Alopecia areata in C3H/HeJ mice involves leukocyte-mediated root sheath disruption in advance of overt hair loss.

Alopecia areata (AA) can be induced in C3H/HeJ mice by grafting full-thickness AA-affected skin. An 8- to 12-week delay between surgery and overt hair loss onset provides an opportunity to examine disease pathogenesis. Normal haired C3H/HeJ mice were sham-grafted or grafted with AA-affected skin. Mice were euthanatized 2, 4, 6, 8, 10, and 12 weeks after surgery along with chronic AA-affected mice as a positive control. Until 6 weeks after grafting, inflammation was only evident around anagen-stage hair follicles in host skin adjacent to but not distant from the AA-affected graft. From 8 weeks on, AA-grafted but not sham-grafted mice exhibited a diffuse dermal inflammation at distant sites that progressively focused on anagen-stage hair follicles at 10 and 12 weeks. Perifollicular inflammation was primarily composed of CD4+ and CD8+ cells associated with follicular epithelium intercellular adhesion molecule -1 expression. Only CD8+ cells penetrated intrafollicularly by 12 weeks after surgery, although both CD4+ and CD8+ intrafollicular cells were observed in chronic AA-affected mice. Under electron microscopy, intrafollicular lymphocyte and macrophage infiltration associated with hair follicle dystrophy was prominent 10 weeks after surgery, primarily within the differentiating outer and inner root sheaths. This study shows that focal follicular inflammation develops some time in advance of overt hair loss and focuses on the differentiating root sheaths in C3H/HeJ mice. The severity of inflammation and the degree of hair follicle dystrophy induced by the infiltrate appear to reach a threshold level before overt hair loss occurs.

Increased expression of type 2 cytokines in chronic proliferative dermatitis (cpdm) mutant mice and resolution of inflammation following treatment with IL-12.

Chronic proliferative dermatitis (cpdm) is a spontaneous mutation that results in eosinophilic inflammation in multiple tissues, including the skin. To determine the mechanisms underlying the eosinophilic inflammation, the expression of cytokines in the skin was determined. There was increased expression of IL-4, IL-5, IL-13, and granulocyte-macrophage colony-stimulating factor in the skin of cpdm/cpdm mice, and no change in IL-10 and TNF expression. Supernatants of cultured spleen cells of cpdm/cpdm mice contained an increased amount of IL-5 and IL-13, and a decreased amount of IFN-gamma. The ability of the cpdm/cpdm mice to mount a delayed-type hypersensitivity response was greatly reduced. These data are consistent with impaired type 1 and excessive type 2 cytokine production in cpdm/cpdm mice. The significance of this imbalanced cytokine production was evident in the efficacy of systemic treatment of cpdm/cpdm mice with IL-12. Mutant mice treated for 3 weeks with IL-12 had minimal changes in the skin as opposed to the severe dermatitis in mice treated with the vehicle. Treatment with IL-11, which opposes the effect of IL-12, had no effect.

Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.

Flaky tail (gene symbol ft) is an autosomal recessive mutation in mice that results in a dry, flaky skin, and annular tail and paw constrictions in the neonatal period. Previous studies demonstrated that the ft mutation maps to the central region of mouse chromosome 3, in the vicinity of the epidermal differentiation complex, a gene locus that includes many nonkeratin genes expressed in epidermis. In this study we report a detailed characterization of the flaky tail mouse. Affected homozygous ft/ft mice exhibit large, disorganized scales on tail and paw skin, marked attenuation of the epidermal granular layer, mild acanthosis, and orthokeratotic hyperkeratosis. Biochemical analysis demonstrated that ft/ft mice lacked normal high molecular profilaggrin (approximately 500 kDa), and instead expressed a lower molecular weight form of profilaggrin (220 kDa) that is not proteolytically processed to profilaggrin intermediates or filaggrin. Mutant mice lacked the large, irregular F-type keratohyalin granules that contain profilaggrin, and filaggrin was absent from the cornified layers of ft/ft epidermis. The expression of epidermal keratins was unchanged, whereas the cornified envelope proteins involucrin and loricrin were increased in ft/ft epidermis. Cultured ft/ft keratinocytes also synthesized reduced amounts of profilaggrin mRNA and protein, demonstrating that the defect in profilaggrin expression is intrinsic to epidermal cells. These findings demonstrate that flaky tail mice express an abnormal profilaggrin polypeptide that does not form normal keratohyalin F-granules and is not proteolytically processed to filaggrin. We propose that the absence of filaggrin, and in particular the hygroscopic, filaggrin-derived amino acids that are thought to function in epidermal hydration, underlies the dry, scaly skin characteristic of ft/ft mice. This animal model provides a tool for understanding the role of filaggrin in normal epidermal function and may provide insight into the molecular basis of the filaggrin-deficient human skin disorder ichthyosis vulgaris. J Invest Dermatol 115:1072-1081 2000

Asebia-2J (Scd1(ab2J)): a new allele and a model for scarring alopecia.

A spontaneous, autosomal, recessive mouse mutation exhibiting mild scaly skin, progressive scarring alopecia, slightly runted growth, and photophobia arose at The Jackson Laboratory in 1993 in the inbred mouse strain DBA/1LacJ. Because this mutant mouse showed genetic, anatomical, and laboratory similarities to the asebia mutation, crosses were done between the new mutant and mice carrying the asebia-J allele. Because the F1 offspring were affected, indicating the two mutants were allelic, the new mutation was named asebia-2J. Careful histological analysis of skin development of mice homozygous and heterozygous for either asebia-J or asebia-2J revealed that both types of mutant mice are very similar regardless of their background. Notable histopathological features of mice homozygous for either allele included extreme sebaceous gland hypoplasia, abnormally long anagen follicles, retained inner root sheath, hair fiber perforation of the anagen follicle base, and progressive follicular replacement by scarring. In this article we present a new pathogenetic hypothesis based on the importance of the sebaceous gland in hair fiber sheath dissociation: in the absence of a functional sebaceous gland the hair follicle is destroyed. The cutaneous pathology of this mutant mouse underscores the importance of the sebaceous gland to follicular biology and presents an animal model for studying the human scarring alopecias, which characteristically begin with sebaceous gland ablation.

Lanceolate hair-J (lahJ): a mouse model for human hair disorders.

Lanceolate hair-J (lahJ) arose spontaneously in 1994 on the DBA/1LacJ inbred background at The Jackson Laboratory. Mutant mice were runted, alopecic, and lacked vibrissae. As they aged, their skin wrinkled. Affected mice developed a noninflammatory, proliferative skin disease with follicular dystrophy. Hair fibers developed a number of abnormalities including periodic nodules along the shaft (trichorrhexis nodosa), compaction resembling trichorrhexis invaginata, spiral fractures, broken tips, and lance-shaped tips. This mutation exhibits some characteristics that resemble an autosomal recessive ichthyosiform disease that occurs in humans characterized in part by peculiar, invaginating, multinodal, hair shaft abnormalities known as Netherton's syndrome. Periodic nodules also resemble the human genetic based disease monilethrix. This autosomal recessive mouse mutation, allelic with lanceolate hair (lah), based on breeding studies, is located on mouse Chromosome 18, within a cluster of genes coding for adhesion molecules. Homozygotes for either of these allelic mouse mutations have elevated serum IgE levels, a feature also common with human Netherton's syndrome.

Successful treatment of alopecia areata-like hair loss with the contact sensitizer squaric acid dibutylester (SADBE) in C3H/HeJ mice.

A type of hair loss closely resembling human alopecia areata has been described in C3H/HeJ mice. In order to test the assumed analogy with human alopecia areata, we investigated the efficacy of treatment with the contact allergen squaric acid dibutylester. In 12 C3H/HeJ mice with alopecia areata an allergic contact dermatitis was induced and elicited weekly on one side of the back by topical applications of squaric acid dibutylester. Overt hair regrowth was observed only on the treated side of the back in nine of 12 mice. Histopathologic examination revealed a change in the distribution of the inflammatory infiltrate from a dense perifollicular lymphocytic infiltrate around the mid and lower regions of hair follicles in untreated skin to a uniform presence in the upper dermis in treated skin. Immunohistomorphometric studies revealed that treatment with squaric acid dibutylester increased the CD4+/CD8+ ratio from approximately 1:2 in untreated alopecia areata to 1:1 in treated alopecia areata. Additional immunohistochemical investigations showed an aberrant expression of major histocompatibility complex class I, major histocompatibility complex class II and intercellular adhesion molecule 1 on keratinocytes of the mid and lower parts of hair follicles in untreated alopecia areata. In successfully treated skin ectopic major histocompatibility complex class I and II expression was clearly reduced, whereas intercellular adhesion molecule 1 expression showed only minor changes. In conclusion, alopecia areata-like hair loss in C3H/HeJ mice responded to treatment with the contact sensitizer squaric acid dibutylester analogous to human alopecia areata. Moreover, successful treatment changes the aberrant expression of major histocompatibility complex class I and II in a way similar to that observed in human alopecia areata. These observations support the concept that alopecia areata-like hair loss in C3H/HeJ mice can be utilized as an appropriate model for the study of human alopecia areata.

Absence of Peyer's patches and abnormal lymphoid architecture in chronic proliferative dermatitis (cpdm/cpdm) mice.

The chronic proliferative dermatitis (cpdm) mutation causes inflammation in multiple organs, most prominently in the skin. Examination of the immune system revealed severe abnormalities in the architecture of lymphoid tissues. Peyer's patches were absent. In contrast, the spleen, lymph nodes, and nasal-associated lymphoid tissues were present. The spleen had normal numbers of T and B cells, but the spleen, lymph nodes, and nasal-associated lymphoid tissues had poorly defined follicles and lacked germinal centers and follicular dendritic cells. The marginal zone in the spleen was absent. The total concentration of serum IgG, IgA, and IgE in cpdm/cpdm mice was significantly decreased, whereas serum IgM was normal. Fecal IgA was low to undetectable in mutant mice, and the concentration of fecal IgM was increased. The titer of DNP-specific Abs following immunization with DNP-keyhole limpet hemocyanin was significantly decreased for all IgG subclasses. In contrast, T cell function appeared normal as assessed by evaluation of the contact hypersensitivity response in cpdm/cpdm mice. The cpdm mutation causes a complex phenotype that is characterized by multiorgan inflammation and the defective development of lymphoid tissues. The cpdm/cpdm mouse may be a useful model to study the factors that control the development of lymphoid tissues, in particular the Peyer's patches, and the mechanisms that control the humoral immune response.

Changes in keratin and filaggrin expression in the skin of chronic proliferative dermatitis (cpdm) mutant mice.

The expression of keratins and filaggrin by keratinocytes is a highly regulated process and depends on their state of differentiation and proliferation. As such, these proteins can be used as markers to determine if keratinocyte differentiation is normal. Mutant cpdm/cpdm mice develop a chronic skin disease characterized by epidermal hyperplasia and inflammation. Immunohistochemical staining for the basal keratins K5 and K14 revealed expression in the basal and suprabasal cell layers. The expression of K1 and K10 was reduced and limited to the outer layers of the stratum spinosum. Keratin 6 was expressed in the suprabasal layers of affected skin, and throughout all layers in severely affected skin. Filaggrin was present in the stratum granulosum which had variable thickness. These results indicate that the differentiation of keratinocytes in cpdm/cpdm mice was normal. The altered distribution and expression of keratins in comparison with the skin of control mice was the result of hyperproliferation.

Spontaneous alopecia areata-like hair loss in one congenic and seven inbred laboratory mouse strains.

Alopecia areata (AA) research has been hampered by the lack of suitable animal models for use in experimental procedures. AA-like hair loss has been observed in several species, including dogs, cats, horses, cattle, and nonhuman primates; however, these examples are isolated cases in outbred species of large size, limiting their use in AA research. Inbred rodent strains are ideal research models. C3H/HeJ mice can develop spontaneous AA-like hair loss and have previously been advanced as a suitable experimental model. The search for additional mouse strains with AA-like hair loss has continued. Nonscarring, inflammatory, spontaneously reversible hair loss has been observed in individual mice from several inbred mouse strains. Aside from C3H/HeJ mice, an AA-like phenotype has been observed in the substrain C3H/HeJBir, with an expression frequency of 5%. Up to 10% of individuals in an A/J mouse colony have been confirmed to develop patchy AA-like hair loss. Isolated examples of AA have also been identified in C3H/HeN/J mice, C3H/OuJ mice, HRS/J+/hr heterozygous normal mice, CBA/CaHN-Btk(xid)/J mice, and BALB.2R-H2h2/Lil mice, each with a colony frequency of less than 1%. BALB.2R-H2h2/Lil mice may also have severe nail defects. AA is regarded as rare in nonhuman species; however, nonscarring inflammatory based alopecia has been identified in several mouse strains. These examples may represent different subtypes of the heterogeneous AA phenotype. Pathologic and genetic analysis of different AA affected mouse strains may contribute to understanding AA pathogenesis and elucidating susceptibility genes.

Experimental induction of alopecia areata-like hair loss in C3H/HeJ mice using full-thickness skin grafts.

Alopecia areata (AA)-like hair loss in C3H/HeJ mice provides an excellent model for human AA disease research. The potential to induce mouse AA in normal haired C3H/HeJ mice at an early age or serially passage the AA phenotype was investigated by exchange of full-thickness skin grafts. Skin grafts from normal male and female C3H/HeJ, or severe combined immunodeficient C3H/SmnC Prkdc(scid)/J, mice onto AA-affected C3H/HeJ mice became inflamed and lost hair (28 of 28). Successful grafts from AA-affected C3H/HeJ mice induced hair loss in histocompatible C3H/OuJ mice (four of 13) and normal C3H/HeJ mice dependent on age (four of 17 at <31 d and 15 of 15 at >70 d). The AA phenotype was serially transmitted from induced AA mice to normal C3H/HeJ mice (nine of nine). Grafts from AA-affected C3H/HeJ mice onto C3H/SmnC Prkd(scid)/J mice resulted in depigmented hair fiber regrowth and perifollicular neutrophil and eosinophil infiltrates but no hair loss (15 of 15). Sham grafting did not induce AA (none of 10). The finding that AA can be serially transferred from AA-affected C3H/HeJ mice to normal littermates and C3H/ OuJ mice, indicates that an immune response against hair follicles can be induced with suitable stimuli. Conversely, skin grafts from normal C3H/HeJ, or C3H/SmnC Prkd(scid)/J, mice rapidly lose hair due to lymphocyte, but not neutrophil and eosinophil, mediated inflammation. This AA induction method reproducibly provides large numbers of AA-affected mice to study the pathogenesis and treatment of human AA.

Rhino-9J (hr(rh9J)): a new allele at the hairless locus.

A spontaneous mutation arose in the MRL/MpJ-Fas(lpr)/Fas(lpr) colony in which mice lost hair after completion of the first hair cycle. Hair loss was progressive from head to tail. As these mice aged, they developed wrinkled skin and long curved nails. Histologically, affected skin developed comedones (pilary cysts), deep dermal cysts, and dermal foreign body granulomas associated with rupture of the follicles. The alopecic epidermis was acanthotic. The mutation was autosomal recessive. Crosses with RHJ/Le +/hr(rhJ) (rhino-J) mutant mice yielded affected offspring with the same phenotype, verifying the presence of a new allele of rhino, a mutation at the hairless (hr) locus on mouse chromosome 14.

Comparison of alopecia areata in human and nonhuman mammalian species.

Alopecia areata (AA) is a nonscarring form of inflammatory hair loss in humans. AA-like hair loss has also been observed in other species. In recent years the Dundee experimental bald rat and the C3H/HeJ mouse have been put forward as models for human AA. AA in all species presents with a wide range of clinical features from focal, locally extensive, diffuse hair loss, to near universal alopecia. Histologically, all species have dystrophic anagen stage hair follicles associated with a peri- and intrafollicular inflammatory cell infiltrate. Autoantibodies directed against anagen stage hair follicle structures are a consistent finding. Observations on AA pathogenesis suggest nonhuman species can provide excellent models for the human disease. Ultimately, animal models will be used to determine the genetic basis of AA, potential endogenous and/or environmental trigger(s), mechanism(s) of disease initiation and progression, and allow rapid evaluation of new and improved disease treatments.

Autoantibodies to hair follicles in C3H/HeJ mice with alopecia areata-like hair loss.

We have previously described spontaneous but reversible hair loss that clinically and histologically resembles human alopecia areata in a colony of C3H/HeJ mice. Alopecia areata in humans is associated with antibodies to hair follicles. This study was conducted to determine whether C3H/HeJ mice with hair loss have a similar abnormal antibody response to hair follicles. Eighteen C3H/HeJ mice with alopecia, 12 unaffected littermates, and 15 control mice were examined for circulating antibodies to C3H/HeJ anagen hair follicles by indirect immunofluorescence and against extracts of isolated C3H/HeJ and human anagen hair follicles by immunoblotting. Using both procedures, antibodies to anagen hair follicles were present in all C3H/HeJ mice with alopecia but in none of the control mice. The antibodies were also present in some unaffected C3H/HeJ littermates but were absent in mice of an unrelated strain with inflammatory skin disease and alopecia, indicating that their appearance did not result from the hair loss. These antibodies reacted to hair follicle-specific antigens of 40-60 kDa present in murine and human anagen hair follicles. These antigens were also reactive with human alopecia areata antibodies. Some of the antibodies in both C3H/HeJ mice and humans with alopecia areata reacted to antigens of 44 and 46 kDa, which were identified as hair follicle-specific keratins. This study indicates that C3H/HeJ mice with hair loss have circulating antibodies to hair follicles similar to those present in humans with alopecia areata. These findings confirm that these mice are an appropriate model for human alopecia areata and support the hypothesis that alopecia areata results from an abnormal autoimmune response to hair follicles.

Harlequin ichthyosis (ichq): a juvenile lethal mouse mutation with ichthyosiform dermatitis.

The harlequin ichthyosis (ichq) mouse mutation arose spontaneously in 1989 in a colony of BALB/cJ mice at The Jackson Laboratory. Affected mice developed thick skin due to formation of compact, orthokeratotic scales that fractured over articular surfaces, secondary to bending. Harlequin ichthyosis mice on the inbred BALB/cJ background died between 9 and 12 days of age. Onset of the clinical phenotype corresponded with emergence of hair fibers from follicles at 5 days of age. There was marked proliferation of the root sheaths of anagen hair follicles, limited to the region within the dermis. Sebaceous glands were present but small compared with those of littermate controls. Emerging hair fibers were surrounded by a thick, compact sheath of cornified cells. Mutant skin contained large mitochondria with lamellar-shaped, electron-dense structures at the ultrastructural level. Keratohyalin granules were smaller and less pleomorphic than those in control mice. Lamellar bodies were not evident in either mutant or littermate control mice. Using a panel of antibodies to evaluate changes in keratinocyte differentiation, mouse-specific keratin 6 was overexpressed in the suprabasilar, hyperplastic epidermis. Loricrin expression, within the cytoplasm of cells in the stratum granulosum, decreased rapidly postmortem, unlike that in normal mice where it was stable for over 24 hours postmortem. Filaggrin expression, within granules of cells in the stratum granulosum, was prominent, corresponding to hypergranulosis evident by light microscopy in mutant mouse skin. Skin grafts from harlequin ichthyosis mice grafted onto immunodeficient nude mice maintained the phenotype for the 10-week observation period. The mutant gene locus mapped to the proximal end of mouse chromosome 19 and is inherited as a fully penetrant autosomal recessive gene. The harlequin ichthyosis mouse mutation is very similar to human type 2 harlequin ichthyosis for which it may be a good model.

Angora mouse mutation: altered hair cycle, follicular dystrophy, phenotypic maintenance of skin grafts, and changes in keratin expression.

Angora is an autosomal recessive mouse mutation caused by a deletion of approximately 2 kilobases in the fibroblast growth factor 5 (Fgf5) gene. Phenotypically, homozygous angora (Fgf5go/Fgf5go) mice have excessively long truncal hair and can be differentiated from heterozygous (+/Fgf5go) and wild-type (+/+) littermates by 21 days of age. Abnormal hair length is due to a prolongation of the anagen phase of the hair cycle of approximately 3 days. In addition, widely scattered hair follicles produce structurally defective hair shafts that twist within the follicle, presumably causing secondary hyperplasia of the outer root sheath and epidermis adjacent to the follicle. These follicular abnormalities were accentuated by immunohistochemical detection of mouse specific keratin 6, a nonspecific marker of epidermal hyperplasia. These abnormalities could be identified from birth throughout life in angora mice genotyped by polymerase chain reaction techniques. Moreover, the long truncal hair phenotype was maintained in skin grafted onto C.B-17/Sz-scid/scid mice that had normal pelage hairs and hair cycles, suggesting that circulating or diffusible humoral factors regulating the mouse hair cycle are not involved in this mutation. The angora mutation provides another useful mouse model for studying the hair cycle and its modulation.

Development and progression of psoriasiform dermatitis and systemic lesions in the flaky skin (fsn) mouse mutant.

Flaky skin (fsn) mutant mice were originally described as a mouse model for psoriasis accompanied by hematological abnormalities. However, homozygous (fsn/fsn) mice develop a number of other pathological changes. Systematic evaluation of over 300 fsn/fsn and normal littermate control (+/+ or +/fsn) mice was carried out to characterize these changes. Psoriasiform skin lesions were first evident as focal epidermal hyperplasia and inflammation at 2 weeks of age. These lesions became confluent and diffuse by 3-4 weeks of age and were associated with marked dermal infiltration of lymphocytes and small numbers of neutrophils and macrophages. Mast cell numbers increased significantly in the dermis from 2 weeks of age onward. Diffuse dermal neovascularization accompanied these cutaneous changes. Systemic lesions included progressive and massive papillomatosis of the stratified squamous epithelium of the forestomach, hyperplasia and dysplasia of the glandular stomach, increased apoptosis of cecal enterocytes, renal glomerulopathy associated with immune complex and complement deposition, testicular degeneration, mixed inflammatory cell infiltrates and fibrosis around portal triads in the liver, splenomegaly due to massive erythropoiesis, and granulomatous lymphadenitis. This spontaneous mouse mutation provides a useful model for modulating neovascularization and keratinocyte hyperproliferation, especially since the cutaneous changes resemble some forms of psoriasis in humans.

Epidermal dendritic cell populations in the flaky skin mutant mouse.

Flaky skin (gene symbol: fsn) is an autosomal recessive mouse mutation that causes pathologic changes in the skin yielding a papulosquamous disease resembling human psoriasis. Preliminary studies of epidermal sheets from foot pads of fsn/fsn mice stained for Ia+ Langerhans cells (LC) or Thy-1+ dendritic epidermal cells (Thy-1+ DEC) indicated a rise in LC numbers at the time of weaning, when the skin lesion becomes clinically evident. To further investigate this observation, epidermal sheets were obtained from the ear, dorsal skin, and foot pads from replicates of 6 female mice (both mutants and normal littermates) on weekly intervals from birth to 8 weeks of age. Dorsal skin epidermal thickness was quantitated by computer assisted image analysis and found to be significantly thickened from one week onward in the mutant mice. Using immunofluorescence microscopy, epidermal dendritic cell numbers were determined following staining with antibodies for the following markers: Ia, NLDC-145, and S-100 (for LC) or Thy 1.2 and asialo-GM1 (for Thy-1+ DEC). Use of all 5 markers to evaluate skin from 3 different locations yielded a subtle but significant increase in LC and Thy-1+ DEC in flaky skin mice. Of the three sites evaluated, the dorsal skin and ear epidermal sheets were most informative, which corresponded to the degree of pathological involvement. Mice doubly homozygous for fsn and for the severe combined immunodeficiency (scid) mutation developed the psoriasiform dermatitis. Bone marrow grafts from fsn/fsn homozygotes to homozygous scid/scid mice reproduce the skin lesion. These studies suggest that the psoriasiform dermatitis in the flaky skin mouse mutation is associated with abnormalities at the level of hematopoietic progenitor cells.