, 50 -TGG GAC TAC ATC AGA AAC TGC-30 and 50 -CTG ATT TCG GCC TGA GGA-30 , 460 bp; bactin, 50 -TGG AAT CCT GTG GCA TCC ATG AAA C-30 and 50 -TAA AAC GCA GCT CAG TAA CAG TCC G-30 , 520 bp. Primers used were designed according to the reported sequences in the GenBank databases. Amplification was performed using Taq polymerase (Invitrogen) over 35 cycles, using an automated thermal cycler. Each cycle consisted of the denaturing at 941C (1 minute), annealing at 601C (45 seconds), and extension at 721C (45 seconds). PCR products were analyzed by agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light (Sharov et al., 2003b, 2004, 2005).

was employed, because this technique allows for the visualization of dermal papilla morphology, a useful marker for staging HF cycling (Muller-Rover et al., 2001). The percentages of HFs in distinct catagen stages were assessed and calculated in SP-treated and control mice. All evaluations were performed on the basis of accepted morphologic criteria of HF classification (Muller-Rover et al., 2001). Only every tenth cryosection was used for analysis in order to exclude the repetitive evaluation of the same HF, and 2–3 cryosections were assessed from each animal. Altogether, 250–300 HFs in 50–60 microscopic fields, derived from five animals (approximately 50–60 follicles per animal) of distinct age were analyzed and compared to a corresponding number of HFs from appropriate, age-matched wild-type mice. Number of SP þ nerve fibers in skin during early and advanced stages of AA was assessed, as described previously (Peters et al., 2001). The number of immune cells (lymphocytes, macrophages, and mast cells) was assessed in skin cryosections of SP-treated and control animals, as described previously (Hoffman et al., 1996; Freyschmidt-Paul et al., 1999b). In total, 40–50 such measurements were performed in 50–60 microscopic fields derived from five animals per experimental and control group. All sections were analyzed at  200 to  400 magnification, and means7SEM were calculated from pooled data. Differences were judged as significant if the P-value was lower than 0.05, as determined by the independent Student’s t-test for unpaired samples. CONFLICT OF INTEREST The authors state no conflict of interest.

Immunohistochemistry Cryostat sections, fixed in acetone (201C, 10 minutes), were used to analyze the expression of NEP, NK-1R, lymphocyte markers CD4/ CD8, macrophage marker MOMA-2, and granzyme B antigen in non-affected skin of control C3H/HeJ mice and in AA-affected skin. Perfusion fixation with formaldehyde was used for visualizing SPimmunoreactive nerve fibers in skin, as described previously (Botchkarev et al., 1997a, b). Rabbit polyclonal antibodies were used for the analysis of SP, NEP and NK-1R expression (Chemicon, Temecula, CA). CD4 þ and CD8 þ T cells were detected by rat mAbs (Pharmingen, San Diego, CA), and rabbit polyclonal antisera were used to visualize MOMA-2 (Nibbering et al., 1987; Serotec, Kidlington, Oxford, UK) and granzyme B (Lab Vision, Fremont, CA). All sections were incubated with primary antisera overnight at room temperature, followed by incubation with TRITC- or FITC-conjugated F(ab)2 fragments of goat IgG (Jackson ImmunoResearch, West Grove)for 45 minutes at 371C. Double immunovisualization of the NK-1R and CD8, CD4 or MOMA-2, or granzyme B and CD8 antigens was performed according to the protocols described previously (Botchkarev et al., 1999a, 2000; Sharov et al., 2003a). Mast cells were visualized by FITC–avidin staining, as described previously (Botchkarev et al., 1997b). Sections were counterstained with Hoechst 33342 (Sigma). The same investigator analyzed all sections used for the evaluation of cell numbers in a blinded manner. Photo-documentation was performed by multicolor confocal microscopy (Zeiss, Thornwood, NY) and digital image analysis system (Pixera, Los Gatos, CA).

Histomorphometry and statistical analysis For the precise identification of the defined stages of HF cycling, histochemical detection of endogenous alkaline phosphatase activity

ACKNOWLEDGMENTS This study was supported in part by Areata Foundation (to VAB and JPS), Society (to VAB), NIH (RR000173 Forschungsgemeinschaft and the Otto Stiftung (to MM).

grants from the National Alopecia the North-American Hair Research to JPS), and from the Deutsche Eppenauer und Lieselotte Gutzeit-

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