title: Expression of vascular endothelial growth factor, apoptosis inhibitors (survivin and p16) and CCL27 in alopecia areata before and after diphencyprone treatment: an immunohistochemical study.
abstract: BACKGROUND
  Alopecia areata (AA) is a relatively common inflammatory form of nonscarring hair loss of unknown pathogenesis, but possibly of autoimmune origin. Topical immunotherapy, using a potent contact allergen such as diphencyprone (DPC), is currently considered the most effective mode of treatment. However, the way in which DPC operates on hair follicles in AA still remains to be elucidated. Vascular endothelial growth factor (VEGF), essential for angiogenesis and vascular permeability, may be responsible for maintaining proper vasculature around hair follicles, and several studies provide evidence that apoptosis is a central element in the regulation of hair follicle and vascular regression. The cutaneous lymphocyte-associated antigen (CLA) and the skin-associated chemokine CCL27 highlight an important role for epithelial cells in controlling homeostatic lymphocyte trafficking.
  
  
  OBJECTIVE
  To determine the expression pattern of VEGF, factor (F)VIII, survivin, p16, CD4, CD8, CLA and CCL27 in alopecic skin before and after treatment with DPC. Methods Immunohistochemical staining methods were applied to skin biopsy specimens obtained from alopecic areas of 14 patients before and after DPC treatment and from five healthy subjects. Sections were incubated with monoclonal antibodies against VEGF, FVIII, survivin, p16, CCL27, CLA, CD4 and CD8, and their immunohistochemical expression was evaluated by light microscopy.
  
  
  RESULTS
  The intensity of VEGF staining in alopecic human hair follicles was significantly lower than in healthy scalp tissue. FVIII immunostaining showed a significantly reduced development of the microvasculature in AA in comparison with healthy scalp tissue. After DPC therapy, cells of alopecic hair follicles showed a significant increase of VEGF immunopositivity, and the number of capillary vessels expressing FVIII was markedly increased in comparison with untreated scalp tissue. The increase in microvessels was associated with strong survivin expression in endothelial cells after treatment. All alopecic specimens showed expression of p16 in the hair follicle outer root sheath (ORS), with a significant increase after therapy. After treatment we observed a significantly decreased number of CD4+ cells and an increase of CD8+ cells (CD4/CD8 ratio 0.85) in alopecic skin compared with untreated scalp tissue (CD4/CD8 ratio 3.45). Most of the T lymphocytes found in inflammatory skin lesions expressed CLA antigen and after therapy we observed a significantly higher CLA positivity in hair follicles (50% or more) in comparison with untreated alopecic scalp tissue. Alopecic patients showed a CCL27 immunopositivity significantly lower than in normal scalp tissue. After DPC therapy the labelling intensity for CCL27 showed a significant increase both in the ORS and in the inner root sheath; similarly, in the basal interfollicular keratinocytes we observed a moderate increase in CCL27 expression.
  
  
  CONCLUSIONS
  Topical immunotherapy exerts an important role in angiogenesis, upregulating VEGF in human hair follicle keratinocytes and upregulating survivin to preserve endothelial cell viability. Moreover, it considerably alters the peribulbar CD4/CD8 ratio, restoring a condition close to normal scalp skin. Our study could contribute to explaining some aspects of AA pathogenesis that are still unknown and aid understanding of how DPC could act in this complex disease.
authors.affiliation: Clinica Dermatologica, Università Politecnica delle Marche, Ospedale Regionale, Via Conca 20, 60020 Torrette, Ancona, Italy. o.simonetti@umbertoprimo.marche.it
authors.forename: O
authors.initials: O
authors.lastname: Simonetti
authors.forename: G
authors.initials: G
authors.lastname: Lucarini
authors.forename: M L
authors.initials: ML
authors.lastname: Bernardini
authors.forename: C
authors.initials: C
authors.lastname: Simoncini
authors.forename: G
authors.initials: G
authors.lastname: Biagini
authors.forename: A
authors.initials: A
authors.lastname: Offidani
mesh_terms: D000328:Adult; D000506:Alopecia Areata / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; D017209:Apoptosis; D015496:CD4-Positive T-Lymphocytes / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000473:pathology; D054425:Chemokine CCL27; D019742:Chemokines, CC / Q000378:metabolism; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D003521:Cyclopropanes / Q000627:therapeutic use*; D005169:Factor VIII / Q000378:metabolism; D005260:Female; D018859:Hair Follicle / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D051034:Inhibitor of Apoptosis Proteins; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008875:Middle Aged; D009363:Neoplasm Proteins; D000077022:Survivin; D042442:Vascular Endothelial Growth Factors / Q000378:metabolism
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; D000951:Antigens, Neoplasm; C107489:BIRC5 protein, human; C492276:CCL27 protein, human; C063419:CTAGE1 protein, human; D054425:Chemokine CCL27; D019742:Chemokines, CC; D019941:Cyclin-Dependent Kinase Inhibitor p16; D003521:Cyclopropanes; D051034:Inhibitor of Apoptosis Proteins; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D000077022:Survivin; D042442:Vascular Endothelial Growth Factors; D005169:Factor VIII; C029402:diphenylcyclopropenone