Search for Susceptibility Genes in Alopecia Areata

10 : 3 DECEMBER 2005

PLENARY WORKSHOP ON ALOPECIA AREATA

expression and production of the pro-inflammatory cytokine IL-12 in addition to the expected increased expression of regulatory cytokine IL-10. As such, some of the CD4 þ / CD25 þ cells in AA patients’ PBMC probably do not retain true regulatory cell properties. Further research is required to determine whether there is an effective functional deficiency of CD4 þ /CD25 þ regulatory cells. Using the mouse model, the CD4 þ /CD25 cell subset has been defined as the primary candidate pro-inflammatory pathogenic cell subset with the capacity to induce AA (McElwee et al, 2005). Examination of this cell subset in humans with progressive AA reveals an activated state with significantly increased expression of co-stimulatory ligands, such as CD28, and production of pro-inflammatory cytokines IL-12, IFN-g, and TNFa (Zo¨ller et al, 2004a, b). In addition, CD4 þ /CD25 cells from AA patients are relatively resistant to apoptosis, as defined by a reduction in annexin V binding, compared with the continued susceptibility to apoptosis observed in CD4 þ /CD25 þ cells. Similar features have been demonstrated in other autoimmune disease mechanisms. These properties of the CD4 þ / CD25 cell population may raise the threshold of activity that regulatory cells must attain to successfully promote anergy or activation-induced cell death in CD4 þ /CD25 cells and prevent autoimmunity. Boosting the number and function of CD4 þ /CD25 þ regulatory cells and reducing apoptosis resistance in the CD4 þ /CD25 candidate pathogenic cell population may be beneficial in the treatment of AA. Although still very much a theoretical option, research to develop such therapeutic approaches for other autoimmune diseases is being conducted and might be adapted for treating AA (Tang et al, 2004). References Gershon RK, Kondo K: Cell interactions in the induction of tolerance: The role of thymic lymphocytes. Immunology 18:723–735, 1970 Green DR, Webb DR: Saying the ‘‘S’’ word in public. Immunol Today 14:523–525, 1993 Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH: Identification and functional characterization of human CD4( þ )CD25( þ ) T cells with regulatory properties isolated from peripheral blood. J Exp Med 193:1285–1294, 2001 Matzinger P: The danger model: A renewed sense of self. Science 296:301–305, 2002 McElwee K, Freyschmidt-Paul P, Ziegler A, Happle R, Hoffmann R: Genetic susceptibility and severity of alopecia areata in human and animal models. Eur J Dermatol 11:11–16, 2001 McElwee KJ, Freyschmidt-Paul P, Hoffmann R, Kissling S, Hummel S, Vitacolonna M, Zo¨ller M: Transfer of CD8( þ ) cells induces localized hair loss whereas CD4( þ )/CD25() cells promote systemic alopecia areata and CD4( þ )/CD25( þ ) cells blockade disease onset in the C3H/HeJ mouse model. J Invest Dermatol 124:947–957, 2005 McElwee KJ, Hoffmann R, Freyschmidt-Paul P, Wenzel E, Kissling S, Sundberg JP, Zoller M: Resistance to alopecia areata in C3H/HeJ mice is associated with increased expression of regulatory cytokines and a failure to recruit CD4 þ and CD8 þ cells. J Invest Dermatol 119:1426–1433, 2002 Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M: Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor a-chains. J Immunol 155:1151–1164, 1995 Tang Q, Henriksen KJ, Bi M, et al: In vitro-expanded antigen specic regulatory T cells suppress autoimmune diabetes. J Exp Med 199:1455–1465, 2004 Zo¨ller M, McElwee KJ, Engel P, Hoffmann R: Transient CD44 variant isoform expression and reduction in CD4( þ )/CD25( þ ) regulatory T cells in C3H/ HeJ mice with alopecia areata. J Invest Dermatol 118:983–992, 2002 Zo¨ller M, McElwee KJ, Vitacolonna M, Hoffmann R: Apoptosis resistance in peripheral blood lymphocytes of alopecia areata patients. J Autoimmun 23:241–256, 2004a

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Zo¨ller M, McElwee KJ, Vitacolonna M, Hoffmann R: The progressive state, in contrast to the stable or regressive state of alopecia areata, is reflected in peripheral blood mononuclear cells. Exp Dermatol 13:435–444, 2004b

Search for Susceptibility Genes in Alopecia Areata Amalia Martinez-Mir, Abraham Zlotogorski,w and Angela M. Christianoz 

Department of Dermatology, Columbia University, New York, New York, USA; wDepartment of Dermatology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; zDepartment of Genetics and Development, Columbia University, New York, New York, USA

Alopecia areata (AA) is a multifactorial disorder, in which genetic and environmental factors combine to result in the phenotype (Green and Sinclair, 2000). The prognosis of AA is unpredictable and there is no definitive treatment. Several lines of evidence support the polygenic inheritance of AA, including the high prevalence of the trait, the Gaussian curve of distribution of the phenotype, and heritability among first-degree relatives (Aita and Christiano, 2001). Genetic studies have been limited to association analyses, which suggest that a permissive HLA status may potentiate the development of AA (Welsh et al, 1994). With the hypothesis that there is a genetic basis underlying the susceptibility to develop AA, we initiated a search for susceptibility genes by performing a genomewide scan in multiplex AA pedigrees. The genetic dissection of complex traits has traditionally been focused on large collections of small families (affected sib pairs, for example). But it has recently been shown that a small sample of larger pedigrees can potentially derive more robust results, because of a reduced level of genetic heterogeneity (‘‘noise’’), enrichment of genetic factors within families, and the availability of the pedigree structure (Terwilliger and Goring, 2000). The ascertainment and diagnosis of the families studied was undertaken by a dermatologist (Dr Abraham Zlotogorski), and using the diagnostic questionnaire developed by the NIH AA Registry. We collected DNA from a total of 22 multiplex AA pedigrees, comprising 69 unaffected and 78 affected family members. We performed a genome-wide scan using a panel of 324 microsatellite markers, with an average marker spacing of 10 cM in all DNA samples. Because of the complex nature of traits, it is expected that a number of genetic components will be contributing to the final presentation of the phenotype. For this reason, we subjected the dataset derived from the genome-wide scan to a combination of different statistical tests (Terwilliger and Ott, 1994): (i) the heterogeneity LOD score, maximized over four settings of the penetrance parameters (MAXHLOD); (ii) the mean test for affected sib-pairs, as implemented in the ANALYZE program (ASP); a test of allele sharing that uses all sibs (ALLSIBS); and a likelihood version of the transmission disequilibrium test (TDT-LIKE). This strategy represents a combination of parametric or model-based (MAXHLOD) and non-parametric or model-free (ASP, ALLSIBS, and TDTLIKE) tests.

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We identified several chromosomal regions yielding suggestive LOD scores, including the HLA region on chromosome 6. These intervals could harbor potential susceptibility loci for alopecia areata. In order to exclude those regions that represent spurious positive scores and to confirm and refine the true susceptibility loci, we are currently undertaking a fine-mapping study in a larger group of AA families. We anticipate that these studies will lead to the identification of AA susceptibility genes, and provide a foundation for understanding the interactions of these gene(s) with each other and with other variables such as the immune system and environmental factors. References Aita VM, Christiano AM: The genetics of alopecia areata. Dermatol Ther 14:329– 339, 2001 Green J, Sinclair RD: Genetics of alopecia areata. Australas J Dermatol 41:213– 218, 2000 Terwilliger JD, Goring HH: Gene mapping in the 20th and 21st centuries: Statistical methods, data analysis, and experimental design. Hum Biol 72:63–132, 2000 Terwilliger JD, Ott J: Handbook of Human Genetic Linkage. Baltimore: Johns Hopkins, 1994 Welsh EA, Clark HH, Epstein SZ, Reveille JD, Duvic M: Human leukocyte antigenDQB103 alleles are associated with alopecia areata. J Invest Dermatol 103:758–763, 1994

Neurotrophins in Autoimmune Diseases: Possible Implications for Alopecia Areata T. N. Palkina,w A. A. Sharov, T. Y. Sharova, and Vladimir A. Botchkarev 

Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA; wN.N. Blokhin’s Memorial Cancer Center, Russian Academy of Medical Sciences, Moscow, Russia

Neurotrophins are a family of structurally and functionally related polypeptides that include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (for a review see Roux and Barker, 2002). They bind a 75kDa transmembrane neurotrophin receptor (p75NTR), and also each neurotrophin binds a distinct receptor of the Trk family. When p75NTR and Trk are coordinately bound by neurotrophins, survival signal is initiated through Trk. But in absence of the Trk, p75NTR is activated alone to induce a variety of cellular responses including apoptosis. Increased evidence of data suggests that neurotrophins play an important role during the development of autoimmune disorders: NGF levels are significantly increased in the synovium of patients affected by rheumatoid arthritis, in the cerebrospinal fluid of patients with multiple sclerosis, in plasma of patients affected by lupus erythematosus, and in the skin of patients with systemic scleroderma (for a review see Aloe and Tuveri, 1997). The mechanisms of neurotrophin involvement in pathogenesis of autoimmune disorders, however, are largely unknown. Because neurotrophins are expressed in skin affected by alopecia areata (AA) and promote hair follicle regression in normal non-affected skin

(Botchkarev et al, 2000; Botchkarev, 2003), we asked if neurotrophins may play a role in AA pathogenesis. CD8 lymphocytes were isolated from involved skin and peripheral blood of AA-affected C3H/HeJ mice as well as from non-affected control mice. In contrast with peripheral blood CD8 cells that displayed low p75 levels, cells from AA involved skin expressed significantly higher p75 level (po0.001, FACScan analysis). Multi-color immunofluorescence microscopy showed that p75-positive CD8 cells were assembled specifically around the hair follicles as well as inside the follicular epithelium. To identify the phenotype of CD8 cells expressing p75NTR in skin affected by AA, the expression of interferon-g (IFN-g) and granzyme B as markers of effector CD8 cells was studied by FACS analysis and immunofluorescence. p75NTR-positive CD8 cells also showed the expression of IFN-g and granzyme B suggesting their effector phenotype. By RT-PCR, however, these cells were negative for Trk (TrkA, TrkB, TrkC) receptor expression, suggesting that neurotrophin administration could induce apoptosis of these inflammatory cells. Consistent with this hypothesis, NGF- and BDNFinduced apoptosis in CD8 cells isolated from skin of mice affected by AA and cultured in vitro, as compared with diluent alone. Furthermore, in vivo administration of agarose beads soaked with NGF or BDNF resulted in significant reduction of CD8 cells (po0.01) in AA-affected skin. These data suggest that neurotrophin-stimulated apoptosis in CD8 cells may play a role as a part of the protective response mechanisms limiting the development of AA and that p75NTR agonists could be used as a novel therapeutic intervention to arrest the development of AA. References Aloe L, Tuveri MA: Nerve growth factor and autoimmune rheumatic diseases. Clin Exp Rheumatol 15:433–438, 1997 Botchkarev VA: Neurotrophins and their role in pathogenesis of alopecia areata. J Investig Dermatol Symp Proc 8:195–198, 2003 Botchkarev VA, Botchkareva NV, Albers KM, Chen L-H, Welker P, Paus R: A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression. FASEB J 14:1931–1942, 2000 Roux PP, Barker PA: Neurotrophin signaling through the p75 neurotrophin receptor. Progr Neurobiol 67:203–233, 2002

The Functional Relevance of the Type 1 Cytokines IFN-c and IL-2 in Alopecia Areata of C3H/HeJ Mice P. Freyschmidt-Paul, M. Zo¨ller,w K. J. Mcelwee, J. Sundberg,z R. Happle, and R. Hoffmann

Department of Dermatology, Philipp University Marburg, Marburg, Germany;

wGerman Cancer Research Centre, Heidelberg, Germany; zThe Jackson Laboratory, Bar Harbor, Maine, USA

Alopecia areata (AA) was regarded as a type 1 (Th1, Tc1)mediated autoimmune disease of the hair follicle, because type 1 cytokines interferon-g (IFN-g) and interleukin (IL)-2 are expressed in lesional AA skin. Recently we have shown that the type 2 (Th2, Tc2) cytokine IL-10 also is expressed in