Pemphigus vulgaris (PV) is a potentially fatal autoimmune mucocutaneous blistering disease. It was assumed that PV is caused by anti-desmoglein (Dsg) 3 autoimmunity because absorption of PV sera with a chimeric baculoprotein containing the Dsg 3 and IgG1 portions, rDsg3-Ig-His, eliminated disease-causing antibodies. In this study we demonstrate that rDsg3-Ig-His adsorbs out autoantibodies to different keratinocyte antigens, including a non-Dsg 3 130-kd polypeptide. Because the pool of disease-causing PV IgGs contains antibodies against the keratinocyte acetylcholine receptor (AChR), we sought to identify the targeted receptor(s). Preincubation of monkey esophagus with PV antibodies blocked specific staining of the keratinocyte cell membrane with rabbit monoepitopic antibody to alpha9 AChR, indicating that this first of its kind AChR with dual, muscarinic and nicotinic pharmacology is targeted by PV autoimmunity. Anti-alpha9 antibody stained keratinocytes in a fishnet-like intercellular pattern, and visualized a single band at approximately 50 kd in Western blots of keratinocyte membrane proteins. Using step-by-step reverse transcription polymerase chain reactions with primers based on known alpha9 sequence regions, we identified the complete reading frame of human alpha9. Its amino acid sequence showed 85% similarity with rat alpha9. Treatment of keratinocyte monolayers with anti-alpha9 antibody induced pemphigus-like acantholysis, which could be reversed either spontaneously or by using the cholinergic agonist carbachol. We conclude that alpha9 is coupled to physiological regulation of keratinocyte adhesion, and its interaction with PV IgG may lead to blister development.

IntroductionIn pemphigus vulgaris (PV), blisters develop on oral mucosa. Mucosal lesions are often followed by skin involvement. The deep intraepidermal cleft occurs between the basal cells and the overlaying spinous keratinocytes. In pemphigus foliaceus (PF), the oral mucosa is usually not involved, and cutaneous erosive lesions develop owing to a superficial epidermal split localized to the stratum granulosum. The pathophysiological mechanism causing autoimmune pemphigus is unknown and still being intensively investigated. To date, a catalogue of self-antigens, demonstrated by various authors and detection techniques to react uniquely with pemphigus IgGs, includes approximately 20 molecules with different relative molecular masses, namely: 12,18,33,47,50,52,55,59,66, 67, 68, 75, 78, 80, 85, 102, 105, 160, 180, 185/190, and 210 kDa (reviewed in ref. 1). The number of detectable target molecules varies from patient to patient and depends on the sensitivity of the detection technique, i.e., immunoblotting versus immunoprecipitation. Hypothetically, some of these bands may represent degradation products of pemphigus antigens having higher native molecular weights. The number of detectable pemphigus antigens can be substantially reduced by altering the sensitivity of the technique, as is performed when the keratinocyte protein suspension, the source of antigens, is first preabsorbed with normal human serum and then used in an immunoprecipitation assay with PV and PF sera (2, 3). Only a few protein bands remain, including the pairs of 85/130 and 85/160 kDa that were considered to represent the pathophysiologically important targets of PV and PF autoimmunity, respectively (4). Likewise, the number of clones detected in a λgt 11 keratinocyte cDNA library by PV IgG was reduced by substituting the whole PV IgG fraction with the affinity-purified IgG from a single band, the 130-kDa keratinocyte polypeptide (5).The 130-kDa PV antigen was reported to be a novel keratinocyte desmoglein (Dsg) 3 (5); the 160-kDa PF antigen, Dsg 1 (6); and the 85-kDa antigen, recognized by both PV and PF IgGs, was identified as the adhesion molecule plakoglobin (7). Autoantibodies to these adhesion molecules in pemphigus were interpreted as direct, cause-and-effect pathogenesis with autoantibody binding to an adhesion molecule inducing a dis- Pemphigus is an autoimmune disease of skin adhesion associated with autoantibodies against a number of keratinocyte antigens, such as the adhesion molecules desmoglein (Dsg) 1 and 3 and acetylcholine receptors. The notion that anti-Dsg antibodies alone are responsible for blisters in patients with pemphigus vulgaris (PV) stems from the ability of rDsg1 and rDsg3 to absorb antibodies that cause PV-like skin blisters in neonatal mice. Here, we demonstrate that PV IgGs eluted from rDsg1-Ig-His and rDsg3-Ig-His show similar antigenic profiles, including the 38-, 43-, 115-, and 190-kDa keratinocyte proteins and a non-Dsg 3 130-kDa polypeptide present in keratinocytes from Dsg 3 knockout mouse. We injected i...

Several ganglionic nicotinic acetylcholine receptor (nAChR) types are abundantly expressed in nonneuronal locations, but their functions remain unknown. We found that keratinocyte α7 nAChR controls homeostasis and terminal differentiation of epidermal keratinocytes required for formation of the skin barrier. The effects of functional inactivation of α7 nAChR on keratinocyte cell cycle progression, differentiation, and apoptosis were studied in cell monolayers treated with α-bungarotoxin or antisense oligonucleotides and in the skin of Acra7 homozygous mice lacking α7 nAChR channels. Elimination of the α7 signaling pathway blocked nicotine-induced influx of 45Ca2+ and also inhibited terminal differentiation of these cells at the transcriptional and/or translational level. On the other hand, inhibition of the α7 nAChR pathway favored cell cycle progression. In the epidermis of α7−/− mice, the abnormalities in keratinocyte gene expression were associated with phenotypic changes characteristic of delayed epidermal turnover. The lack of α7 was associated with up-regulated expression of the α3 containing nAChR channels that lack α5 subunit, and both homomeric α9- and heteromeric α9α10-made nAChRs. Thus, this study demonstrates that ACh signaling through α7 nAChR channels controls late stages of keratinocyte development in the epidermis by regulating expression of the cell cycle progression, apoptosis, and terminal differentiation genes and that these effects are mediated, at least in part, by alterations in transmembrane Ca2+ influx.

SUMMARY:Smoking is associated with aberrant cutaneous tissue remodeling, such as precocious skin aging and impaired wound healing. The mechanism is not fully understood. Dermal fibroblasts (DF) are the primary cellular component of the dermis and may provide a target for pathobiologic effects of tobacco products. The purpose of this study was to characterize a mechanism of nicotine (Nic) effects on the growth and tissue remodeling function of DF. We hypothesized that the effects of Nic on DF result from its binding to specific nicotinic acetylcholine receptors (nAChRs) expressed by these cells and that downstream signaling from the receptors alters normal cell functioning, leading to changes in skin homeostasis. Using RT-PCR and Western blotting, we found that a 24-hour exposure of human DF to 10 M Nic causes a 1.9-to 28-fold increase of the mRNA and protein levels of the cell cycle regulators p21, cyclin D1, Ki-67, and PCNA and a 1.7-to 2-fold increase of the apoptosis regulators Bcl-2 and caspase 3. Nic exposure also up-regulated expression of the dermal matrix proteins collagen type I␣1 and elastin as well as matrix metalloproteinase-1. Mecamylamine (Mec), the specific antagonist of nAChRs, abolished Nic-induced alterations, indicating that they resulted from a pharmacologic stimulation of nAChRs expressed by DF. To establish the relevance of these findings to a specific nicotinergic pathway, we studied human DF transfected with anti-␣3 antisense oligonucleotides and murine DF from ␣3 nAChR knockout mice. In both cases, lack of ␣3 was associated with alterations in fibroblast growth and function that were opposite to those observed in DF treated with Nic, suggesting that the nicotinic effects on DF were mostly mediated by ␣3 nAChR. In addition to ␣3, the nAChR subunits detected in human DF were ␣5, ␣7, ␤2, and ␤4. The exposure of DF to Nic altered the relative amounts of each of these subunits, leading to reciprocal changes in [ 3 H]epibatidine-binding kinetics. Thus, some of the pathobiologic effects of tobacco products on extracellular matrix turnover in the skin may stem from Nic-induced alterations in the physiologic control of the unfolding of the genetically determined program of growth and the tissue remodeling function of DF as well as alterations in the structure and function of fibroblast nAChRs. (Lab Invest 2003, 83:207-225).

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease of skin adhesion associated with IgG autoantibodies against keratinocytes (KC).