Keratinocyte Antigens Research Articles

Stress-induced Oral Lichen Planus Immunopathogenesis and Potential Therapy: A Narrative Review

Background: Oral lichen planus is a chronic inflammatory condition that only involves in the oral cavity and is mediated by the cellular immune system. The etiology of OLP is unclear but autoreactive T lymphocytes are considered to play an important role in the development of OLP. Factors like stress and psychological anxiety have been associated with OLP but their roles in the development of OLP is yet explored. Purpose: To describe stress-induced OLP immunopathogenesis and therapeutic potential. Reviews: In the early stages, the mechanism of OLP involves the expression of keratinocyte antigens or exposure to an antigen in the form of self-peptide or heat shock protein (HSP). HSP90 is the most expressed heat shock protein in the basal layer of keratinocytes and plays a role in recruitment of cellular immune cells through the production of cytokines due to TLR2/4 and CD91 activation, inflammatory cell migration due to 4 integrin activation, and increased antigen presentation due to HSP90-peptide binding to MHC class I/II. Stress as a physiological response triggers the release of the hormone cortisol from the adrenal cortex and catecholamine hormones such as epinephrine/ adrenaline and norepinephrine/noradrenaline from the adrenal medulla. Catecholamines increase the migration of T lymphocyte cells through the interaction of integrins and integrin ligands on the endothelium through the expression of 2-integrin after binding to adrenergic receptors on the cell membrane. Conclusion: Oral lichen planus is a chronic inflammatory condition caused by various factors where stress increases the migration of T lymphocyte cells on the side that expresses self-peptides and antigens through the interaction of immune cells with catecholamines. Topical nonselective beta blockers can be supporting therapy in reducing pain and size of OLP lesions.

Serum and tissue levels of coenzyme Q10 in pemphigus vulgaris.

Pemphigus vulgaris (PV) is a debilitating autoimmune blistering disease of the skin and mucous membranes. It occurs due to the action of autoantibodies against various keratinocyte self-antigens. Anti-mitochondrial autoantibodies are detected in patients with PV. Coenzyme Q10 (CoQ10) is a member of the mitochondrial respiratory chain involved in cellular metabolism, including apoptosis. This study aimed to assess the serum and tissue levels of CoQ10 of patients with PV and healthy controls to determine its relevance to the disease pathogenesis. In this case-control study, 20 patients with PV and 20 healthy controls were included. Blood and skin samples were collected for the measurement of CoQ10 levels using ELISA. CoQ10 was significantly lower in both serum and tissue of patients with PV compared with controls (p=0.001). Similar results were found when gender subgroups were separately compared. A significant positive correlation was found between serum and tissue CoQ10 levels in controls (p=0.019, r=0.521), but not in patients with PV. CoQ10 appears to be one of the parameters affected by the autoimmune response in PV, which may contribute to the tissue damage caused by autoantibodies. The absence of a significant correlation between CoQ10 level and disease severity or duration may be caused by the complex pathophysiological process in PV with multiple autoantibodies against different keratinocyte antigens.

Autoeczematization: A Strange Id Reaction of the Skin.

Autoeczematization, or id reaction, is a disseminated eczematous reaction that occurs due to a release of antigen(s) after exposure to a primary stimulus, with the eczema spreading to a site distant from the original one. This seemingly perplexing generalization most commonly is associated with stasis dermatitis or tinea pedis, though it may be caused by a wide variety of other disorders. We postulate that T cells are reactive to keratinocyte antigens that are produced during keratinocyte damage, which induce this autoeczematization. Studies with current technology are needed to facilitate further understanding of this phenomenon.

Patients with pemphigus vulgaris under pathology and on the background of prolonged use of high doses of glucocorticosteroid therapy – the possibility of influencing the glutathione system

Introduction. Pemphigus vulgaris (PV) is a life-threatening disease of the skin and mucous membranes that is associated with IgG antibodies that target several types of keratinocyte antigens and cause epidermal lysis (acantholysis) via intracellular signaling that activates apoptotic enzymes (apoptolysis).The aim of the study was to increase the effectiveness of treatment of patients with PV with the substantiation and development of modern methods of corrective therapy based on the study of indicators of changes in the balance of the thiol-disulfide system in the body of patients under pathological conditions and with prolonged use of high doses of glucocorticosteroid hormones (GCS).Materials and research methods. There were examined 30 patients with PV (4 men and 26 women), who were hospitalized in KU «Zaporizhzhya Regional Skin and Venereal Clinical Dispensary» ZOR, Zaporizhzhia. At the time of observation, most of the patients were aged 61-70 years. As a comparison group, 20 practically healthy people were examined, constituting a control group.Our studies were four-phase: before treatment; 2-3 weeks of maximum doses of glucocorticosteroids (stage I); 1.5-2 months before discharge from the hospital, when the patient was gradually reduced dose of systemic glucocorticosteroids and the selection of the optimal daily dose (stage II); after 5-6 months, when doses of hormones were minimal (2-3 tablets) in the absence of clinical manifestations of vesicles (stage III).Results. Our pathogenetic therapy by GCS significantly led to an increase in the level of reduced glutathione (GSH), so in particular after the third treatment stage it was at 1.52 ± 0.13 mkm / mg protein, exceeding the same rate of the first and second stages of GCS administration by 87.65 and 61.70%, respectively (p <0.05). The course of GCS therapy in patients with PV contributed to the fact that the level of reduced thiols increased significantly during each stage of therapy of the examined patients, in particular in the third stage of maintenance GCS therapy reduced thiols were determined at 15.64 ±1.23 mM / mg protein, exceeding this marker of the second stage by 36.95%, and the value of reduced thiols of the first stage by 83.78% (p <0.05). The value of reduced thiols of the group of patients with PV in the second stage was 11.42 ±1.08 mM / mg protein, and in the first stage – 8.51 ±0.92 mM / mg protein, the percentage difference between these therapeutic stages was determined in 34.19% (p <0.05).Prescribing systemic GCS to patients with PV for three stages led to the restoration of the balance of the thiol-disulfide system in patients, which manifested itself in the form of a decrease in markers: oxidized glutathione and oxidized thiols; and also in the form of an increase in the level of markers of restorative processes – glutathione reductase, glutathione peroxidase, reduced glutathione, reduced thiols, exerting a systemic positive effect on the course of the pathological process, which was reflected both in the normalization of laboratory parameters in patients and clinically in the form of stable remission.Conclusion. GCS therapy helps to normalize the activity of the antioxidant system of the human body under conditions of PV pathology, which prevents deprivation of the glutathione chain of the thiol-disulfide system during activation of oxidative and nitrosative stress processes and prevents the development of decompensation of the antioxidant system as a whole with the development of damage to key cells and target organs.

Pulse therapy: Opening new vistas in treatment of pemphigus

Pemphigus is comprised of a group of life-threatening autoimmune diseases that is characterized by circulating IgG antibodies targeting several types of keratinocyte antigens. After introduction of systemic steroids, survival has improved dramatically. However, mortality and morbidity were still very high due to side effects of steroids. Pulse therapy is defined as discontinuous/intermittent intravenous infusion of very high doses of corticosteroids along with certain immunosuppressive agents over a short period. This therapy was introduced to minimize the side effects of conventional corticosteroid therapy. The target is to achieve a faster response and stronger efficacy and to decrease the need for long-term use of systemic corticosteroids. As a result, this therapy has gained its popularity since three decades. The purpose of this article is to review the various available pulse therapy regimens with dosage, indications and contraindications and side effects.

049 Development of active mouse models to recapitulate pemphigus subtypes and to evaluate response to therapy

Pemphigus is an autoimmune bullous disease characterized by loss of adhesion caused by autoantibodies (PVIgG) against keratinocyte antigens, desmogleins (Dsg1 and Dsg3) and non-Dsg proteins, such as acetylcholine receptors (AChR) or Desmocollin (Dsc3). Different PVIgG result in diverse clinical phenotypes. In order to recapitulate the features of human pemphigus, to evaluate the role of different antigens, and to test the response to therapy, we generated an active mouse model. We produced murine recombinant proteins (rDsg1, rDsg3, rDsc3, rAChRM3, rAChRNα7 and rAChRNα9). We then immunized Dsg3-/- mice with mouse rDsg3, and adoptively transferred their splenocytes into C57BL/6 Rag2-/- (Rag2) immunodeficient mice, that express Dsg3. Recipient mice stably produce the pathogenic anti-Dsg3 IgG and exhibit the features of pemphigus vulgaris, as shown by clinical score and Dsg3 levels (ELISA). We also generated the pemphigus foliaceous active mouse model, by breaking the immunological tolerance in WT mice with rDsg1. Similarly, we created a Dsg1/Dsg3 mouse model by transferring the combined splenocytes from mice immunized with rDsg1 and mice immunized with rDsg3 into Rag2 mice. The Dsg1/Dsg3 mouse model displays the most severe phenotype, reproduces symptoms of mucocutaneous PV in patients, as well as the same level of intraepithelial detachment and inter-keratinocyte deposits of IgG. Moreover, only the Dsg1/Dsg3 mice significantly responded to treatment with methylprednisolone in a time-dependent manner (clinical score and ELISA). In conclusion, we created new tools that could 1. improve the knowledge on pemphigus pathomechanisms; 2. serve for the identification of new therapeutic targets; 3. allow a long-term pre-clinical observation with or without current and novel therapies

Autoantibody Signaling in Pemphigus Vulgaris: Development of an Integrated Model.

Pemphigus vulgaris (PV) is an autoimmune skin blistering disease effecting both cutaneous and mucosal epithelia. Blister formation in PV is known to result from the binding of autoantibodies (autoAbs) to keratinocyte antigens. The primary antigenic targets of pathogenic autoAbs are known to be desmoglein 3, and to a lesser extent, desmoglein 1, cadherin family proteins that partially comprise the desmosome, a protein structure responsible for maintaining cell adhesion, although additional autoAbs, whose role in blister formation is still unclear, are also known to be present in PV patients. Nevertheless, there remain large gaps in knowledge concerning the precise mechanisms through which autoAb binding induces blister formation. Consequently, the primary therapeutic interventions for PV focus on systemic immunosuppression, whose side effects represent a significant health risk to patients. In an effort to identify novel, disease-specific therapeutic targets, a multitude of studies attempting to elucidate the pathogenic mechanisms downstream of autoAb binding, have led to significant advancements in the understanding of autoAb-mediated blister formation. Despite this enhanced characterization of disease processes, a satisfactory explanation of autoAb-induced acantholysis still does not exist. Here, we carefully review the literature investigating the pathogenic disease mechanisms in PV and, taking into account the full scope of results from these studies, provide a novel, comprehensive theory of blister formation in PV.

Humoral and Cellular Autoreactivity to Epidermal Proteins in Atopic Dermatitis.

Atopic dermatitis (AD), a chronic relapsing inflammatory disease of the skin, is an important public health concern affecting 10-20% of children worldwide. The etiology and pathogenesis of AD involve the interplay of genetic and environmental factors, including abnormalities in skin integrity and a skewed immune system usually driven by a Th2 phenotype in childhood with a switch to Th1 in the chronic phase of disease. Children and adults with AD commonly have elevated IgE levels directed to multiple different antigens, including aeroallergens, food allergens, and microbial proteins. IgE targeting self-antigens from epidermal proteins have been detected in up to 91% of patients, particularly in severe persistent AD. It has been suggested that the occurrence of autoreactivity develops in early childhood. However, it is not clear yet if autoreactive IgEs in patients with AD are pathogenic or just an epiphenomenon. The fact that these autoantibodies are associated with severity and are not present in other allergic or skin diseases favors the pathogenicity of IgE-mediated autoreactivity in AD. In this review, we evaluate the pathogenesis of AD and the emerging role of autoreactivity to various keratinocyte antigens involving both the humoral and cellular components of the immune system.

Identification of Autoantigen Epitopes in Alopecia Areata

Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA.

Human papillomavirus 16 E7 protein inhibits interferon‐γ‐mediated enhancement of keratinocyte antigen processing and T‐cell lysis

Infection of epithelium with human papillomavirus (HPV) 16 is generally prolonged, suggesting an ineffective virus-specific immune response, and prolonged infection promotes anogenital cancer. To determine whether poor antigen presentation by HPV-infected keratinocytes (KCs) contributes to prolonged HPV infection, KCs and KCs expressing HPV 16 E7 protein (E7-KCs) were compared for susceptibility to T-cell-mediated lysis directed to ovalbumin (OVA) processed for presentation by the KCs. Interferon (IFN)-γ efficiently enhanced susceptibility to lysis of KCs presenting OVA, but not of E7-KCs similarly presenting OVA. E7-KCs also exhibited impaired IFN-γ-induced upregulation of transcription of major histocompatibility complex class I antigen processing and presentation-associated genes, and of membrane SIINFEKL-H-2K(b) complexes. Thus, expression of HPV 16 E7 protein in KCs may inhibit enhancement by IFN-γ of KC sensitivity to T-cell lysis, by impairing antigen presentation.

Anti-desmoplakin antibodies in erythema multiforme and Stevens-Johnson syndrome sera: pathogenic or epiphenomenon?

The pathophysiology of erythema multiforme (EM), Stevens-Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN) is unclear. Whether autoantibodies against desmoplakin (Dp) I and II play a pathogenic role or result from an epitope spreading phenomenon is uncertain. Our aim was to characterize the keratinocyte antigens recognized in EM, TEN and SJS. Of 33 patients studied, 2 had TEN, 1 SJS, 9 EM major and 21 EM minor, according to Roujeau's criteria. All sera were studied by indirect immunofluorescence (IIF), immunoblotting and immunoprecipitation. Twenty normal sera were used as controls. 10/33 sera reacted with polypeptides of 215 and/or 250-kDa molecular mass, which co-migrate with Dp I and II as assessed by an anti-Dp I and II monoclonal antibody on IB. In IP, none of the anti-Dp I and -Dp II 10 patient sera immunoprecipitated Dp I and/or II from radiolabeled keratinocyte extracts. Two of 10 patient sera (SJS, EM minor) reacted with DpI and II when denaturated by the IB procedure. The reactivity against intracellular antigens DpI and II as denaturated proteins may result from the epidermal damage produced by aggressive autoreactive T cells, playing therefore only a secondary role in the pathogenesis of the disease.

Narrow‐band UVB‐induced Externalization of Selected Nuclear Antigens in Keratinocytes: Implications for Lupus Erythematosus Pathogenesis†

The aim of this study was to analyze whether sera obtained from patients with lupus erythematosus (LE) react with membrane structures found on keratinocytes irradiated with narrow-band ultraviolet B (NB-UVB). We applied atomic force microscopy (AFM) to visualize cell surface structures expressing nuclear antigens upon apoptosis following NB-UVB irradiation. Immortalized human keratinocytes (HaCaT) were cultured under standard conditions, irradiated with 800 mJ cm(-2) NB-UVB light and imaged by AFM mounted on an inverted optical microscope. It was observed that NB-UVB irradiation provoked significant alterations of the keratinocyte morphology and led to the membrane expression of antigens recognized by anti-La and anti-Ro 60 kDa sera but not by antidouble-strand DNA sera. The presence of La and Ro 60 kDa antigens on keratinocyte surfaces after NB-UVB irradiation was limited mainly to the small bleb-like protrusions found on the keratinocytes by AFM. A closer investigation by AFM also revealed that some structures positively stained with anti-Ro 60 kDa serum were also located submembranously. We hypothesize that the externalization of some nuclear antigens because of NB-UVB exposure might be responsible for exacerbation of skin symptoms in patients suffering from LE.

Betulinic acid induces apoptosis in skin cancer cells and differentiation in normal human keratinocytes

Betulinic acid (BA), a pentacyclic triterpene of plant origin, induces cell death in melanoma cells and other malignant cells of neuroectodermal origin. Little is known about additional biological effects in normal target cells. We show, in this study, that BA induces differentiation as well as cell death in normal human keratinocytes (NHK). Cytotoxicity profiles of BA are compared among normal human keratinocytes, HaCaT cells, IGR1 melanoma cells and normal melanocytes. As expected, BA is toxic to all cell types, normal and malignant, but varies in its cytotoxic potency and in the extent of induction of apoptotic vs. necrotic cell death in the four different skin cell types. Apoptosis is proved by annexin V and Apo2.7 binding and by DNA fragmentation. Induction of differentiation-associated antigens in keratinocytes--filaggrin and involucrin--is demonstrated, together with specific morphological changes in treated cell cultures. BA, apart from its cytotoxic activities in cellular systems, is capable of inducing differentiation of NHK into corneocytes without immediately provoking apoptotic cell death.

Epidermal cells accelerate the restoration of the blood flow in diabetic ischemic limbs.

Epidermal progenitor cells (EpPCs) were long thought to be unipotent, giving rise only to other keratinocytes but recent studies question this assumption. Here, we investigated whether mouse EpPCs can adopt other antigenic and functional phenotypes. To test this, we injected freshly isolated and cultured EpPCs and transient amplifying cells into diabetic and non-diabetic mouse ischemic hindlimb and followed the cells' fate and the recovery of the ischemic limb blood flow over time. Both freshly isolated and cultured EpPCs and transient amplifying cells were incorporated into the vasculature of the ischemic limb 2 and 5 weeks post-injection, and some expressed endothelial cell but not keratinocyte antigens. Additionally, in the non-diabetic animals, first transient amplifying cells and then EpPCs accelerated the restoration of the blood flow. By contrast, in diabetic animals, only injected EpPCs or unsorted epidermal cells accelerated the restoration of the blood flow. These data indicate that epidermal cells can adopt non-skin phenotypes and functions, and that this apparent pluripotency is not lost by differentiation of EpPCs into transient amplifying cells. They also suggest that epidermal cell therapy might be of therapeutic value in the treatment of diabetic ischemia. Finally, because epidermal cells are readily accessible and expandable, they appear to be ideally suited for use as a non-viral gene delivery therapy.

Identification of keratinocyte-specific markers using phage display and mass spectrometry.

Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity.

Mohs Micrographic Surgery-Induced Pemphigus

BACKGROUND Pemphigus is an autoimmune blistering disease that presents with flaccid intraepidermal blisters, erosions of the skin and mucous membranes, acantholysis, and in vivo bound and circulating autoantibodies against keratinocyte antigen. Currently a handful of reports incriminate surgical trauma as an initiating factor in this disease. OBJECTIVE To document pemphigus evolving in a wound after Mohs micrographic surgery. METHODS Case report. RESULTS We present a case of pemphigus that started in a Mohs surgical wound after the excision of a squamous cell carcinoma (SCC) from a 49-year-old woman. Biopsy of the preoperative lesion did not reveal pemphigus. Biopsy of the postoperative lesion revealed pemphigus with no residual SCC. CONCLUSION We suggest that Mohs surgery, like any other skin surgery, may nonspecifically activate pemphigus. This change must be differentiated from postoperative wound infection and other causes of poor wound healing.

A localized variant of paraneoplastic pemphigus: acantholysis associated with malignant melanoma

We report a 72-year-old male patient with a nodular malignant melanoma that was associated with focal suprabasal acantholysis (FSA). This phenomenon, which is regarded as an incidental finding by dermatopathologists, may be associated with inflammatory and also neoplastic skin diseases. Haematoxylin and eosin stained sections from an erythematous plaque surrounding the patient's tumour showed FSA, direct immunofluorescence (DIF) and indirect immunofluorescence (IIF) on normal human skin, monkey oesophagus and rat urinary bladder were negative. On electron microscopy few desmosomes could be detected in the basal cell layer of the acantholytic areas and there was a nearly complete loss of these structures in the spinous cell layer. Only remnants of cytoplasmic plaques and keratin filaments could be observed in those areas. In contrast, adherens junctions appeared to be well preserved. An enzyme-linked immunosorbent assay (ELISA) using recombinant fusion proteins as antigens did not show circulating autoantibodies against desmoglein 1 (Dsg1) or desmoglein 3 (Dsg3). In contrast, immunoblotting revealed autoantibodies directed against keratinocyte antigens with a molecular weight of 85 kDa and 250 kDa, the first band corresponding to the molecular weight of comigrating plakoglobin. Immunoprecipitation with patient serum also revealed a 85-kDa band. We conclude that these autoantibodies, probably in conjunction with cofactors produced by the tumour, could play a part in the pathogenesis of this variant of FSA, for which we propose the term 'localized paraneoplastic pemphigus.'

De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction. Isolation of a human single chain antibody fragment against human keratin 14.

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly non-differentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.

Novel Human α9 Acetylcholine Receptor Regulating Keratinocyte Adhesion is Targeted by Pemphigus Vulgaris Autoimmunity

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.

Expression of SS-A/Ro and SS-B/La antigens in skin biopsy specimens of patients with photosensitive forms of lupus erythematosus.

The reason that only some patients with lupus erythematosus (LE) develop autoantibodies to SS-A/Ro and SS-B/La antigens and photosensitivity is unknown. One hypothesis is that both events are related to the level of expression of these antigens in the skin. To test this hypothesis, we measured the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in normal sun-protected and sun-exposed skin in 14 patients with LE with photosensitivity, 12 patients with LE without photosensitivity, and 4 normal individuals. The presence of circulating antibodies to these antigens was measured in all patients. Outpatient clinic in an academic medical center. We found that the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in skin biopsy specimens obtained from the same site was 4- to 10-fold higher in patients with LE with photosensitivity than in those patients with LE without photosensitivity (P<.001). Antigen expression was highly correlated with the presence and titer of circulating anti-SS-A/Ro and anti-SS-B/La antibodies (P<.001). These findings indicate that photosensitivity and the presence and titer of circulating anti-SS-A/Ro and anti-SS-B/La antibodies are both directly correlated with the expression of accessible and immunoreactive SS-A/Ro and SS-B/La antigens in the skin specimens of patients with LE. Thus, the expression of these antigens in keratinocytes may be an important determinant of the development of both SS-A/Ro and SS-B/La autoantibodies and of photosensitive forms of LE.