Anita C. Gilliam

A Brg1 null mutation in the mouse reveals functional differences among mammalian SWI/SNF complexes.

Mammalian SWI/SNF complexes utilize either brahma (Brm) or brahma-related gene 1 (Brg1) catalytic subunits to remodel nucleosomes in an ATP-dependent manner. Brm was previously shown to be dispensable, suggesting that Brm and Brg1 are functionally redundant. To test this hypothesis, we have generated a Brg1 null mutation by gene targeting, and, surprisingly, homozygotes die during the periimplantation stage. Furthermore, blastocyst outgrowth studies indicate that neither the inner cell mass nor trophectoderm survives. However, experiments with other cell types demonstrate that Brg1 is not a general cell survival factor. In addition, Brg1 heterozygotes are predisposed to exencephaly and tumors. These results provide evidence that biochemically similar chromatin-remodeling complexes have dramatically different functions during mammalian development.

Murine Sclerodermatous Graft-Versus-Host Disease, a Model for Human Scleroderma: Cutaneous Cytokines, Chemokines, and Immune Cell Activation

Murine sclerodermatous graft-vs-host disease (Scl GVHD) models human scleroderma, with prominent skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA. Fibrosis in Scl GVHD may be driven by infiltrating TGF-β1-producing mononuclear cells. Here we characterize the origin and types of those cutaneous effector cells, the cytokine and chemokine environments, and the effects of anti-TGF-β Ab on skin fibrosis, immune cell activation markers, and collagen and cytokine synthesis. Donor cells infiltrating skin in Scl GVHD increase significantly at early time points post-transplantation and are detectable by PCR analysis of Y-chromosome sequences when female mice are transplanted with male cells. Cutaneous monocyte/macrophages and T cells are the most numerous cells in Scl GVHD compared with syngeneic controls. These immune cells up-regulate activation markers (MHC class II I-Ad molecules and class A scavenger receptors), suggesting Ag presentation by cutaneous macrophages in early fibrosing disease. Early elevated cutaneous mRNA expression of TGF-β1, but not TGF-β2 or TGF-β3, and elevated C-C chemokines macrophage chemoattractant protein-1, macrophage inflammatory protein-1α, and RANTES precede subsequent skin and lung fibrosis. Therefore, TGF-β1-producing donor mononuclear cells may be critical effector cells, and C-C chemokines may play important roles in the initiation of Scl GVHD. Abs to TGF-β prevent Scl GVHD by effectively blocking the influx of monocyte/macrophages and T cells into skin and by abrogating up-regulation of TGF-β1, thereby preventing new collagen synthesis. The Scl GVHD model is valuable for testing new interventions in early fibrosing diseases, and chemokines may be new potential targets in scleroderma.

Hair Follicle Stem Cell-Specific PPARγ Deletion Causes Scarring Alopecia

Primary cicatricial or scarring alopecias (CA) are a group of inflammatory hair disorders of unknown pathogenesis characterized by the permanent destruction of the hair follicle. The current treatment options are ineffective in controlling disease progression largely because the molecular basis for CA is not understood. Microarray analysis of the lymphocytic CA, Lichen planopilaris (LPP), compared to normal scalp biopsies identified decreased expression of genes required for lipid metabolism and peroxisome biogenesis. Immunohistochemical analysis showed progressive loss of peroxisomes, proinflammatory lipid accumulation, and infiltration of inflammatory cells followed by destruction of the pilosebaceous unit. The expression of peroxisome proliferator-activated receptor (PPAR) gamma, a transcription factor that regulates these processes, is significantly decreased in LPP. Specific agonists of PPARgamma are effective in inducing peroxisomal and lipid metabolic gene expression in human keratinocytes. Finally, targeted deletion of PPARgamma in follicular stem cells in mice causes a skin and hair phenotype that emulates scarring alopecia. These studies suggest that PPARgamma is crucial for healthy pilosebaceous units and it is the loss of this function that triggers the pathogenesis of LPP. We propose that PPARgamma-targeted therapy may represent a new strategy in the treatment of these disorders.

Inactivation of the Vitamin D Receptor Enhances Susceptibility of Murine Skin to UV-Induced Tumorigenesis

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is the biologically active ligand for the vitamin D receptor (VDR). VDR(-/-) mice have a hair follicle-cycling defect resulting in alopecia. However, mice lacking 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1(-/-)), and having no circulating 1,25(OH)(2)D(3), have normal follicular function. These mouse models indicate that VDR functions independently of 1,25(OH)(2)D(3) in regulating hair-follicle cycling. Here, we show that VDR(-/-) mice rapidly develop chemically induced skin tumors, whereas CYP27B1(-/-) and wild-type mice do not, indicating that VDR, and not the 1,25(OH)(2)D(3) ligand, is essential for protection against skin tumorigenesis. Because the majority of human skin cancer results from exposure to UV, the susceptibility of VDR(-/-) mice to this carcinogen was also evaluated. VDR(-/-) mice developed UV-induced tumors more rapidly and with greater penetrance than did VDR(+/+) mice. p53 protein levels were upregulated at similar rates in UV-treated keratinocytes of VDR(-/-) and VDR(+/+) mice. However, rates of thymine-dimer repair and UV-induced apoptosis were significantly lower in VDR(-/-) epidermis compared with the wild type epidermis. UV-induced epidermal thickening was also attenuated in VDR(-/-) skin, indicating that VDR plays a critical role in the repair and removal of severely damaged keratinocytes and adaptation of the skin to chronic UV exposure.

Disruption of EphA2 Receptor Tyrosine Kinase Leads to Increased Susceptibility to Carcinogenesis in Mouse Skin

EphA2 receptor tyrosine kinase is frequently overexpressed in different human cancers, suggesting that it may promote tumor development and progression. However, evidence also exists that EphA2 may possess antitumorigenic properties, raising a critical question on the role of EphA2 kinase in tumorigenesis in vivo. We report here that deletion of EphA2 in mouse led to markedly enhanced susceptibility to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. EphA2-null mice developed skin tumors with an increased frequency and shortened latency. Moreover, tumors in homozygous knockout mice grew faster and were twice as likely to show invasive malignant progression. Haploinsufficiency of EphA2 caused an intermediate phenotype in tumor development but had little effects on invasive progression. EphA2 and ephrin-A1 exhibited compartmentalized expression pattern in mouse skin that localized EphA2/ephrin-A1 interactions to the basal layer of epidermis, which was disrupted in tumors. Loss of EphA2 increased tumor cell proliferation, whereas apoptosis was not affected. In vitro, treatment of primary keratinocytes from wild-type mice with ephrin-A1 suppressed cell proliferation and inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) activities. Both effects were abolished in EphA2-null keratinocytes, suggesting that loss of ERK inhibition by EphA2 may be one of the contributing mechanisms for increased tumor susceptibility. Interestingly, despite its tumor suppressive function, EphA2 was overexpressed in skin tumors compared with surrounding normal skin in wild-type mice, similar to the observations in human cancers. EphA2 overexpression may represent a compensatory feedback mechanism during tumorigenesis. Together, these results show that EphA2 is a novel tumor suppressor gene in mammalian skin.

T cells reactive to a single immunodominant self-restricted allopeptide induce skin graft rejection in mice.

Alloreactive T lymphocytes can respond to foreign MHC complexed with foreign peptides through the direct pathway of allorecognition and can additionally recognize allopeptides expressed in the context of recipient (self) MHC through the indirect pathway. To better elucidate how indirect pathway-responsive CD4(+) T cells mediate allograft rejection, we isolated and characterized a TH1 T cell line from BALB/c recipients of B10.A skin that responds to a defined immunodominant, self-restricted allopeptide, I-Abetak58-71. When transferred into BALB/c severe combined immunodeficiency recipients of B10.A skin allografts, this cell line specifically induced a form of skin graft rejection characterized by the presence of TH1 cytokines, macrophage infiltration, and extensive fibrosis. Recall immune responses and immunofluorescence of the rejecting skin revealed only the presence of the peptide-specific T cells within the recipient animals, with no evidence of a direct pathway alloresponse. These studies demonstrate that T cells reactive to a single self-restricted allopeptide can mediate a form of allogeneic skin graft rejection that exhibits characteristics of a chronic, fibrosing process.

Cutaneous lymphoid hyperplasias.

Benign hyperplastic lymphoid infiltrates of the skin (pseudolymphoma, older term) simulate lymphoma clinically and histologically. They can be divided into B-cell predominant (typical cutaneous lymphoid hyperplasia (CLH), angiolymphoid hyperplasia, Kimuras disease, and Castlemans disease) and T-cell predominant (T-cell CLH, lymphomatoid contact dermatitis, and lymphomatoid drug eruption). Both types may represent exaggerated reactions to diverse external antigens (insect bite, tattoo, zoster, trauma, among others). A composite assessment of clinical presentation and behavior, routine histology, immunophenotyping, and molecular studies is essential for the diagnosis of benign cutaneous lymphoid infiltrates. Treatment includes antibiotics, intralesional and systemic corticosteroids, excision, radiotherapy, and immunosuppressants. Treatment depends on the assessment and biologic behavior, which is usually benign. Molecular biologic analysis has shown that a significant proportion of cases harbor occult B- or T-cell clones (clonal CLH). Progression to overt cutaneous lymphoma has been observed in a minority of cases. Patients with clonal populations of B or T cells and persistent lesions should be closely observed for emergence of a lymphoma.

Keratinocyte but Not Endothelial Cell-Specific Overexpression of Tie2 Leads to the Development of Psoriasis

Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4(+) T cells, infiltrating epidermal CD8(+) T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-gamma, tumor necrosis factor-alpha, and interleukins 1alpha, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, beta-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis.

A Definitive Role of Ornithine Decarboxylase in Photocarcinogenesis

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UVB component, to human skin is the major cause for more than a million new cases of cutaneous malignancies diagnosed annually in the United States. Photocarcinogenesis, like other cancers, is a multistep process that includes initiation and promotion. A proper understanding of the molecular events occurring during the tumor promotion phase of photocarcinogenesis could lead to the development of novel approaches for the management of skin cancer. Using a transgenic mouse model (K5/ODC mice), which overexpresses the enzyme ornithine decarboxylase (ODC) in hair follicle keratinocytes, we studied the role of this gene in photocarcinogenesis. A single UVB-exposure of 180 mJ/cm(2) to the transgenic mice resulted in a minimal increase in bifold skin thickness and ODC activity. However, in SKH-1 hairless mice, the most common and highly sensitive model for photocarcinogenesis, and in littermate nontransgenic mice, increases in skin thickness and ODC activity were substantial. In long-term experiments, mice were exposed to 180 mJ/cm(2) of UVB radiation three times a week for 2 weeks (tumor-initiating dose). At 30 weeks after this treatment, in two independent experiments, 40% of the K5/ODC transgenic mice exposed to UVB were found to develop epidermal tumors. The tumors were histologically verified as benign papillomas and squamous cell carcinomas. Interestingly, 100% of the transgenic mice also developed >20 pigmented cysts/mouse, which contained keratinocyte material with increased keratinocytic melanization. Under similar UVB-exposure protocol, the nontransgenic littermates or SKH-1 hairless mice did not develop tumors or pigmented cysts for up to 50 weeks. Oral consumption of alpha-difluoromethylornithine, an irreversible specific inhibitor of ODC, in the drinking water (1% w/v) to the transgenic mice resulted in complete prevention of UVB-mediated tumorigenesis and a substantial decrease in the formation of pigmented cysts (<10 per mouse). These data establish a definitive role of ODC in the promotion phase of photocarcinogenesis.

Folliculotropic mycosis fungoides with large-cell transformation presenting as dissecting cellulitis of the scalp.

Follicular mycosis fungoides (MF) is a rare variant of cutaneous T‐cell lymphoma (CTCL) in which malignant lymphocytes preferentially infiltrate hair follicles. This report describes a patient with follicular mycosis fungoides presenting in a manner similar to dissecting cellulitis of the scalp with nonhealing, draining nodular lesions. Follicular mucinosis associated with folliculotropic mycosis fungoides resulted in follicular disruption and deep dissecting cellulitis. Large‐cell transformation of CTCL was present in the initial diagnostic scalp and axillary lymph node specimens. The patient died from progressive CTCL 9 months following initial diagnosis despite electron beam radiation, topical mechlorethamine, interferon‐α, and systemic chemotherapy. This case indicates that large‐cell transformation of folliculotropic mycosis fungoides is an aggressive form of CTCL, and that folliculotropic mycosis fungoides can give rise to lesions which resemble dissecting cellulitis of the scalp. Upregulation of intercellular adhesion molecule‐1 (ICAM‐1) on follicular epithelium adjacent to lymphocyte function‐associated antigen‐1 (LFA‐1)‐positive folliculotropic lymphoma cells in this report provides insight into lymphocyte homing mechanisms in folliculotropic MF.