CXCL10 produced from hair follicles induces Th1 and Tc1 cell infiltration in the acute phase of alopecia areata followed by sustained Tc1 accumulation in the chronic phase.

BACKGROUND: Alopecia areata (AA) is an organ-specific and cell-mediated autoimmune disease. T lymphocytes densely surround lesional hair bulbs, which is histologically referred to as "swarm of bees". However, pathomechanisms of "swarm of bees" are still uncertain. OBJECTIVE: We investigated the pathological mechanisms of "swarm of bees", focusing on T-cell chemotaxis so that inhibition of chemotaxis may be strong candidate of novel treatments for AA. METHODS: We investigate the expression of chemokine receptors on T cells obtained from peripheral blood mononuclear cells (PBMCs) and skin infiltrating cells in AA patients. In addition, real-time chemotaxis assay was also demonstrated. RESULTS: In PBMCs, the frequency of CXCR3+CD4+ T cells (Th1) was significantly higher in acute-phase AA than in chronic-phase AA or healthy control, while CXCR3+CD8+ T cells (Tc1) were significantly increased in chronic-phase AA. In the skin lesions of acute-phase AA, CXCR3+CD4+ and CXCR3+CD8+ T cells infiltrated in the juxta-follicular area. In chronic-phase AA, CXCR3+CD8+ T cells dominated the infiltrate around hair bulbs, possibly contributing to the prolonged state of hair loss. Lymphocytes obtained from a lesional skin of acute-phase AA contained CXCR3+CD4+ and CXCR3+CD8+ T cells at higher percentages than those of PBMCs, suggesting preferential emigration from the blood. Immunohistochemical and real-time RT-PCR studies demonstrated that hair follicles of acute-phase AA expressed a high level of Th1-associated chemokine CXCL10. By chemotaxis assay, freshly isolated PBMCs from acute-phase AA patients had a strong velocity of chemotaxis toward CXCL10 with increased expression of F-actin. CONCLUSIONS: These results suggest that the increased production of CXCL10 from hair follicles induces preferential infiltrates of highly chemoattracted Th1 and Tc1 cells in the acute phase of AA, and Tc1 infiltration remains prolonged in the chronic phase.

Corticotropin-releasing hormone stimulates the in situ generation of mast cells from precursors in the human hair follicle mesenchyme.

Hair follicles (HFs) maintain a peripheral, functional equivalent of the hypothalamic-pituitary-adrenal (HPA) axis, whose most proximal element is corticotropin-releasing hormone (CRH). The mast cell (MC)-rich connective-tissue sheath (CTS) of mouse vibrissa HFs harbors MC precursors. Differentiation of these MC precursors into mature MCs can be induced by stem cell factor (SCF). We have investigated whether the MC progenitors of normal human scalp HF CTS respond to stimulation with CRH. Microdissected anagen HFs and full-thickness scalp skin were treated with CRH (10(-7) M). CRH treatment induced the degranulation of CTS MCs, in addition to increasing the number of CTS MCs in full-thickness skin and HF organ cultures in situ. In the latter, cells with characteristic MC features emigrated from the CTS. CRH-receptor protein expression in the CTS was colocalized with Kit expression on some CTS MCs in situ. CRH treatment upregulated SCF mRNA and protein expression within the HF epithelium. In skin organ culture, CRH-induced degranulation of CTS MCs was abolished by anti-SCF antibody. We demonstrate that human skin is an extramedullary reservoir for MC precursors, and we have identified a regulatory loop between CRH and SCF signaling. This highlights a previously unpublished finding about neuroendocrine control of human MC biology.

Combination therapy with oral PUVA and corticosteroid for recalcitrant alopecia areata.

Alopecia areata (AA) is regarded as a tissue-specific autoimmune disease for which several therapies have been suggested to modify the immune reaction against HFs, such as contact immunotherapy, psoralen plus ultraviolet A (PUVA), corticosteroids, cyclosporine, minoxidil, and dithranol. However, severe type AA, such as alopecia totalis (AT) and alopecia universalis (AU), often show resistance against these therapies. We applied a combination therapy with oral corticosteroid and oral PUVA for intractable cases of AT and AU. These patients took 20 mg/day corticosteroid and were irradiated with UVA on the whole body 2 h after taking methoxsalen for 1 month. In all patients, the terminal hair on the whole scalp regrew after 2 months. Two patients had a relapse of hair loss 3 months after the termination of the treatment. FACS analysis revealed that the CD4+CD25(high) and CD4+CD25+FOXP3+ Treg population in PBMC was increased after the combination therapy. Furthermore, the number of infiltrating cells decreased and FOXP3+ cells were often found in lesion skin after the combination therapy. Mitogen-induced proliferation tests showed low responses against PHA and Con A after the combination therapy. Taken together, the combination therapy may modify the systemic immune system and increase the number of Treg cells, resulting in improvement of recalcitrant AA.

Roxithromycin inhibits chemokine-induced chemotaxis of Th1 and Th2 cells but regulatory T cells.

BACKGROUND: Roxithromycin (RXM), a 14-member macrolide antibiotic, has a variety of bioregulatory functions such as anti-inflammatory effects, anti-oxidant effects, and modulation of immune responses. OBJECTIVES: In this study, we analyzed the effect of RXM on chemokine-induced chemotaxis of Th1, Th2, and regulatory T (Treg) cells established from three normal human peripheral blood lymphocytes by the reported methods. METHODS AND RESULTS: Incubation with 10 microM RXM for 18 h did not alter the expression profile of CXCR3 on Th1 cells and CCR4 on Th2 and Treg cells. However, upon RXM preincubation, the migration of Th1 cells to IP-10 and Th2 cells to TARC was partially suppressed, although RXM did not influence Treg cell migration. Erythromycin and clarithromycin at the same concentration did not exert such effects. F-actin polymerization and Ca(++) influx induced by IP-10 and TARC in Th1 and Th2 cells, respectively, was down-regulated by RXM pretreatment. CONCLUSION: These results imply that RXM exhibits bioregulatory function by influencing chemotaxis of Th1 and Th2 cells while leaving Treg cell migration unaffected.

Roxithromycin antagonizes catagen induction in murine and human hair follicles: implication of topical roxithromycin as hair restoration reagent.

Roxithromycin (RXM) is a 14-member macrolide antibiotics, with a variety of bioregulatory functions including anti-apoptotic activity to keratinocytes. Therefore, RXM has been used for many kinds of skin diseases. In this study, human and murine hair follicles were treated with RXM in order to find the possibility to cure hair loss disease such as androgenetic alopecia (AGA). In AGA, dihydrotestosterone signals apoptosis in dermal papilla cells in susceptible individuals, resting in premature termination of anagen and early entry into catagen. Therefore, anti-apoptotitic drug has a possibility of new candidate for AGA. This study revealed RXM antagonized the in vitro inhibitory effect of IFN-gamma on proliferation of keratinocytes and induction of apoptosis in murine and human hair bulb. RXM increases hair elongation and inhibits catagen-like changes induced in vitro with IFN-gamma in murine and human hair follicles. Furthermore, topical 5% RXM solution effectively restores hair growth in about half of individuals with AGA without any local and systemic adverse effects. Therefore, RXM is new candidate as a hair restoration drug for AGA.

Immune privilege and the skin.

This chapter summarizes the evidence that defined compartments of the hair follicle (HF) and nail epithelium maintain an area of relative immune privilege (IP). HF and nail IP is chiefly characterized by absent or very low level of expression of major histocompatibility complex class Ia antigens, complemented by a number of factors, such as the local production of potent immunosuppressive agents, dysfunction of professional antigen-presenting cells and inhibition of natural killer cell activities. In the hair bulb, IP is seen only in the anagen stage of HF cycling, while the nail apparatus continuously maintains an IP site in its proximal nail matrix, since the nail apparatus does not cycle. Possibly, the (non-cycling) bulge area of human scalp HFs also enjoys some relative, stably maintained IP, even though it is not as pronounced as the IP of the anagen hair bulb. A collapse of HF and nail IP likely plays a key role in the pathogenesis of one of the most common organ-specific autoimmune diseases, alopecia areata. Therefore, the therapeutic restoration of IP collapse promises to be a particularly effective future strategy for the treatment of alopecia areata.

Maintenance of hair follicle immune privilege is linked to prevention of NK cell attack.

Hair follicles (HFs) enjoy a relative immune privilege (IP) that is characterized by downregulation of major histocompatibility complex (MHC) class I and local expression of potent immunosuppressants. Normally, natural killer (NK) cells attack cells with absent/low MHC class I expression. However, because few perifollicular NK cells are found around healthy human anagen HFs, we asked how HFs escape from NK cell attack. This study suggests that this happens via an active NK cell suppression. Alopecia areata (AA), an organ-specific autoimmune disease thought to result from a collapse of HF-IP, in contrast, shows striking defects in NK cell inhibition/containment. We show that the NK cell inhibitor macrophage migration inhibitory factor is strongly expressed by the HF epithelium, and very few CD56(+)/NKG2D(+) NK cells are observed in and around normal anagen HFs compared to AA with prominent aggregations of CD56(+)/NKG2D(+) NK around AA-HFs. By flow cytometry, many fewer NK function-activating receptors (NKG2D, NKG2C) and significantly more killer cell Ig-like receptors-2D2/2D3 were found to be expressed on peripheral blood CD56(+) NK cells of healthy controls than on those of AA patients. In addition, only weak immunoreactivity for MHC class I chain-related A gene was observed in normal anagen HFs compared to AA. To our knowledge, this defect is previously unreported and must be taken into account in AA pathogenesis and its management.

Immunology of the human nail apparatus: the nail matrix is a site of relative immune privilege.

The nail apparatus is constantly exposed to environmental damage. It requires effective immune responses to combat infection, while avoiding the loss of nail production and regeneration by autoaggressive immunity. By immunohistology, we define here previously unknown characteristics of the normal human nail immune system (NIS). Compared with other regions of nail epithelium, human leukocyte antigen (HLA)-A/B/C expression is prominently down regulated on both keratinocytes and melanocytes of the proximal nail matrix (PNM), whereas HLA-G(+) is upregulated here. Together with the expression of macrophage migration inhibitory factor in PNM, this may serve to inhibit an natural killer (NK) cell attack on major histocompatibility complex (MHC) class Ia-negative PNM. PNM also displays strong immunoreactivity for potent, locally generated immunosuppressants such as transforming growth factor-beta1, alpha-melanocyte stimulating hormone, insulin-like growth factor-1, and adrenocorticotropic hormone, exhibits unusually few CD1a(+), CD4(+), or CD8(+), NK, and mast cells. Finally, MHC class II and CD 209 expression on CD1a(+) cells in and around the PNM is reduced, indicating diminished antigen-presenting capacity. Thus, the NIS strikingly differs from the skin immune system, but shows intriguing similarities to the hair follicle immune system, including the establishment of an area of relative immune privilege in the PNM. This nail immune privilege may offer a relative safeguard against autoimmunity. But, the localized intraepithelial defect of innate and adaptive immunity in the PNM revealed here also may impede effective anti-infection defense.

Human hair follicles display a functional equivalent of the hypothalamic-pituitary-adrenal axis and synthesize cortisol.

The skin and its major appendages are prominent target organs and potent sources of key players along the classical hypothalamic-pituitary axis, such as corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and alpha melanocyte stimulating hormone (alpha-MSH), and even express key steroidogenic enzymes. Therefore, it may have established local stress response systems that resemble the hypothalamic-pituitary-adrenal (HPA) axis. However, functional evidence that this is indeed the case in normal human skin in situ has still been missing. We show that microdissected, organ-cultured human scalp hair follicles respond to CRH stimulation by up-regulating proopiomelanocortin (POMC) transcription and immunoreactivity (IR) for ACTH and alpha-MSH, which must have been processed from POMC. CRH, alpha-MSH, and ACTH also modulate expression of their cognate receptors (CRH-R1, MC1-R, MC2-R). In addition, the strongest stimulus for adrenal cortisol production, ACTH, also up-regulates cortisol-IR in the hair follicles. Isolated human hair follicles secrete substantial levels of cortisol into the culture medium, and this activity is further up-regulated by CRH. CRH also modulates important functional hair growth parameters in vitro (hair shaft elongation, catagen induction, hair keratinocyte proliferation, melanin production). Finally, human hair follicles display HPA axis-like regulatory feedback systems, since the glucocorticoid receptor agonist hydrocortisone down-regulates follicular CRH expression. Thus, even in the absence of endocrine, neural, or vascular systemic connections, normal human scalp hair follicles directly respond to CRH stimulation in a strikingly similar manner to what is seen in the classical HPA axis, including synthesis and secretion of cortisol and activation of prototypic neuroendocrine feedback loops.

Collapse and restoration of MHC class-I-dependent immune privilege: exploiting the human hair follicle as a model.

The collapse of major histocompatibility complex (MHC) class-I-dependent immune privilege can lead to autoimmune disease or fetal rejection. Pragmatic and instructive models are needed to clarify the as yet obscure controls of MHC class I down-regulation in situ, to dissect the principles of immune privilege generation, maintenance, and collapse as well as to develop more effective strategies for immune privilege restoration. Here, we propose that human scalp hair follicles, which are abundantly available and easily studied, are ideally suited for this purpose: interferon-gamma induces ectopic MHC class I expression in the constitutively MHC class-I-negative hair matrix epithelium of organ-cultured anagen hair bulbs, likely via interferon regulatory factor-1, along with up-regulation of the MHC class I pathway molecules beta(2)microglobulin and transporter associated with antigen processing (TAP-2). In the first report to identify natural immunomodulators capable of down-regulating MHC class I expression in situ in a normal, neuroectoderm-derived human tissue, we show that ectopic MHC class I expression in human anagen hair bulbs can be normalized by treatment with alpha-MSH, IGF-1, or TGF-beta1, all of which are locally generated, as well as by FK506. These agents are promising candidates for immune privilege restoration and for suppressing MHC class I expression where this is clinically desired (eg, in alopecia areata, multiple sclerosis, autoimmune uveitis, mumps orchitis, and fetal or allograft rejection).

Febrile ulceronecrotic Mucha-Habermann's disease managed with methylprednisolone semipulse and subsequent methotrexate therapies.

Febrile ulceronecrotic Mucha-Habermann's disease is an unusual severe form of pityriasis lichenoides et varioliformis acuta characterized by abrupt onset of ulceronecrotic eruption associated with a high fever and systemic symptoms. To our knowledge, 19 cases of this disease have been reported in the literature, and 4 of them were fatal. We report the case of a 12-year-old boy with this disorder who had abdominal pain, hypoproteinemia, and anemia. Although these associated symptoms are considered life-threatening factors according to reported cases, our patient was successfully treated with methylprednisolone semipulse and subsequent methotrexate therapies. From a review of the literature and the present case, we propose that when patients have these systemic symptoms, therapeutic choices include methotrexate, high-dose corticosteroids, and 4,4-diamino-diphenyl-sulfone, which may depress early development of this disease.

The hair follicle and immune privilege.

This essay reviews the available evidence that the proximal hair follicle epithelium generates and maintains an area of relative immune privilege during a defined segment of the hair cycle (i.e., during anagen). This immune privilege is chiefly characterized by a very low level of expression of MHC class Ia antigens and by the local production of potent immunosuppressive agents, such as alpha-MSH and TGF-beta1. We discuss the putative functions of immune privilige of the anagen hair bulb, favoring the view that immune privilege serves mainly to sequester anagen- and/or melanogenesis-associated autoantigens from immune recognition by autoreactive CD8+ T cells. On this basis, we develop how the "immune privilege collapse model" of alopecia areata pathogenesis was conceived. In our discussion of the clinical implications of immune privilege, we outline the currently available evidence in support of this still hypothetical scenario to explain the initiation, progression, and termination of alopecia areata lesions. We review the most recent evidence from our laboratory that alpha-MSH, IGF-1, and TGF-beta1 can downregulate IFN-gamma-induced ectopic MHC class I expression in human anagen hair bulbs in vitro. Finally, we suggest that hair follicle-derived alpha-MSH, IGF-gamma, and TGF-beta1 form part of a constitutively active "IP restoration machinery" of the anagen hair bulb, which we propose to be recruited whenever the hair follicle suffers immune injury. Finally, we sketch some particularly promising avenues for future investigation into the far too long ignored hair follicle immune privilege.