OBSERVATION Pemphigus Vulgaris Acantholysis Ameliorated by Cholinergic Agonists Vu Thuong Nguyen, PhD; Juan Arredondo, PhD; Alexander I. Chernyavsky, PhD; Mark R. Pittelkow, MD; Yasuo Kitajima, MD, PhD; Sergei A. Grando, MD, PhD, DSc Background: Pemphigus vulgaris (PV) is an autoim- phorylation level of E-cadherin and plakoglobin was in- mune, IgG autoantibody–mediated disease of skin and mu- creased by PV IgG, whereas this effect of PV IgG was at- cosa leading to progressive blistering and nonhealing ero- tenuated in the presence of 0.5mM carbachol. sions. Patients develop autoantibodies to adhesion Pyridostigmine bromide, an acetylcholinesterase inhibi- molecules mediating intercellular adhesion and to kerat- tor, produced effects similar to those of carbachol, which inocyte cholinergic receptors regulating cell adhesion. helps explain its clinical efficacy in a patient with active PV that was resistant to treatment with systemic gluco- Observations: To determine whether a cholinergic ago- corticosteroids. Treatment with pyridostigmine bro- nist can abolish PV IgG–induced acantholysis, litter mates mide (360 mg/d) in a patient with PV allowed to keep of neonatal athymic nude mice were injected with PV IgG his disease under control at a lower dose of prednisone together with carbachol (0.04 µg/g body weight). None than that used before starting pyridostigmine bromide of these mice developed skin lesions. Through in vitro treatment. experiments, we measured the expression of adhesion molecules in monolayers of normal human keratino- Conclusion: Elucidation of the cholinergic control of ke- cytes incubated overnight in the presence of 0.25mM car- ratinocyte adhesion merits further consideration be- bachol using semiquantitative Western blot and immu- cause of a potential for the development of novel anti- nofluorescence. Carbachol caused an elevation of the acantholytic therapies using cholinergic drugs. relative amount of E-cadherin in keratinocytes (PϽ.05) without changing that of plakoglobin (PϾ.05). The phos- Arch Dermatol. 2004;140:327-334 EMPHIGUS VULGARIS (PV) IS antholytic effects of glucocorticosteroids an autoimmune-mediated in vitro.7 We report that stimulation of the disease of skin and mucosa keratinocyte cholinergic receptors con- leading to progressive blis- trols PV IgG–induced acantholysis in neo- tering and nonhealing ero- natal mice and ameliorates the natural Psions. Therapy for patients with PV relies course of disease in a patient with PV. Cho- on the long-term use of systemic gluco- linergic agonists stimulated expression of corticosteroids in relatively large doses, E-cadherin and abolished phosphoryla- which, although lifesaving, may cause se- tion of E-cadherin and plakoglobin in ke- vere adverse effects, including death. Ac- ratinocytes caused by PV IgG. Thus, novel tive disease state is characterized by the antiacantholytic therapies may be devel- presence of serum IgG autoantibodies oped based on the antiacantholytic ef- binding to the keratinocyte cell mem- fects of cholinomimetic drugs. brane proteins. Although the antiacan- tholytic effect of glucocorticosteroids is at- METHODS tributed to immunosuppression, high doses PASSIVE TRANSFER OF PV From the Departments of of glucocorticosteroids can directly block Dermatology, University of PV IgG–induced acantholysis in vitro1,2 and Neonatal athymic nude mice were used to test California Davis, Sacramento rapidly (within 48 hours) stop blistering direct antiacantholytic effects of cholinergic ago- (Drs Nguyen, Arredondo, in patients with pemphigus without alter- nists. At the third day of life, athymic nude mice Chernyavsky, and Grando), ing the titer of autoantibodies or block- weigh approximately 1.5 g and can develop gross Mayo Clinic, Rochester, Minn skin blisters on passive transfer of PV antibod- ing antibody binding to keratinocytes (Dr Pittelkow), and Gifu 3-5 ies. This study had been approved by the Uni- University, Gifu City, Japan (“pulse therapy”). Patients develop au- versity of California Davis Review Committee (Dr Kitajima). The authors toantibodies to keratinocyte cholinergic on the Use of Animals in Research, Sacra- have no relevant financial receptors regulating cell adhesion.6 Acti- mento, Calif. We injected 52 mice intraperito- interest in this article. vation of these receptors mimics antiac- neally through a 30-gauge needle with PV (ex- (REPRINTED) ARCH DERMATOL / VOL 140, MAR 2004 WWW.ARCHDERMATOL.COM 327 ©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 periment) or normal human IgG (negative control). One subgroup PHOSPHORYLATION ASSAY of mice received PV IgG alone and the other subgroup received Quantitative phosphorylation assay was designed based on the PV IgG together with a test drug. The patients’ and control hu- 13 man IgG serum fractions were obtained using 40% ammonium established protocols, as detailed elsewhere. Briefly, cul- tured DJM-1 cells were grown to approximately 75% conflu- sulfate followed by dialysis against phosphate-buffered saline (PBS; 2 Gibco BRL, Gaithersburg, Md). The fractions were then lyophi- ence in 75-cm flasks (Corning Costar, Cambridge, Mass) in 8 serum-free keratinocyte growth medium (Clonetics Corp, San lized and reconstituted in PBS as detailed elsewhere. The nega- 2+ tive control IgG was isolated from normal human serum pur- Diego, Calif) containing 0.09mM Ca at 37°C in a humid 5% chased from Sigma Chemical Co, St Louis, Mo. carbon dioxide incubator. The cells were then treated for 14 hours with phosphate-free minimal essential medium contain- 2+ ASSESSMENT OF ACANTHOLYSIS ing 1.8mM Ca and 5% dialyzed fetal calf serum (Gibco BRL) and then treated with the same medium containing 200 µCi/mL Gross skin lesions were observed approximately 24 to 40 hours of 32P orthophosphate (Amersham Pharmacia Biotech, Inc). The after the first injection of PV IgG. Histological evaluation of le- cells were labeled for 8 hours and then exposed for 1 hour to 1 sional and perilesional skin in mice with clinical symptoms of mg/mL of pooled normal human IgG (Sigma Chemical Co) or induced PV revealed extensive intraepidermal split and nonspe- PV IgG pooled from 5 PV sera samples in the absence or pres- cific changes. To allow accurate analysis of the extent of acan- ence of 0.5mM carbachol or pyridostigmine bromide. After in- tholysis, we euthanized the mice at the preclinical stage of the cubation, the cells were lysed and the lysates were cleared by disease (ie, approximately 20 hours after the injection). The ani- centrifugation at 14000g for 10 minutes. The adhesion mol- mals were cross-sectioned at the umbilicus level and freshly fro- ecules under consideration were immunoprecipitated, re- zen. The need to compare skin specimens from the same body solved by 7.5% sodium dodecyl sulfate–polyacrylamide gel region stemmed from the fact that acantholysis in neonatal mice electrophoresis, electroblotted, and analyzed with the Phos- may vary between different anatomical regions.9 Three 6-µm thick phorImager feature of the Storm system (Molecular Dynam- cross sections of each mouse body were placed on the glass slide ics, Mountain View, Calif). To determine protein concentra- and stained with hematoxylin-eosin. The images (original mag- tion of each precipitated adhesion molecule, the samples were nification ϫ100) of 5 randomly selected longitudinal skin areas analyzed by Western blotting, as described above. The results in each specimen were photographed using a computer-linked were expressed as ratios of the radioactivity value of each ad- microscope and printed. We determined the extent of acan- hesion molecule to its protein quantity in each sample, com- tholysis in the photographs by measuring the length of the in- pared with the ratios obtained in control cultures (taken as 1.00). traepidermal split (at least 4 basal cells long) and expressed the results as percentage of the total length of epidermis in the field, STATISTICS taken as 100%. The results of quantitative experiments were expressed as IMMUNOHISTOCHEMISTRY mean±SD. Significance was determined using the t test. The results were deemed significant if the P value was less than .05. Direct and indirect immunofluorescence assays were per- formed as detailed elsewhere.8 For the direct immunofluores- cence assay, a tissue specimen was incubated for 1 hour at room REPORT OF A CASE temperature with a fluorescently labeled goat antihuman IgG an- A recently reported case of PV that had improved by ciga- tibody (Pierce, Rockford, Ill; dilution 1:200 in PBS). For the in- 14 direct immunofluorescence experiments, a tissue substrate was rette smoking and a study showing successful use of 15 first treated with a primary antibody to E-cadherin or plakoglo- nicotinamide as a steroid-sparing agent in pemphigus, bin (both from BD Transduction Laboratories, Lexington, Ky; suggested that pharmacological regulation of keratino- dilution, 1:200) and then exposed to a secondary fluorescently cyte acetylcholine (ACh) axis may be a novel antiacan- labeled antibody (Pierce). Semiquantitative indirect immuno- tholytic therapy for pemphigus because (1) cigarette fluorescence assay was performed as detailed previously,10,11 us- smoke contains the cholinomimetic agent nicotine and ing a computer-assisted image analysis with a software package (2) nicotinamide exhibits cholinomimetic effects16 ow- purchased from Scanalytics (Fairfax, Va). For each tissue speci- ing to the stimulation of ACh release17 and inhibition