AA IN RODENT MODELS

In addition to isolated case reports of AA in outbred mammals, several inbred mouse strains and substrains have been identified with AA-like hair loss of variable expression frequency (^{McElwee et al, 1998a;McElwee et al, 1999b}). However, C3H/HeJ mouse AA is the most extensively characterized and most commonly utilized model. AA-like hair loss was first observed in a C3H/HeJ mouse colony with a frequency of 0.25% for females and 0.035% for males, at five months of age (^{Sundberg et al, 1994a;Sundberg et al, 1994b}). By 18 months, the frequency of AA increased to 20%. Typically first onset of AA develops from four months of age in females compared to six or more months in males. The lesion is often first expressed as a large, uniform thinning of hair or as multi focal sites often with symmetrical distribution on the ventrum later progressing to the dorsum.

The Dundee experimental bald rat (DEBR) is derived from a spontaneous alopecia trait first observed in BD-IX inbred rats (^{Michie et al, 1991}). To improve the poor fecundity of these alopecic animals, the BD-IX rats were crossed with Wistar rats and the DEBR descendants of this cross were derived from full sib-matings. Two separate sublines, one brown and one black hooded, have been developed subsequent to the Wistar cross, each isolated and now inbred to generation F₃₈+0. Each subline has different genetic haplotypes, but common disease expression.

DEBR rats develop a full coat of hair within two weeks of birth and hair growth continues until the rats are approximately 5 months old. First onset of AA occurs from 5 to 8 months of age with female rats twice as likely as males to express the lesion and more likely to develop extensive AA. Up to 42% of individuals in a DEBR rat colony (70% female) can express a lesion that may persist as a slight thinning of the pelage or extensive patches of hair loss. Near total loss of hair is attained in 15% of affected rats, almost exclusively female. Typically, alopecia on the head spreads backwards around the eyes to merge with alopecia on the flank and reaches a chronic stable phase at 10–14 months of age. Approximately 25% of lesional DEBR rats have nail deformities with the frequency of expression equally distributed between males and females. The nails typically present as exceptionally long and twisted (onchogryphosis) with minor irregularities on the nail surface. There is no apparent correlation between hair loss extent and nail deformities.

Histologically, both AA-affected mouse and rat skin initially exhibit increased anagen stage hair follicle activity as the lesions aggressively develop and expand. Later, in the chronic, stable disease phase, hair follicles are observed predominantly in a telogen state. Focal peri- and intrabulbar mononuclear cell infiltrates are associated with only anagen stage hair follicles; telogen hair follicles are unaffected. The infiltrates consist primarily of CD4⁺, CD8⁺ cells and macrophages (^{Michie et al, 1990;Sundberg et al, 1994a;Sundberg et al, 1994b;Zhang and Oliver, 1994}). The peri-follicular infiltrate may be located from the region of the bulbar follicular epithelium up to the level of the sebaceous glands. Pigmentary incontinence and follicular dyskeratosis are particularly prominent in rodents with lesions of prolonged duration. Research has revealed hair follicle specific autoantibodies to be present in significantly elevated concentrations in affected DEBR and C3H/HeJ mice. As with humans, hair follicle specific autoantibodies are also occasionally present in rats and mice without hair loss (^{McElwee et al, 1996a;McElwee et al, 1996b;Tobin et al, 1997a;Tobin et al, 1997b}).

DISEASE MODELS FOR ALOPECIA AREATA

Previously, it has been hypothesized that AA may be modeled on one of three basic mechanisms. (1) AA is the result of infection localized to the hair follicle activating an immune response against the infectious agent with secondary disruption of hair follicle growth (^{Skinner et al, 1995a;Skinner et al, 1995b}); (2) A fundamental defect in the immune system leads to failure of tolerance towards hair follicle specific antigens and subsequent development of a classic autoimmune disease scenario (^{Mitchell and Krull, 1984}); (3) A fundamental defect in hair follicle function exists in patients with inflammation being a secondary event (^{Goldsmith, 1991}; Nutbrown et al, 1996).

In each of the above models the AA inciting antigen(s) targeted by the immune system are apparently located within hair follicles and must be exposed to immuno-surveillance for onset of AA to occur. Whether the antigenic target within hair follicles is endogenous or exogenous has yet to be defined. Exogenous antigen activation of AA such as hair follicle localization of cytomegalovirus has been suggested (^{Skinner et al, 1995a;Skinner et al, 1995b}). However, mouse CMV and other candidate infectious agent contamination of rodent model skin could not be confirmed (^{McElwee et al, 1998b}). There is no circumstantial evidence of direct infectious agent involvement, as AA is not transferred between cohabiting rodent breeding pairs and littermates even after more than a year of continuous contact (personal observations). AA could not be described as an autoimmune disease if hair follicle localized exogenous antigens were identified as the direct activator of AA (^{McElwee et al, 1999a}). Equally, an endogenous hair follicle expressed target antigen does not preclude the involvement of environmental antigens in AA onset. Theoretically, exogenous antigens may activate AA by mimicry of endogenous epitopes eliciting an autoimmune reaction. In addition, general viral and vaccine load may be a susceptibility or severity modifier of AA.

Transient inflammation of AA affected hair follicles is readily apparent in rodents, restricted to anagen stage hair follicles. The inciting factor for hair follicle inflammation may be similarly transiently expressed. Inflammation could potentially be a purely secondary phenomenon in response to a defect of anagen stage hair follicles or a transient infectious agent that directly promotes AA. However, whether the inciting hair follicle antigen is truly endogenous or exogenous, and AA is truly autoimmune in nature, there is research evidence from rodent models indicating inflammation is the primary promoter and maintainer of hair loss during development and chronic, persistent phases of AA:

1. Immunosuppressive agents have been used successfully in AA affected rodents to promote hair regrowth (^{Sainsbury et al, 1991;Oliver and Lowe, 1995;McElwee et al, 1997}). However, such drugs are nonspecific in their action and may have a direct hair growth promoting action as well as immunosuppressive properties. This is the case for Cyclosporin A and Tacrolimus (^{Yamamoto et al, 1994;Jiang et al, 1995}) and so the beneficial effects of these agents on AA can only be taken as circumstantial evidence of an inflammatory, controlling mechanism for AA.
2. Of greater significance, CD4⁺ and CD8⁺ cell depletion using monoclonal antibodies in both AA affected C3H/HeJ mice and DEBR permitted hair regrowth whereas isotype matched irrelevant MoAbs had no effect on AA (^{McElwee et al, 1996c;McElwee et al, 1999c;Carroll et al, 2002}). This suggests CD4⁺ and CD8⁺ cells are significantly involved in maintaining the AA lesions.
3. In addition, inhibiting inflammatory cell migration using monoclonal antibodies (MoAbs) against CD44v10, commonly expressed on activated lymphocytes, blocks development of AA (^{Freyschmidt-Paul et al, 2000}).
4. Inhibition of AA onset has also been demonstrated using MoAbs against costimulatory antigen B7 commonly expressed on antigen presenting cells (APCs) (^{Carroll et al, 2002}).
5. Grafting AA affected skin to histocompatible C3Smn.CB17-Prkdc^scid/J (C3H SCID) mice induced neutrophil inflammation of anagen stage hair follicles in graft and host skin, but in the absence of CD4⁺ and CD8⁺ cells hair regrowth was observed from grafted skin and no hair loss developed in host skin. (^{McElwee et al, 1998c}).
6. AA can be transferred from AA affected mice to unaffected mice by injection of skin draining lymph node or spleen derived cells (^{Carroll et al, 2002}).
7. Further confirmation of CD4⁺ and CD8⁺ cell importance in an inflammatory mediated mechanism of AA may come from a human –Prkdc^scid/Prkdc^scid mouse xenograft model. Human AA affected skin grafted to Prkdc^scid/Prkdc^scid mice permits hair regrowth. AA can be re-induced in the skin graft after injection of in vitro activated lymphocytes (^{Gilhar and Krueger, 1987;Gilhar et al, 1998;Gilhar et al, 1999}). However, whether the SCID model approach induces hair loss mimicking AA or whether the hair loss is a scarring alopecia remains to be elucidated. Rodent AA perpetuation is inflammatory cell mediated and targets anagen stage hair follicles, but the studies described above reveal little about the initial events in AA pathogenesis.

Hypotheses for alopecia areata pathogenesis

Several hypotheses for AA development have been suggested from focal infection and toxic substance exposure to reflex nerve irritation, emotional disturbance and endocrine hormone action (^{McElwee et al, 1998a}). Recently, studies revealed gonadal hormones may play a role in susceptibility to AA onset in C3H/HeJ mice. While estrogen supplementation apparently accelerated the rate of AA progression, dihydrotestosterone supplementation entirely inhibited onset of AA (^{McElwe et al, 2001}). However, while such epigenetic factors may play a significant role in disease susceptibility and severity, they are likely secondary disease modifiers to the primary disease mechanism. In light of current evidence, recent AA pathogenesis hypotheses have focused on inflammatory cell mediation with autoimmune disease as an underlying principle. The most detailed hypothesis to date (^{Paus et al, 1993}) suggested onset of AA may involve a two stage process whereby inciting antigens within the hair follicle are abnormally exposed by subclinical immune system activity and subsequent presentation of these antigens activates a second, much larger immune system response resulting in the overt, clinical hair loss disease. In part this hypothesis relies upon the putative transient immune privilege attributed to anagen stage hair follicles and its breakdown in AA (^{Christoph et al, 2000}).

Small skin grafts from rat AA lesions transferred to prelesional DEBR rats enabled regrowth of normal hair from initially dystrophic hair follicles. Reciprocal studies in both rat and mouse models resulted in normal haired skin grafts swiftly developing a dystrophic state when transferred to mature, lesional animals. AA affected rat skin grafts to nude mice and AA affected mouse skin grafts to C3H SCID mice enabled the dystrophic hair follicles in the grafts to regrow hair (Oliver, unpublished observations;^{McElwee et al, 1998c}). This demonstrated the importance of an immune component in the perpetuation of hair loss and the importance of systemic immune influence over the localized affected hair follicles. The studies suggest that the AA-like lesions in rodents are a true, nonscarring form of alopecia that can potentially be reversed.

Intriguingly, grafting skin from AA affected mice to unaffected C3H/HeJ mice transfers or induces onset of AA typically 8–10 weeks after grafting (^{McElwee et al, 1998c}). Prior to overt hair loss there is a gradual buildup of inflammation in skin, comparatively unfocused at first, but later forming a peri- and intrafollicular distribution. This time delay between surgery and hair loss may be consistent with the Paus hypothesis (^{Paus et al, 1993}). Analysis of this time delay from skin grafting, as the disease activation event, to onset of hair loss should provide significant clues as to the mechanisms involved in AA onset.

Alopecia areata pathogenesis in rodent models

The anagen stage hair follicle has been suggested as a transient immune privileged site (^{Barker and Billingham, 1977;Westgate et al, 1991;Christoph et al, 2000}). Normal anagen hair follicles express very low or no MHC class I or class II antigens, but during human and rodent AA expression significantly increases (^{Messenger and Bleechen, 1985;Bröcker et al,1987;Khoury et al, 1988}). Increased MHC expression may result in presentation of hair follicle located antigens not normally exposed to the immune system. However, it has been indicated that MHC expression in hair follicles is a secondary phenomenon, developing subsequent to inflammatory cell infiltration. The hair follicle also incorporates a physical and biochemical barrier to immune surveillance, the basement, or glassy, membrane. For inflammatory cells to penetrate to intrafollicular positions in AA, these barriers must first be breached.

In the mouse model of AA, the putative immune privilege of hair follicles provides little resistance to activated inflammatory cells. Normal haired C3H/HeJ, histocompatible C3H SCID or C3H/OuJ skin grafted to AA affected skin is rapidly infiltrated by inflammatory cells with almost immediate hair follicle dystrophy and inhibition of visible hair fiber growth (^{McElwee et al, 1998c}). This suggests that the exciting antigen targets for activated inflammatory cells are normally expressed in anagen hair follicles of C3H mouse strains. However, it is possible that C3H/HeJ mice and histocompatible substrains inherently express antigens that are not normally expressed in unrelated mouse strains. Equally, it is possible that C3H mouse strains have a defect in the hair follicles' immune privilege resulting in a greater general susceptibility towards AA development. While inflammatory cells are apparently the controlling mediators of AA lesion development, the possibility of hair follicle defects cannot be ruled out. The commentary of Goldsmith (^{Goldsmith, 1991}) may yet be applicable to AA with some modification.

These and other evidence demonstrate rodent AA is an inflammatory mediated, "organ" specific disease and are consistent with an autoimmune pathogenesis. Although the inciting agents within anagen hair follicles have yet to be defined, a basic principle of disease development emerges from rodent model research (Figure 2). Both genetic and environmental factors can contribute to the general degree of AA susceptibility in any one individual. The combined effects may potentially act on the hair follicle and/or on aspects of the immune system. As environmental factors change with time, gene activity may be modified through gene–environment interaction and additively affect an individual's susceptibility to AA onset. Once AA has developed, genetic and environment interaction may still influence the rate of lesion progression, end stage extent of hair loss, lesion duration, and resistance to treatment.

Figure 2.

Alopecia areata pathogenesis and perpetuation.

Full figure (40K)

Onset of AA requires exposure of unknown inciting epitopes within hair follicles subsequently taken up by antigen presenting cells (APCs) and presented to lymphocytes in draining lymph nodes and possibly in other immune system organs such as the spleen (Figure 2). Activated inflammatory cells migrate from organs of the immune system and infiltrate the skin and anagen stage hair follicles. Their disruptive effects, mediated in part through Fas – Fas ligand binding, result in hair follicle dystrophy and continued antigen exposure in association with MHC expression. This may expose numerous antigenic epitopes for uptake by APCs and further stimulation of autoreactive lymphocytes. With repeated cycles of hair follicle disruption, antigen exposure, and lymphocyte activation, epitope spreading may occur and a wider range of antigens and hair follicle structures may be targeted (^{Chan et al, 1998}). This disease cycle is self-perpetuating and the initial disease activating agents need not persist for AA lesions to continue. Hypothetically, the activating antigenic epitopes may only be transiently expressed.

Practical and hypothetical treatment approaches

If AA pathogenesis is founded upon an inflammation mediated concept, then there are several possible approaches to new treatment development (Figure 3):

1. Drug treatments for AA are predominantly immunosuppressive or immunomodulatory in their effect. Immunosuppressive agents restrict infiltration of the skin and hair follicles by activated inflammatory cells (^{Gupta et al, 1990;Oliver and Lowe, 1995;McElwee et al, 1997}), while contact sensitizing agents alter the skin environment such that the action of inflammatory cells on hair follicle growth is down-regulated (^{Hoffmann et al, 1994;Hoffmann and Happle, 1999;Freyschmidt-Paul et al, 1999}).

   Remission of mouse and rat AA has been induced using several immunomodulatory compounds. Intralesional injection of triamcinolone acetonide, PUVA (methoxysporalen plus UV A light), and treatment with the topical sensitizers dinitrochlorobenzene (DNCB), diphenylcyclopropenone (DPCP), and squaric acid dibutyl ester (SADBE) have each induced hair growth in one or both models (^{Sundberg et al, 1994a;Sundberg et al, 1994b;Oliver and Lowe, 1995;Sundberg et al, 1995;McElwee et al, 1997;Freyschmidt-Paul et al, 1999;Shapiro et al, 1999;Gardner et al, 2000}). These and other evidence demonstrate a good correlation between rodent models and human AA and indicate hair loss may similarly be due to inflammatory autoimmune mediated, hair follicle specific mechanisms.

2. Minoxidil, and also in part cyclosporine and tacrolimus, have direct hair follicle growth promoting effects (^{Yamamoto et al, 1994;Jiang et al, 1995}). As such, an additional secondary AA therapeutic approach is to treat the symptom of hair loss using agents with direct action on the affected hair follicles (^{Fiedler-Weiss, 1987;Price, 1987}). However, the relatively nonspecific action, potential for side-effects, and limited response in many individuals to current treatments means new therapeutic approaches are required. In the short to mid term, new and improved drug treatments will become available, primarily derived from pharmaceutical research and developments in other autoimmune and inflammatory diseases. With a greater understanding of immunosuppressive agents, contact sensitizers and hair growth promoting drugs and their modes of action, more refined and potent drugs may become available that are suitable for use in AA.
3. Theoretically, autoimmune or inflammatory disease mechanisms can be modulated in several ways. By extension of our understanding of inflammatory mechanisms and drug action upon them, specific pathways may be targeted. Observations on contact sensitization treatment have led to speculation that future treatments could involve modifying the skin and hair follicle biochemical environment. Injection of interleukins or application of their respective cDNA sequences may be used to block or modulate inflammatory cell infiltration of the skin and hair follicles. Alternatively, similar approaches may directly promote hair growth in spite of hair follicle inflammation.
4. Potentially, the most challenging therapeutic approach is to define lymphocyte clones reactive for hair follicle antigen epitopes and either block their production (clonal deletion) or promote tolerance (clonal anergy) (^{Theofilopoulos, 1995}). Oral tolerance has been one suggested approach in autoimmune disease treatment; however, without knowledge of the antigenic targets involved in AA, such treatment approaches are not yet viable.
5. The blockade or modulation of antigen presentation and costimulation by antigen presenting cells to the responsive lymphocyte clones involved in AA may be a further focus of new treatment approaches.
6. Once the lymphocytes are activated, it may still be possible to block cell migration from sites of activation to the skin and hair follicles. Preliminary investigations in which CD44v10 was targeted with MoAbs suggest cell migration inhibition may have potential AA treatment benefit (Freyschmit-Paul et al, 2000). Other activated cell surface markers and variants expressed during migration may be additional targets. These approaches may be employed as both treatment and preventative measures in AA susceptible individuals.
7. Once hair follicle inflammation is underway, several treatment approaches are still possible. A potentially complicated therapeutic intervention may involve blocking or modulating antigens expressed within anagen stage hair follicles, that are the apparent target for inflammatory cells, or masking their expression. In addition to modifying the inflammation inciting antigen expression, inhibition of MHC expression within hair follicles may also block AA lesion perpetuation.
8. Finally, the mechanisms by which the inflammatory cells adversely affect hair follicle activity may be targeted, including disruption of Fas – Fas ligand interaction, prevention of granzyme and perforin action, oxygen radical neutralization, and alteration of the cytokine receptor and cytokine milieu.

Figure 3.

Practical and theoretical therapeutic interventions in alopecia areata pathogenesis.

Full figure (37K)

CONCLUSION

Human AA is heterogeneous in its clinical presentation and also probably in the pathogenic mechanisms of development. Multiple factors may contribute to disease susceptibility, activation, progression, and perpetuation. Influence on disease may come from the environment and from the genetics of the affected individual, and changes in the gene–environment interaction over time may modify the nature of the disease. Different factors may be important for individual cases; while genetics may be the primary determinant in one individual, the environment may be the major disease factor in another. It is possible that what we currently collectively describe as AA may be a common clinical symptom of several different mechanisms of disease pathogenesis. Consequently, rodent models may not be the equivalent of each and every human case, but may represent a subset of the human population. It is possible that different rodent models represent different human subgroups making comparative research of multiple models desirable. Where the mechanisms of human and rodent disease are similar, so information gained from rodent models will help define how AA is initiated and progresses and will provide clues as to how AA can be treated better.

Defining what comes first in the onset of spontaneous AA, hair follicle aberration or inflammatory cell infiltration may not be practical. However, research to understand the downstream cycle of disease pathogenesis after the activation event and disease perpetuation is highly practical when using rodent models. While the initiation of rodent model AA may involve a succession of events, the perpetuation of AA is likely to be a cycle of events and not a cascade. This is particularly relevant when the morphological target, the hair follicle, follows a cycle of proliferation and regression. Within the cycle there are four key events: (1) hair follicle located antigen exposure (be it exogenous, endogenous, normal or aberrant expression) to the immune system; (2) antigen presentation, costimulation, and activation of responsive lymphocytes by APCs; (3) activated inflammatory cell migration to and infiltration of hair follicles; and (4) the action of the inflammatory cell infiltrate on the hair follicles. Each of these events is vulnerable to therapeutic intervention. In addition, peripheral to the disease cycle mechanism, the reactive lymphocyte clones may be tolerized, deleted, or otherwise modulated.

Therapeutic intervention in the short term will involve new and improved drug treatments to modulate hair follicle inflammation and to promote hair growth. In the mid term, aspects of the disease cycle may be targeted with cytokines, antigens, antibodies, and other factors. In the long term, treatment may target genes directly involved in the disease cycle mechanism or the contribution of general susceptibility and severity modifying genes. Rodent models will remain vital to understand AA and to develop such treatments.

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