Pemphigus Vulgaris IgG Research Articles

Distinguishing the antigen specificities of pemphigus vulgaris and pemphigus foliaceus using the biotin-avidin immunofluorescence method

To compare the specificities of autoantibodies in sera from patients with pemphigus vulgaris (PV) and those with pemphigus foliaceus (PF), blocking-immunofluorescence studies were carried out using the biotin-avidin immunofluorescence technique. First cryostat sections of bovine muzzle epidermis were incubated with one of either the unlabeled PV or PF serum samples for 2 h at room temperature, then rinsed and overlayered for 30 min with serially diluted corresponding or other biotin-labeled PV (or PF) IgG fractions containing their autoantibodies. The sections were then incubated for 30 min in fluorescein-labeled avidin. The blocking abilities of PV (or PF) sera for the reaction of labeled PV (or PF) IgG on the membranous part of keratinocytes were compared. The following results were obtained: the titers of biotin-labeled PF IgG decreased considerably more in sections preincubated with PF sera than sections preincubated with PV or normal sera. The titers of biotin-labeled PV IgG decreased considerably more in sections preincubated with PV sera than sections preincubated with PF or normal sera. These results suggest that there may be a distinction in antigenic specificities between PV and PF sera.

Defining the role of complement in experimental pemphigus vulgaris in mice.

Parenteral passive transfer of human pemphigus vulgaris IgG (PV IgG) into neonatal mice reproduces the cutaneous disease. We used this model to study the role of complement in the development of acantholysis in three steps. Peptic F(ab')2 fragments were prepared from PV IgG and were injected into seven newborn mice, and all animals developed acantholytic skin blisters without local complement activation, as shown by direct immunofluorescence. These fragments were reduced and alkylated to produce Fab' fragments with equivalent in vitro binding activity. The monovalent fragments were given in an identical fashion to five littermates but failed to produce disease even though they were bound in the epidermis in vivo. Intact PV IgG was injected in 20 genetically C5-deficient neonates (B10-D2-OSN strain) and 20 control neonates (B10-D2-NSN, normal complementemic). Extensive blistering, with a positive Nikolsky sign, was produced in all 40 animals. PV IgG was given to 34 BALB/c neonates that were complement depleted by pretreatment with cobra venom factor (CoF) for 24 hr, and to 38 untreated neonates from the same litters. There was no difference in the disease produced after CoF treatment in animals that received high doses of PV IgG (5 to 15 mg/g/day). In animals receiving 2.5 mg PV IgG/g/day, blister formation was delayed and the final extent of the cutaneous lesions was less in CoF-treated mice (n = 12) than in normal complementemic controls (n = 12, p less than 0.02). These results show that complement activation is not an essential mechanism in PV IgG-induced acantholysis in vivo, but it does have an amplifying effect on the development of cutaneous lesions under certain conditions, and lesions can be induced in vivo by bivalent F(ab')2 fragments of PV IgG, but not by the monovalent Fab' fragments, suggesting that cross-linking of the cell surface antigen is an initiating signal in acantholysis.

Dexamethasone inhibits plasminogen activator activity in experimental pemphigus in vivo but does not block acantholysis.

In vitro studies have suggested that autoantibody-stimulated increases in epidermal plasminogen activator (PA) may be an important pathogenetic mechanism in pemphigus vulgaris (PV). We measured PA in murine epidermis after i.p. injection of normal human IgG (NH IgG) and PV IgG, with and without exposure to dexamethasone (DEX). BALB/c neonates received i.p. injections of saline control or DEX (20 mg/kg). Twenty-four hours later, they received a second injection of saline or DEX and a single dose of NH or PV IgG (20 mg/gm body weight). After 24 hr, epidermis was obtained and was sequentially extracted in 0.14 M NaCl, pH 6.8, and 0.5% Triton X-100 in 0.1 M Tris, pH 8.1. Epidermal PA was assayed in the Triton-Tris supernatant by a two-stage colorimetric reaction and was expressed as milliPloug units per milligram of protein (mPu/A280). PA in animals injected with NH IgG was 0.21 +/- 0.11 mPu/A280 (n = 8). Epidermal PA was increased in animals with cutaneous lesions of pemphigus to 0.42 +/- 0.29 (n = 15). Treatment with DEX decreased PA levels in both animals receiving NH IgG and PV IgG by 80%, to 0.04 +/- 0.05 (n = 15) and 0.09 +/- 0.07 (n = 7), respectively. Despite the decreased PA activity, all animals in the PV IgG and the PV IgG-plus-DEX group had identical and extensive cutaneous disease, and lesions developed at the same time points. This finding shows that PV autoantibodies can stimulate increases in epidermal PA, but reduction of PA by corticosteroids does not inhibit acantholysis in vivo. There is no clear correlation between PA and disease activity in the murine model of pemphigus.

Specificity and Inhibition of the Epidermal Cell Detachment Induced by Pemphigus IgG in Vitro

IgG isolated from sera of patients with pemphigus vulgaris (PV) has been shown to induce cell detachment when added to primary epidermal cell cultures (PECC). We studied the specificity of this phenomenon. IgG fractions were purified from the sera of five patients with PV and control IgG fractions from the sera of normal donors and patients with bullous pemphigoid (BP), systemic lupus erythematosus (SLE), and anti-AB blood group sera (anti-AB). IgG fractions were added to PECC either at initial plating (0 hours), at media change (48 hours), or sequentially at both times, and cell detachment was quantitated at 72 and 96 hours. Significant cell detachment occurred only when PV IgG was added to the growth media sequentially at 0 and 48 hours (p = 0.001), and this effect was dose-dependent for either dose. Substitution of an unrelated IgG (BP, SLE, or anti-AB) at either time points reduced cell detachment to near control values. Furthermore, cell detachment was inhibited by the addition of the proteinase inhibitors alpha 2 macroglobulin (70% inhibition of detachment), aprotinin (63% inhibition), soybean and lima bean trypsin inhibitor (62 and 64%, respectively), and pepstatin (49%), but not by the inhibitors chymostatin, leupeptin, or antipain. These data confirm that PV IgG induces increased cell detachment in PECC and shows that this effect is specific for PV IgG, is dose-dependent, and may be inhibited by certain proteinase inhibitors.

Anti-cell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells. A mechanism for the loss of epidermal cohesion and blister formation.

Binding of anti-cell surface pemphigus autoantibodies to cultured human epidermal cells stimulates synthesis and secretion of plasminogen activator (PA). Increases in PA activity were detected within 6 h of the addition of IgG and stimulation was dependent upon IgG concentration. Stimulation of PA activity was inhibited by cycloheximide, which indicates that synthesis of protein was necessary. Pharmacological doses of dexamethasone also prevented IgG-induced stimulation of PA. Electrophoretic profiles of PA secreted by cultured human epidermal cells in the presence or absence of pemphigus IgG were similar. The majority of the PA activity comigrated with the higher-molecular-weight species of human urokinase (approximately 55,000). Explants of normal human skin incubated with pemphigus vulgaris IgG displayed loss of epidermal cohesion similar to that observed in patient biopsies. The histologic changes were potentiated by the inclusion of human plasminogen. Loss of epidermal cohesion in normal skin explants incubated with pemphigus foliaceous IgG was dependent upon the addition of plasminogen and was inhibited by aprotinin or lima bean trypsin inhibitor, which indicated that plasmin is the active enzyme in producing acantholysis. These data support the hypothesis that stimulation of PA by the anti-cell surface autoantibodies of pemphigus results in a localized increase in plasmin, which through proteolysis produces the loss of epidermal cohesion characteristic of pemphigus.