T and B cells target identical regions of the non-collagenous domain 1 of type VII collagen in epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is a severe immunobullous disease and is caused by IgG against type VII collagen (Col VII) of anchoring fibrils. In this study, utilizing ELISA and immunoblot, 13/15 EBA sera but 0/20 bullous pemphigoid sera and 0/30 healthy control sera showed IgG reactivity with distinct recombinant subregions of the non-collagenous domain 1 (NC1) of Col VII. In two EBA patients, IgG titers against Col VII-NC1 were grossly correlated to clinical disease activity. Moreover, Col VII-reactive T cells were identified in a representative EBA patient which recognized identical subdomains of Col VII-NC1. These findings strongly suggest that (1) the Col VII-NC1 ELISA is a powerful tool for making the diagnosis of EBA, (2) Col VII-specific IgG grossly relates to disease activity and (3) IgG reactivity is associated with T cell recognition of identical subdomains of Col VII-NC1.

IgG against extracellular subdomains of desmoglein 3 relates to clinical phenotype of pemphigus vulgaris.

Pemphigus vulgaris (PV) is associated with autoantibodies against desmoglein (Dsg) 3 inducing epidermal loss of adhesion. The major pathogenic epitopes of Dsg3 are presumably dependent of their conformation. The aim of this study was to characterize the IgG reactivity of sera from a cohort of clinically well-characterized PV patients against presumably non-conformational subdomains of the Dsg3 ectodomain including recently described NH2-terminal immunodominant epitopes. By ELISA, IgG reactivity against distinct subdomains of Dsg3 was related to disease activity and the clinical phenotype of PV patients. Our findings suggest that (i) autoantibody from PV sera react with non-conformational epitopes of Dsg3; (ii) IgG reactivity against the NH2-terminus and the extracellular domains (EC) 2-4 of Dsg3 was associated with active PV, while IgG titres were not strictly correlated with disease activity and (iii) IgG reactivity against the EC1-4 was associated with mucosal dominant PV and was decreased in cutaneous dominant PV. The findings may help to define more refined serological disease markers of PV.

Diphenylcyclopropenone treatment of alopecia areata induces apoptosis of perifollicular lymphocytes.

Alopecia areata (AA) is a T cell-mediated autoimmune disease that can be treated with the contact sensitizer diphenylcyclopropenone (DCP). Peripheral blood leukocytes from AA patients are relatively resistant to apoptosis which might be due to decreased Fas Ligand (FasL) expression, or to an increase in CD44v7 expression. Moreover it has been suggested in a murine model of AA that contact allergen treatment might interfere with the emigration of Langerhans cells into the draining lymph node, thus hampering autoreactive T-cell activation. To assess whether and which of these mechanisms is of clinical relevance, immunohistochemistry was performed in scalp biopsies of successfully DCP-treated AA patients in the early phase of hair regrowth. In line with recent studies in a murine model of AA, there was no evidence that DCP treatment would interfere with extravasation and skin homing of activated leukocytes. Perifollicular infiltrates of DCP-treated as compared to untreated AA patients actually showed an increased number of perifollicular CD8(+) and CD1a(+) cells. Furthermore, the expression of CD44 and CD49d, which are of major importance in leukocyte extravasation, was even increased in DCP-treated as compared to AA patient infiltrates. The same accounted for the skin homing receptor CD44v10. When we evaluated the leukocyte subpopulations in DCP-treated as compared to untreated AA patients' skin biopsies, there was an undue increase in CD1a(+) cells, that could well be indicative of hampering of the emigration of antigen presenting cells (APC) by allergen treatment. Most importantly, the number of perifollicular TUNEL- and FasL-positive cells was strikingly increased, whereas the number of CD44v7(+) cells remained unaltered. Taken together, this study provides strong evidence that long term treatment with a contact sensitizer allows for the recovery of hair follicle by driving autoreactive T cells into activation-induced cell death. In addition the replacement with newly activated autoreactive T-cells might be impaired due to a DCP-mediated hindrance of APC emigration.

Cultured peribulbar dermal sheath cells can induce hair follicle development and contribute to the dermal sheath and dermal papilla.

Green fluorescent protein (GFP)-expressing wild-type, and nontransgenic mouse vibrissa follicle cells were cultured and implanted to mouse ears and footpads. Dermal papiller (DP)-derived cells and cells from the peribulbar dermal sheath "cup" (DSC) induced new hair follicles in both implanted ears and footpads, while nonbulbar dermal sheath cells did not. Confocal microscopy revealed that GFP-expressing DP and DSC cells induced hair growth associated with the formation of DP exclusively comprised of fluorescent cells. In mouse ears, but not footpads, fluorescent DP and DSC cells could also be identified in DP along with nonfluorescent cells. DSC cells were characterized in vivo and in vitro by low alkaline phosphatase activity in contrast to high alkaline phosphatase in DP cells. The results indicate transplanted DP and DSC cells were equally capable of DP formation and hair follicle induction. This suggests the DP and peribulbar DSC may be functionally similar. In addition to observing papillae exclusively composed of GFP-expressing cells, DP and DSC cells may also have combined with resident cells to form papillae composed of implanted GFP-expressing cells and host-derived non-GFP-expressing cells. Alkaline phosphatase expression may be utilized as a simple marker to identify hair follicle mesenchyme derived cells with hair follicle inductive abilities.

Resistance to alopecia areata in C3H/HeJ mice is associated with increased expression of regulatory cytokines and a failure to recruit CD4+ and CD8+ cells.

Grafting alopecia areata affected C3H/HeJ mouse skin to littermates induces alopecia areata, but high dietary soy oil reduces alopecia areata susceptibility. Alopecia areata affected and resistant mice were characterized to evaluate possible mechanisms involved in alopecia areata resistance. Of 44 mice that received alopecia areata affected skin grafts but failed to develop alopecia areata, only two of 22 receiving further alopecia areata affected skin grafts developed alopecia areata, whereas 39 of 44 controls developed alopecia areata. Alopecia areata affected skin contained increased numbers of CD4+ and CD8+ cells, increases in pro inflammatory T helper 1 and T helper 2 type cytokines, and upregulation of CD28, CD40L, and their ligands. In draining lymph nodes, a relatively high number of antigen-presenting cells was recovered, whereas several CD44v variants were downregulated. In contrast, alopecia areata resistant mouse skin did not display increased numbers of CD4+ and CD8+ cells, whereas counter-regulatory cytokines interleukins 4 and 10 were upregulated. High expression of CD28, CD80, CD86, CD40, CTLA4, CD44v variants, and FasL occurred in alopecia areata resistant mouse spleens. In vitro, lymph node cells of susceptible and resistant mice responded equally to a mitogenic stimulus, but only lymph node cells from alopecia areata affected mice displayed an increased response with T cell receptor stimulation via anti-CD3 cross-linking. These results suggest alopecia areata is a cell-mediated autoimmune disease, but alopecia areata affected skin graft hosts may resist alopecia areata onset through active counter-regulatory mechanisms. Because alopecia areata resistant mice showed unimpaired responsiveness and a transient inflammatory response towards the graft, it is suggested that alopecia areata develops as a consequence of an inappropriate immune response regulation.

Targeted immunotherapy with rituximab leads to a transient alteration of the IgG autoantibody profile in pemphigus vulgaris.

In pemphigus vulgaris (PV), IgG autoantibodies against the ectodomain of desmoglein 3 (Dsg3) have been shown to be directly responsible for the loss of keratinocyteadhesion. The aim of the present study was to study the effect of the B cell depleting anti-CD20 monoclonal antibody, rituximab, on the profile of pathogenic IgG against distinct regions of the Dsg3 ectodomain in 22 PV patients who were followed up clinically and serologically by Dsg3 ELISA over 12-24 months. Prior to rituximab, all the 22 PV patients showed IgG against Dsg3 (Dsc3EC1-5). Specifically, 14/22 showed IgG reactivity against the Dsg3EC1 subdomain, 5/22 patients against Dsg3EC2, 7/22 against Dsg3EC3, 11/22 against Dsg3EC4, and 2/22 against Dsg3EC5. Within 6 months after rituximab, all the patients showed significant clinical improvement and reduced IgG against Dsg3 (5/22) and the various subdomains, that is, Dsg3EC1 (7/22), Dsg3EC2 (3/22), Dsg3EC3 (2/22), sg3EC4 (2/22), and Dsg3EC5 (0/22). During the entire observation period, 6/22 PV patients experienced a clinical relapse which was associated with the reappearance of IgG against previously recognized Dsg3 subdomains, particularly against the Dsg3EC1. Thus, in PV, rituximab only temporarily depletes pathogenic B cell responses against distinct subdomains of Dsg3 which reappear upon clinical relapse.

IgG reactivity against non-conformational NH-terminal epitopes of the desmoglein 3 ectodomain relates to clinical activity and phenotype of pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune disease caused by immunoglobulin G (IgG) autoantibodies against the desmosomal adhesion molecules, desmoglein (Dsg)3 and Dsg1. The aim of the study was to relate IgG reactivity of 123 PV sera and 40 control sera against NH(2)-terminal non-conformational epitopes of Dsg3 and Dsg1 with disease activity and clinical phenotype by enzyme-linked immunosorbent assay. The results show that (i) the overall reactivity and the titres of IgG reactive with the Dsg3 ectodomain, Dsg3(1-566), significantly correlated with the disease activity of the PV patients; (ii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with active PV while there was no direct correlation between the IgG titres and the disease activity; (iii) IgG reactivity against the NH(2)-terminus of Dsg3, Dsg3(1-161), was associated with mucosal and mucocutaneous PV; (iv) IgG titres against a small stretch of the NH(2)-terminus of Dsg3, Dsg3(25-88), were associated with active PV; and (v) IgG in the PV sera detected non-conformational epitopes in addition to the previously identified conformation-dependent epitopes of the Dsg3 and Dsg1 ectodomains.

Fas-deficient C3.MRL-Tnfrsf6(lpr) mice and Fas ligand-deficient C3H/HeJ-Tnfsf6(gld) mice are relatively resistant to the induction of alopecia areata by grafting of alopecia areata-affected skin from C3H/HeJ mice.

Alopecia areata is suspected to be a T cell-mediated autoimmune disease of the hair follicle, where Fas is expressed on hair follicles and Fas ligand on perifollicular infiltrates. To elucidate whether the Fas/Fas ligand pathway is of pathogenetic significance in alopecia areata, we investigated whether alopecia areata can be induced in Fas-deficient and Fas ligand-deficient mice and whether alopecia areata develops in Fas-deficient and Fas ligand-deficient skin. Therefore, we induced alopecia areata by grafting alopecia areata-affected C3H/HeJ mouse skin on to C3H/HeJ mice (control), on to Fas ligand-deficient C3H/HeJ-Tnfsf6(gld) mice or Fas-deficient C3.MRL-Tnfrsf6(lpr) mice. All control mice developed alopecia areata, whereas no Fas-deficient mice showed hair loss and two of seven Fas ligand-deficient mice developed only transitory, limited alopecia areata. Moreover, skin from C3H/HeJ mice (control), C3H/HeJ-Tnfsf6(gld) mice, and C3.MRL-Tnfrsf6(lpr) mice was grafted on to C3H/HeJ mice with extensive alopecia areata. Skin grafts from control mice developed hair loss, whereas Fas-deficient and Fas ligand-deficient skin grafts were spared from alopecia areata. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and immunofluorescence studies revealed an increased number of apoptotic cells and expression of Fas on hair follicles as well as expression of Fas ligand on cells of the perifollicular infiltrate in C3H/HeJ mice with alopecia areata, whereas in Fas-deficient and Fas ligand-deficient mice apoptotic cells were virtually absent in hair follicles. The results suggest that the Fas/Fas ligand pathway plays an important pathogenetic role in alopecia areata.