(poursuite du programme 2000-2001)
Auteur : Pr. Amos GILHAR (Skin Research Laboratory Technion Faculty of Medicine P.O.B. 9649 HAIFA 31096 ISRAEL).
Travail subventionné par l’AAA (subvention 2002) à hauteur de 12 000 €.
Subvention débloquée le 27 février 2003.
Compte rendu fourni le 08 juin 2004.
COMPTE RENDU DETAILLE (texte intégral)
ACCELERATION OF ORGAN RESTRICTED AUTOIMMUNE DISEASE BY LOSS OF IMMUNE PRIVILEGE: ALOPECIA AREATA INDUCED IN C3H/HeJ MICE BY INTERFERON-GAMMA
Alopecia areata is an autoimmune condition of the hair follicle which results in hair loss in both humans and mice. Hair follicle epithelium is an immune privileged site, with minimal expression of MHC class I and II. Paus proposed that alopecia areata results from loss of immune privilege with induction of MHC class I and II, followed by presentation of MHC class I restricted autoantigens to CD8+ cells. INF-g is known to induce MHC class I and II on hair follicle epithelium. C3H/HeJ mice develop spontaneous alopecia areata with age (20% at 12 months). It was hypothesized that injection of INF-g would induce alopecia areata in genetically susceptible young C3H/HeJ mice. C3H/HeJ mice (8 wks old) were injected intravenously with INF-g 2 X 104 U or saline for 3 consecutive days, and every 7 days thereafter. Hair loss was observed in (7/9) mice injected with INF-g and in none of the (0/9) mice injected with saline. INF-g injected mice demonstrated peri-follicular CD4+ cells, and intrafollicular CD8+ cells with dystrophic anagen hair follicles, along with expression of MHC class I and II by follicular epithelium. These findings are typical of both murine, and human alopecia areata. It was not possible to induce hair loss in non-susceptible mouse strains (C3H/HeN and C57Bl/6J) by injection of INF-g. Organ restricted autoimmune disease (alopecia areata) can be induced by loss of immune privilege on a genetically susceptible background.
Alopecia areata is an autoimmune condition of the hair follicle resulting in hair loss. Histologic findings include peri-follicular T-cell infiltrates (1). Hair loss may involve the entire scalp (alopecia totalis), or all body hair (alopecia universalis). As the condition often affects young adults, and children, it can result in psychiatric consequences (2,3). With evidence that alopecia areata is T-cell mediated, the condition can used as a model for T-cell mediated autoimmunity.
There is a well characterized model of alopecia areata in C3H/HeJ mice, with many histologic, and immunohistologic features similar to the human condition (4). The mice develop spontaneous alopecia as they age, with 20% of female mice developing alopecia by 12 months (5). It is possible to transfer this alopecia to younger mice by grafting of involved skin (6). It is thought that this transfer is mediated by passenger lymphocytes inducing autoreactivity by the host immune system, since the transfer is not effective in immunodeficient mice. Both CD4+ and CD8+ T-cells have a role in the alopecia (7), in a direct parallel to the human condition (8). The C3H/HeJ mouse model is particularly valuable because many therapies effective in humans, including immunotherapy, and immunosuppression, are effective in this model (9,10,11). The major problem with this model is that only a minority of mice develop alopecia with advanced age, and production of a large cohort of mice for therapeutic studies requires skin grafting.
Hair follicles in both mice, and humans may represent an immune privileged site. The proximal follicular epithelium does not express major histocompatibility complex (MHC) class I or class II molecules (12,13), unless induced by interferon-gamma (INF-g) (14,15). This is the case in alopecia areata in both humans, and C3H/HeJ mice, where MHC class I and II are expressed by the follicular epithelium. Paus et al. (16) have hypothesized that induction of class I MHC in alopecia areata allows an autoaggressive response by melanocyte reactive CD8+ T-cells. It was suggested that the CD8+ cells induce HLA-DR on the affected hair follicles by production of INF-g, resulting in a second wave of CD4+ cells. The goal of this study was to the determine the role of loss of immune privilege in the induction of alopecia areata. A secondary goal was to develop a model of alopecia areata that could generate sufficient numbers of mice for use in therapeutic trials, and which would be available to the general research community.
MATERIALS AND METHODS
Mice. C3H/HeJ, C3H/HeN, and C57Bl/6J mice were obtained from standard production colonies at Jackson Laboratories (Bar Harbor, ME, USA). The mice were raised in the pathogen-free animal facility of the B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology. Animal care and research protocols were in accordance with institutional guidelines, and approved by the institutional committee on animal use.
Interferon-g treatments. Mice were first shaved, to induce the anagen (growing phase) of the hair cycle. The mice were then injected intravenously with INF-g 2 X 104 U (Cytolab/Peprotech) or saline for 3 consecutive days, followed by injections every 7 days. For the first experiment there were 4 mice per group, and for the second experiment there were 5 mice per group (C3H/HeJ females; 8 weeks of age). This treatment was previously shown to induce expression of MHC class I and II on follicular epithelium (15).
Histology and immunohistochemistry. The following monoclonal antibodies to murine markers, and secondary antibodies were used: Rat anti-mouse CD8 (clone 53-6.7; SouthernBiotech, Birmingham, USA), Rat anti-mouse CD4 (clone RM4-5; Biosciences Pharmingen), Goat anti-mouse CRG-2/IP-10 (R&D Systems Inc), Goat anti rat IL-10 (R&D Systems Inc), Rat anti-mouse MHC class I (ER-MP42; BMA Switzerland), Rat anti-mouse MHC class II (ER-TR3; BMA Switzerland), Rat anti-mouse IL-4 (AMS Biotechnology, Switzerland), Biotinylated goat anti-rat IgG (Vector Laboratories, Burlingame,USA), Biotinylated rabbit anti-goat IgG from (Vector Laboratories, Burlingame, USA).
Frozen sections were stained for the above indicated mouse markers (7). For cytokine detection, sections were fixed with formaldehyde, blocked with normal serum, followed by blocking of peroxidase activity and endogenous biotin. Anti-cytokine antibody was applied onto the sections for 1.5 hours at room temperature followed by appropriate second antibody and avidin biotin complex. Color was developed with AEC substrate. PBS-Saponin buffer was used for all the incubations and washing steps. For the remaining antibodies, the same procedure was used except for fixation with acetone and the use of tris buffer for the staining.
Induction of Hair loss. Mice were first shaved, to induce hair follicles to enter anagen (growing phase of hair cycle). This was done because anagen hairs are the target of inflammation in alopecia areata, and presumably express relevant autoantigens. The mice were then injected intravenously with INF-g 2 X 104 U or saline for 3 consecutive days, and every 7 days for 5 weeks. For the first experiment there were 4 mice per group, (C3H/HeJ females; 8 weeks of age) group 1 received PBS, and group 2 received INF-g. Hair regrowth was noted in both groups by day 22. Hair loss was noted in the INF-g injected mice on day 36 (3/4 mice). On day 64 regrowth was observed, with the presence of white hairs. A second wave of hair loss was observed in the same (3/4) mice by day 86 (Figure 1).
Figure 1. Dorsal and ventral views of mice injected with PBS (right), and INF-g (left).
For the second experiment there were 5 mice per group (C3H/HeJ females; 8 weeks of age). Mice were shaved, and injected with INF-g or PBS as above. Hair regrowth was observed by day 23, and by day 46 hair loss was noted on the ventral surface of (4/5) mice. Mice were 3.1 months of age at time of hair loss. Hair loss on the ventral surface is typical for murine alopecia areata (5). The combined data for both experiments was hair loss in (7/9) mice injected with INF-g, and (0/9) mice injected with PBS, for a significance of p < 0.01 by Chi-square.
Strains of mice that are not susceptible to alopecia areata (C57Bl/6J and C3H/HeN) were also shaved, and injected with INF-g or PBS (5 mice per group) by the above protocol. The C57Bl/6J mice received 11 injections over 57 days, and the C3H/HeN mice received 13 injections over 70 days. No hair loss was observed in any mice.
Histology and immunohistochemistry. Mice were biopsied at time of first hair loss for both H&E histology, and immunohistochemistry. INF-g treated mice showed dystrophic hair follicles in anagen, associated with intra- and peri-follicular mononuclear cell infiltrates (Figure 2).
Figure 2. Histology of INF-g treated mice (A; B),
and PBS treated mice (C; D).
Immunohistochemical staining of INF-g treated mice revealed that the intra-follicular infiltrates were predominantly CD8+ cells, and the peri-follicular infiltrates were predominantly CD4+ cells. MHC class I and MHC class II were expressed especially on the outer root sheath, but also on the inner root sheath, and MHC class I was also expressed on the hair bulb. There was strong expression of interferon inducible protein-10 (IP-10) on follicular epithelium. IL-10 was weakly expressed on follicular epithelium. IL-4 was expressed by some mononuclear cells. These findings are consistent with those previously reported for alopecia areata in C3H/HeJ mice (17,18), and are similar to those reported for humans (19). PBS treated control mice exhibited minimal to no infiltrate, and no dystrophic anagen follicles. There was no expression of MHC class I and II on proximal follicular epithelium as expected, and cytokine expression was limited to weak expression of IL-10 by follicular epithelium (Figure 3).
Figure 3. CD8+ cells, CD4+ cells, MHC class I, MHC class II, and
IP-10 in mice treated with INF-g or PBS as indicated.
It was possible to accelerate the development of autoimmune hair loss (alopecia areata) by injection of INF-g into genetically susceptible mice. The histologic, and immunochemical features of the hair loss were typical for alopecia areata of C3H/HeJ mice, and not consistent with telogen effluvium, which is hair loss induced non-specifically by illness or medication. Furthermore, the hair loss could only be induced in genetically susceptible C3H/HeJ mice, and not in C3H/HeN, or C57Bl/6J mice which are not known to develop alopecia areata. Induction of alopecia areata was associated with expression of MHC class I and II on proximal follicular epithelium, and supports the hypothesis that reversal of immune privilege can induce organ specific autoimmune disease.
Up to 20% of female C3H/HeJ mice develop alopecia areata spontaneously by 12 months of age. However, this figure was derived from a selectively breed colony at Jackson Laboratories, and the incidence in the standard production colony at Jackson laboratories is 0.25% (20). The mice for this experiment were derived from the standard production colony, and not from this selectively breed population. Injection of INF-g both accelerated the development of alopecia areata from 12 months to 3 months, and increased the incidence of the condition to (7/9) mice.
In addition to the large body of literature on the role of TH1 cytokines in many autoimmune conditions, there is other direct evidence for a role of INF-g in acceleration of murine autoimmune disease. Administration of IL-12 accelerates the development of autoimmune diabetes in non-obese diabetic mice by an INF-g dependent mechanism (21). IL-12 induction of INF-g has a similar role in accelerating renal disease in MRL-Fas(lpr) mice (22).
Alopecia areata can be transferred to human scalp grafts on CB-17 Prkdcscid (SCID) mice by the injection of scalp infiltrating T-lymphocytes (23,24). T-lymphocytes must first be activated by in vitro culture with hair follicle homogenate in the presence of APC. This suggests that alopecia areata is mediated by T-lymphocytes which recognize a hair follicle antigen. Optimal transfer of hair loss requires both CD4+ and CD8+ T-cells (25). Depletion of either CD8+ T-cells (26), or CD4+ T-cells (27) can reverse alopecia areata in the Dundee experimental bald rat, supporting a synergy or cooperation between CD8+ and CD4+ T-cells. Inflammatory T-cells of human alopecia areata are cytotoxic and possess both the Fas/Fas Ligand and granzyme B cytotoxic mechanisms (28). Hair follicle epithelium expresses Fas, and inflammatory cells are FasL positive. Alopecia areata can not be transferred by skin grafts to Fas ligand-deficient C3H/HeJ-Tnfsf6(gld) mice or Fas-deficient C3.MRL-Tnfrsf6(lpr) mice further demonstrating a role for Fas/FasL in pathogenesis (29).
The proximal anagen hair follicle epithelium has characteristics of an immune privileged site including sparse, or absent expression of MHC class I, and very low density of Langerhans dendritic cells (30). Other mechanisms of immune privilege in the anagen hair follicle include production of immunosuppressive cytokines a-MSH, TGF-b, and IGF-1 (13,31). Langerhans cells are present however, in the distal hair follicle epithelium of anagen hairs. Injection of INF-g can induce both MHC class I and II expression by follicular epithelium, and activate dendritic antigen presenting cells (e.g. Langerhans cells). We do not have kinetic data on the effects of INF-g on Langerhans cell activation, and recruitment, but it is likely that activation of Langerhans cells had a role in the acceleration of autoimmune disease.
The original proposal of Paus was that loss of immune privilege in the hair follicle resulted in recognition of MHC class I restricted antigens by auto-reactive CD8+ T-cells (16). It was further hypothesized that melanocyte associated antigens may serve as autoantigens for alopecia areata. We have previously demonstrated that melanocyte associated HLA-A2 restricted peptide epitopes are pathogenic in a human scalp graft / SCID mouse transfer model of alopecia areata (32).
Injection of INF-g accelerated the development of autoimmune organ specific disease (alopecia areata) in a genetically susceptible mouse strain. This was associated with expression of MHC class I and II in an immune privileged site (proximal hair follicle epithelium), and provides support for the hypothesis that loss of immune privilege can induce organ specific autoimmune disease in a susceptible host. This model has the additional value of providing large numbers of mice with alopecia areata for pathogenesis, and therapeutic studies.
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