Monovalent Fab' immunoglobulin fragments from endemic pemphigus foliaceus autoantibodies reproduce the human disease in neonatal Balb/c mice.

Fogo selvagem (FS) is an autoimmune disease caused by IgG autoantibodies to desmoglein I (DG-I), a desmosomal glycoprotein. We have previously shown that the autoantibodies in these patients are pathogenic and restricted mainly to the IgG4 subclass. The purpose of this study was to determine if the Fc domain or the valence of FS autoantibodies were relevant in the induction of epidermal disease in neonatal mice. IgG4 was prepared from sera of FS patients by anion exchange chromatography, and digested with pepsin to yield F(ab')2 fragments. Monovalent FS Fab' were made by reduction and alkylation of FS F(ab')2. Intact FS IgG4, FS F(ab')2, and FS Fab' fragments were injected into neonatal mice. Intact FS IgG4 and both FS IgG fragments were pathogenic. The disease in the animals was dose dependent, and on the molar basis, FS Fab' fragments were more potent and efficient in producing disease than whole FS IgG. These results suggest: (a) simple binding of FS autoantibodies to DG-I may trigger keratinocyte detachment and epidermal disease; (b) DG-I may represent a keratinocyte cell adhesion molecule; and (c) complement activation and surface cross-linking may not be relevant in keratinocyte detachment.

Herpes gestationis autoantibodies recognize a 180-kD human epidermal antigen.

Herpes gestationis (HG) is a putative autoimmune bullous dermatosis of pregnancy which shares many findings with bullous pemphigoid (BP), a disease of the elderly. This study identifies for the first time the antigen detected by HG autoantibodies and compares it with that recognized by BP autoantibodies. Sera from 16 HG and 17 BP patients, and from normal pregnant women were evaluated by immunofluorescent (IF) studies and immunoblotted against human epidermal extracts. 89% of HG sera with circulating antibodies by IF recognized a 180-kD protein by immunoblotting. 71% of BP sera recognized a 240-kD band, but 47% detected a 180-kD protein that comigrated with the antigen detected by HG sera. None of the control sera recognized any specific bands. These findings suggest that the 180-kD epidermal protein may be the antigen detected by the HG factor and they also define immunologic similarities between HG and BP.

Human autoantibodies against desmoplakins in paraneoplastic pemphigus.

Recently, a previously unrecognized autoantibody mediated blistering disease, paraneoplastic pemphigus has been described. Paraneoplastic pemphigus is associated with lymphoid malignancies, thymomas, and poorly differentiated sarcomas. Serum of affected patients contain pathogenic autoantibodies that immunoprecipitate from normal keratinocytes a characteristic complex of four polypeptides with M(r) of 250, 230, 210, and 190 kD. As our preliminary studies indicated that the 250-kD and the 210-kD antigens comigrated with desmoplakins I and II, we investigated the possibility that autoantibodies against the desmoplakins were a component of this autoimmune syndrome. 11 sera from affected patients were tested by indirect immunofluorescence against desmosome containing tissues, immunoprecipitation of metabolically labeled keratinocytes, and Western immunoblotting of desmoplakins I and II that had been purified to homogeneity from pig tongue epithelium. By indirect immunofluorescence, 9 of 11 sera showed strong binding to epithelial and nonepithelial desmosomes, and 2 were weakly reactive. All 11 immunoprecipitated 250- and 210-kD bands of variable intensity that comigrated with bands identified by a murine monoclonal antidesmoplakin antibody, and immunoblotting confirmed binding of the serum autoantibodies to purified desmoplakins. This demonstrates that paraneoplastic pemphigus is the first human autoimmune syndrome in which autoantibodies against the desmoplakins are a prominent component of the humoral autoimmune response.

Pathogenic effects of bullous pemphigoid autoantibodies on rabbit corneal epithelium.

Bullous pemphigoid (BP) is associated with circulating autoantibodies reactive with an antigen(s) of the basement membrane zone (BMZ) of skin and mucosae. The pathogenicity of these autoantibodies, although suspected, is unconfirmed. We have investigated the effects of BP autoantibodies on a closely related tissue, the corneal epithelium of the rabbit. IgG fractions from the sera of seven patients with BP were purified by (a) ammonium sulfate precipitation, (b) ion exchange chromatography, or (c) gel filtration. Control IgG was prepared by ion exchange chromatography of pooled normal human gamma globulins. 32 rabbits received corneal intrastromal injections of BP IgG fractions (50 microliter, 0.95-2.05 mg total dose) in one eye, and control IgG (50 microliter, 1.8 mg) in the contralateral cornea. 28 of 32 BP IgG injections produced corneal inflammatory lesions, 10 of which developed visible blisters. Histologically, lesions showed polymorphonuclear cells clustering along the BMZ, and subepithelial blister formation. Immunofluorescence showed in vivo bound IgG and C3 at the BMZ. The intensity of inflammation was dose dependent and correlated often with in vitro complement fixation titers of the fractions. None of 32 corneas injected with control IgG became inflamed. BP IgG fractions injected intradermally into the ear skin of rabbits failed to produce inflammation. This may be due to slow clearance of IgG in the cornea, and optimal binding by the corneal epithelium. The intracorneal injections of BP IgG reproduce the clinical, histological, and immunological features of BP. This study provides evidence that BP autoantibodies are pathogenic.

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.

The calcium-sensitive epitope of pemphigus foliaceus antigen is present on a murine tryptic fragment and constitutes a major antigenic region for human autoantibodies.

Recent findings indicate that the pemphigus foliaceus (PF) antigen is involved in epidermal cell adhesion and that characteristic PF lesions result from loss of this function as a consequence of autoantibody binding. In the present communication we present data on the epitopes involved in the human autoantibody binding to an immunologically reactive murine tryptic fragment of the PF antigen (tf-PF). Immunoprecipitation experiments showed that 39 PF sera, obtained from North American, Colombian, and Brazilian patients recognized only calcium-sensitive epitope(s) on the tf-PF. Immunofluorescence blocking experiments showed that preincubation with tf-PF completely blocked the immunofluorescence of 80% of the sera when tested on human skin substrate, and 86% of the sera when tested on murine skin substrate. These results show that the calcium-sensitive epitope(s) originally recognized on human PF complex, is (are) present on the murine tf-PF and constitute(s) a major antigenic region for the human PF autoantibodies. They also implicate this region of the PF antigen in the pathogenesis of PF as well as in epidermal cell adhesion.

Experimentally induced pemphigus vulgaris in neonatal BALB/c mice: a time-course study of clinical, immunologic, ultrastructural, and cytochemical changes.

Pemphigus vulgaris autoantibodies (PV IgG) promote cell detachment in epidermal cell cultures and acantholysis in the epidermis of neonatal BALB/c mice in vivo. We have studied the evolution of the immunologic and ultrastructural changes in the epidermis of BALB/c mice that receive parenteral injections of PV IgG. Neonatal BALB/c mice received a single i.p. injection of PV IgG (10 mg/g body weight) or control IgG from normal humans. The skin and serum of these animals was obtained at 0, 1, 3, 6, 12, 18, and 24 h post injection, and examined by immunofluorescence (IF), electron microscopy (EM), and immunoelectron microscopy (IEM). PV IgG was detected in the mouse serum and bound to the epidermal cells as soon as 1 h after injection by IF and IEM. The intensity of the binding in the skin (by IF) increased sharply between 3 and 6 h, and remained positive at 24 h. Early epidermal cell detachment was demonstrable by EM at 1 h as widening of the epidermal intercellular spaces (ICS), and by 6 h the ICS between desmosomes had detached completely. Desmosomal junctions are the last to separate, occurring at 12-18 h. At this point, complete cell detachment occurred in the suprabasilar layers of the epidermis. Basal cells remain attached to the underlying dermis (tombstone row). Coincident with cell detachment, intracellular tonofilaments retracted from the cell periphery and clustered in a perinuclear position. IEM confirmed the binding of PV antibodies to the surface of epidermal cells in early and established lesions. This study demonstrates that the early immunologic and ultrastructural changes that occur in human pemphigus vulgaris are reproduced in this mouse model of the disease.

A soluble and immunoreactive fragment of pemphigus foliaceus antigen released by trypsinization of viable human epidermis.

Pemphigus foliaceus (PF) antigen is a transmembrane desmosomal glycoprotein (desmoglein I), part of which is located on the keratinocyte surface. Previous studies have shown that after trypsinization of viable human epidermis, this antigen is no longer detected on the surface of detached keratinocytes. It was not known, however, if this loss of antigenic activity was due to destruction, internalization, or cleavage of the antigen itself. In the present study we investigated the fate of the PF antigen after trypsinization of viable human skin. By using Concanavalin-A agarose affinity chromatography, we could partially purify an antigenic glycoprotein fraction that was released by trypsinization into the medium. This antigenic fraction was radiolabeled and tested by immunoprecipitation using sera from endemic pemphigus foliaceus or fogo selvagem (FS), non-endemic pemphigus foliaceus (NEPF), pemphigus vulgaris (PV), and bullous pemphigoid (BP) patients, and sera from normal subjects as controls. Immunoprecipitated labeled proteins were analyzed by SDS-PAGE and autoradiography. All FS sera (20 of 20 FS and five of five NEPF) and 46% of the PV sera (six of 13) immunoprecipitated a band of 45-kD molecular weight. Sera from FS patients in prolonged clinical and serological remission (seven of 10), sera from BP patients (five of five), and sera from normal donors (nine of nine) did not precipitate this 45-kD band. This study showed that a fragment of the PF antigen is released by trypsinization of human skin as a soluble immunoreactive glycopeptide of 45-kD molecular weight. Additionally, this procedure has generated sufficient quantities of the PF antigen for further biochemical characterization.

Purification of immunoprecipitated pemphigus foliaceus antigen fragment and its use in radioimmunoassay.

A major difficulty in biochemical studies of the pemphigus foliaceus (PF) antigen is the lack of a method for its quantitative determination. Immunofluorescence blocking and immunoprecipitation methods are semiquantitative and time consuming. Radioimmunoassay (RIA) methods are quantitative but they require pure and stable antigen preparations that have not been available for PF. The present investigation shows the further purification of a previously described preparation of PF antigen fragment obtained from trypsinization media of mouse skin (Con A Frn) and demonstrates its usefulness in a RIA method for quantitation of the antigen. The major contaminants of the 45-kD tryptic fragment of the PF antigen (tf-PF) in immunoprecipitates of the Con A Frn with PF sera were identified as H and L chains of murine IgG and mannose-binding lectins. The IgG contaminants could be removed by avoidance of blood contamination during preparation of the Con A Frn and/or pre-absorption of the Con A Frn with protein A Sepharose. The lectins could be removed by affinity chromatography of the Con A Frn on asialofetuin column and washing the immunoprecipitates with 0.2 M alpha-methyl-mannoside. Using the purified, labeled Con A Frn in RIA, we showed that standard curves could be established and the amounts of PF antigen could be determined in different extracts without the need for electrophoresis, autoradiography, or scanning. This RIA method is rapid and can be easily used to analyze many samples, e.g., chromatographic fractions and extracts made from different tissues.

Ultrastructural studies of acantholysis induced in vivo by passive transfer of IgG from endemic pemphigus foliaceus (Fogo Selvagem).

Intraperitoneal (IP) injections of IgG from patients with Endemic Pemphigus Foliaceus [Fogo Selvagem (FS)] cause acantholysis in BALB/c mice (JID. 85:538, 1985). The dynamic ultrastructural changes of FS IgG-induced acantholysis in mice are the subject of this study. FS IgG was injected IP into neonatal BALB/c mice. Skin and serum was studied at 0, 1, 3, 6, 12, 18, and 24 h post injection by immunofluorescence (IF), electron microscopy (EM), and immuno-EM. Binding of FS IgG in the intercellular spaces (ICS) of the basal cell layer was seen by IF within 1 h and was strongest at 12 h. IgG binding affected the spinous and granular cell layer by 12 h, then faded and remain localized only in the basal cell layer at 24 h. By immuno-EM, IgG binding was diffuse along the keratinocyte surface. Edema of the ICS in the basal cell layer was present at 1 h by EM. At 12 h, there was microvillous formation with intact desmosomes at the tip of the projections. Splitting of desmosomes (forming half desmosomes) and acantholysis primarily affecting the granular cell layer were most prominent between 12 and 24 h. The plaques of the half desmosomes gradually disappeared and tonofilaments retracted into the cytoplasm. Detaching keratinocytes showed vacuolization, swollen mitochondria, and internalization of intact desmosomes and half desmosomes (remnants of split desmosomes). This investigation shows that the ultrastructural changes observed in the epidermis of patients with FS can be duplicated in experimental animals by IP injection of FS IgG. Further, FS IgG may have direct effects on the assembly/disassembly of desmosomes.

Demonstration of pemphigus antibodies on the cell surface of murine epidermal cell monolayers and their internalization.

The pathogenic effects of pemphigus vulgaris (PV) antibodies on epidermal cells can be demonstrated both in vitro using skin organ culture or primary epidermal cell cultures (PECC) and in vivo by passive transfer of PV antibodies into neonatal BALB/c mice. Although PV antibodies have been localized on the epidermal cell surface by several techniques, little is known about the fate of these autoantibodies subsequent to their surface binding. We have examined this, using murine PECC which express pemphigus antigen on their surface, and followed the fate of the bound antibody molecules. Forty-eight-hour PECC were incubated at 37 degrees C with PV antibodies for 20 min and then with horseradish peroxidase-labelled antihuman IgG. This was considered time 0. The monolayers were fixed with glutaraldehyde after 0, 0.5, 1, 3, 6, 18, and 24 h incubation at 37 degrees C and then processed for electron microscopy. At time 0 hour, PV antibodies is detected bound evenly along the surface of keratinocytes. Within 30 min, the bound PV antibodies becomes clustered, internalized into submembranous vesicles via surface pits, and eventually fused with lysosomes. Widening of the intercellular spaces was also seen in PECC treated with PV antibodies within the first 24 h. PECC treated with normal human IgG in parallel cultures showed no such surface binding, internalization, or cell-cell detachment. Treatment with cytochalasin-D and/or colchicine did not affect the internalization of the PV antibodies, but fusion with lysosomes was not seen in treated cultures. These findings suggest that PV antibodies binds a surface antigen and the complex is internalized and fused with lysosomes in a process that may have pathophysiologic relevance.

Studies of the relationship of the 230-kD and 180-kD bullous pemphigoid antigens.

In bullous pemphigoid (BP), autoantibodies from most patients recognize a high molecular weight 230-kD epidermal antigen (Ag) by immunoprecipitation. By Western immunoblotting, 50-70% of sera recognize the high molecular weight Ag, but 30-50% recognize a low molecular weight, 180-kD epidermal Ag. We examined the specificities of affinity-purified antibodies against these Ag. Antibodies specific for the 230- and 180-kD Ag were prepared by immunoaffinity against Ag immobilized on nitrocellulose and released by acid glycine. IgG eluted from the 230-kD Ag band retained its specific binding to the 230-kD Ag by immunoblotting, and bound to the epidermal basement membrane zone (BMZ) by indirect immunofluorescence (IF) and to hemidesmosomes by indirect immunoelectron microscopy (EM). IgG affinity purified by the 180-kD Ag band bound only the 180-kD Ag in immunoblotting, with no cross reaction to the 230-kD Ag, bound the epidermal BMZ by indirect IF, and also bound to hemidesmosomes in immuno-EM. IgG specific for the 230-kD Ag in immunoblotting immunoprecipitated only the 230-kD Ag, with no apparent precipitation of the 180-kD Ag. Surprisingly, IgG specific for the 180-kD Ag precipitated both the 180- and the 230-kD Ag in immunoprecipitation, and the 230-kD Ag band was much more intense than the 180-kD Ag band. This study shows that apparent cross-reactivity between these Ag by BP autoantibodies can only be detected in native conditions by immunoprecipitation, and cannot be demonstrated using denatured Ag in immunoblotting. The two proteins appear to be distinct Ag, closely associated in the epidermal hemidesmosome, but the exact relationship of these Ag to each other may not be clarified until complete structural data become available.

Brazilian pemphigus foliaceus autoantibodies are pathogenic to BALB/c mice by passive transfer.

Brazilian pemphigus foliaceus (fogo selvagem) is a cutaneous blistering disease endemic to certain areas of South America that has distinctive epidemiologic features suggestive of an infectious disease transmitted by an insect vector. Patients with the disease have antiepithelial autoantibodies, both circulating in the serum and bound to lesional epidermis. In order to examine the possible pathogenic role of these autoantibodies, IgG from the sera of these patients was purified and injected into the peritoneum of neonatal BALB/c mice. Thirty-four of 46 mice (74%) receiving parenteral IgG fractions from these patients developed cutaneous lesions that were identical to the human disease by clinical, histologic, immunologic, and ultrastructural criteria. High-titer Brazilian pemphigus foliaceus sera produced lesions more consistently and rapidly than low-titer sera. When injections were discontinued, new lesions ceased to appear and old lesions resolved. The extent of disease correlated with the titer of human antiepithelial antibodies detected in the mouse serum (z less than 0.01). Similar concentrations of IgG fractions obtained from sera of unaffected Brazilians living in endemic areas and from American donors did not induce disease when injected into littermates. These results establish that the antiepithelial autoantibodies play an important role in the pathogenesis of the cutaneous lesions in Brazilian pemphigus foliaceus.

The use of human pemphigoid autoantibodies to study the fate of epidermal basal cell hemidesmosomes after trypsin dissociation.

It is known that during trypsinization of the skin, the epidermis is first separated from the dermis and individual keratinocytes are dissociated by disruption of the epidermal intercellular spaces. The desmosomal unit is separated at the level of the intercellular space and the split desmosomes are internalized in plasma membrane-limited vesicles; however the fate of the hemidesmosome under such conditions has not been studied. We have recently shown (Mutasim et al: J Invest Dermatol 84:47-53, 1985) that autoantibodies from the sera of patients with bullous pemphigoid bind in vitro to hemidesmosomes but not to desmosomes providing a highly specific marker for these organelles. Utilizing these autoantibodies, we studied the fate of the hemidesmosome during trypsin dissociation of epidermal basal cells derived from the skin of neonatal BALB/c mice. During trypsinization, portions of the dermal face of the plasma membrane which include hemidesmosomes formed pits which pinched off to produce vesicles that moved toward the nucleus. This was accompanied by retraction of the tonofilaments away from the cell periphery. The mechanism of this internalization process is not yet known, but may involve contractile elements of the cytoskeleton. The highly specific binding of bullous pemphigoid autoantibodies to the hemidesmosome may prove helpful in future biochemical and immunocytochemical studies of this organelle.

A pool of bullous pemphigoid antigen(s) is intracellular and associated with the basal cell cytoskeleton-hemidesmosome complex.

Bullous pemphigoid (BP) antibodies are known to react with an antigen of the basement membrane zone (BMZ) of squamous epithelia and produce, by the indirect immunofluorescence technique, linear fluorescence at the BMZ. Direct and indirect immunoelectron microscopy (IEM) have demonstrated BP antigen to be within the lamina lucida, in close association with the basal cell membrane. Trypsin-dissociated epidermal basal cells bind BP antibodies in a polar distribution, presumably because the BP antigen is restricted to the dermal pole of the basal cell membrane. In this study we have utilized newborn BALB/c mouse skin to obtain both dissociated basal cells (by trypsinization) and epidermal sheets (by dithiothreitol treatment). We show that viable basal cells, which are impermeable to IgG molecules, do not react with BP antibodies. When the basal cell plasma membrane is disrupted by cytospin centrifugation, air drying, freezing and thawing, or hypotonic lysis, or permeated by nonionic detergents (saponin), cells become reactive with BP antibodies. Basal cell cytoskeletons, prepared by sequential treatment with Triton X-100, deoxyribonuclease, and 2 M NaCl continue to react with BP antibodies. Similarly, viable epidermal sheets fail to bind BP antibodies. When epidermal sheets are treated with nonionic detergents, water, or freezing and thawing prior to incubation with BP antibodies, linear BMZ fluorescence is observed. IEM study of saponin-treated basal cells shows the immunoreactants to be localized on intracytoplasmic vacuoles which represent internalized hemidesmosomes. IEM of permeated epidermal sheets shows the immunoreactants as aggregates on the inner surface of the dermal pole of the basal cell membrane. These observations suggest that the BP antigen is intracellular and is in close association with the basal cell cytoskeleton and hemidesmosomes.

Definition of bullous pemphigoid antibody binding to intracellular and extracellular antigen associated with hemidesmosomes.

Bullous pemphigoid (BP) antibodies are deposited predominantly in the lamina lucida in vivo; however, circulating BP antibodies bind in vitro to the cytoplasmic plaque of basal cell hemidesmosomes. We examined the ability of IgG in nine BP sera to bind to intracellular or extracellular antigen. On skin cryosections, indirect IF showed IgG bound to basement membrane zone (BMZ) and indirect ImmunoEM confirmed intracellular binding on the cytoplasmic plaque of hemidesmosomes. In contrast, when normal skin was exposed to BP serum in organ culture, direct IF showed fainter linear deposition of IgG along the BMZ, and direct ImmunoEM demonstrated extracellular IgG binding in the lamina lucida, predominantly beneath hemidesmosomes. Four of nine sera showed complement fixation on indirect IF samples (IgG bound to intracellular antigen) and three showed complement fixation on direct IF specimens (IgG bound to extracellular antigen). Three of the nine sera contained complement fixing antibodies detectable only in antibody populations specific for intracellular or extracellular antigen. Western immunoblots showed that five of nine sera recognized a 240-kD protein and four of nine recognized a 180-kD protein. There was no correlation between the presence (or absence) of either band and the detection of complement fixing antibodies specific for intracellular or extracellular antigen. BP autoantibodies bind both intracellular and extracellular antigen, and IgG binding exclusively to extracellular antigen that mimics the in vivo situation can be detected by using organ culture. Complement fixation may be restricted to antibodies specific for intracellular or extracellular antigen. These findings underscore the complexity of the autoantibody-antigen system in BP and have implications regarding the proposed pathogenicity of the autoantibodies.

Pemphigus foliaceus antigen: characterization of a keratinocyte envelope associated pool and preparation of a soluble immunoreactive fragment.

In both the endemic and sporadic forms of pemphigus foliaceus (PF), antiepidermal autoantibodies against desmoglein I are present. Desmoglein I is a highly insoluble 160-kD transmembrane glycoprotein of the desmosomal core. The detailed immunochemical characterization of the epitope(s) recognized by the PF autoantibodies is hampered by its large molecular weight and the insolubility of desmoglein I in nondenaturing buffers. This study was designed to identify alternative methods that could yield soluble immunoreactive PF antigen (Ag) from normal human epidermis. The presence of PF Ag in human epidermis and in its soluble or insoluble fractions was monitored by indirect immunofluorescence, immunoadsorption of PF sera, and immunoprecipitation of radiolabeled fractions. The PF Ag from trypsin-resistant, radiolabeled cell envelope preparations was cleaved by papain and immunoprecipitated by PF sera. A 50-kD peptide, isoelectric at pH 5.5-5.8, was immunoprecipitated by sera from all patients with endemic PF (n = 15) or idiopathic PF (n = 4), and by two of four pemphigus vulgaris sera, but by no control sera (n = 7). This study shows that a significant fraction of the PF Ag is insoluble, trypsin-resistant, and is associated with the cornified cell envelope fraction, but an Ag fragment can be obtained in a small molecular weight, soluble, and immunoreactive form by papain digestion. This 50-kD papain fragment is more amenable to detailed chemical and immunologic characterization than the native molecule.

Pemphigus and pemphigoid antigens are expressed in human amnion epithelium.

The sera of patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP) contain autoantibodies which react with antigens present in a variety of mammalian squamous epithelia. The biologic role of these epithelial antigens is unknown; however, they do appear to be markers of epithelial differentiation. We have examined many human tissues that may be used as sources of large amounts of BP and PV antigens, including the human amnion, which is composed of the amnion reflectum and placentum (epithelial monolayers) and amnion cord (stratified epithelium). Human amnion was obtained from normal term deliveries and specimens of each area of the amnion were processed for light and electron microscopy. Samples of amnion were snap-frozen in liquid nitrogen and 4-micron sections were used as a substrate for BP and PV antibodies by indirect immunofluorescence (IF) techniques. Well-characterized BP serum (indirect IF titer 1:2560), PV serum (indirect IF titer 1:160), and normal human serum (negative indirect IF) were utilized as sources of BP and PV autoantibodies and a negative control, respectively. Linear staining of the BMZ was produced by BP antibodies in 8/8 specimens of amnion reflectum, 8/8 specimens of amnion placentum, and 3/3 specimens of amnion cord. Staining of the epithelial intercellular spaces was produced by PV antibodies of 2/3 specimens of amnion cord and none of the amnion reflectum (0/8) or amnion placentum (0/8) tested. Normal human serum produced no specific staining of the amnion. The light and the ultrastructural features of human amnion basement membrane zone and intercellular spaces resembles closely their epidermal counterparts. The restricted distribution of BP antigen in amnion epithelial basal cells (amnion reflectum, placentum, and cord) and PV antigen in stratified amnion epithelium (amnion cord) reinforces their relationship with epithelial differentiation. The abundance and availability of these tissues facilitate extraction and characterization of BP and PV antigens.

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.

An autoantibody in pemphigus serum, specific for the 59 kD keratin, selectively binds the surface of keratinocytes: evidence for an extracellular keratin domain.

We have identified a novel IgG antikeratin autoantibody in the serum of a Brazilian pemphigus foliaceus patient (Cascas-42). This antibody is specific for the 59 kD acidic murine keratin and its 56.5 kD human counterpart (Moll's catalogue #10), and is distinct from the pemphigus antibody system. Antikeratin autoantibodies present in the Cascas-42 serum were purified by affinity chromatography with a 59 kD murine keratin-agarose column (IAP-Cascas-42 antibodies). The specificity of the IAP-Cascas-42 antibodies was tested by indirect immunofluorescence and immunoelectron microscopy against epidermal cryosections, trypsin-dissociated keratinocytes, and epidermal cell cultures. The serum was also tested with extracts from unlabeled and surface 125I-labeled keratinocytes (Iodo-Gen method) by immunoblot analysis of one- and two-dimensional polyacrylamide gel electrophoresis. The IAP-Cascas-42 antibodies bind the intercellular spaces of murine epidermis, and the cell surfaces of viable, dissociated murine keratinocytes, as well as murine epidermal cells in culture by immunofluorescence and immunoelectron microscopy. These autoantibodies did not stain cytoplasmic keratins and did not react with parallel human epidermal substrates. The Cascas-42 serum identified the 59 kD murine acidic keratin and its 56.5 kD human counterpart in epidermal extracts by two-dimensional polyacrylamide gel electrophoresis and immunoblot analysis. In addition, surface radioiodination of viable murine keratinocytes selectively labeled the 59 kD keratin suggesting that a domain of this molecule is exposed on the cell surface. The 125I-labeled 59 kD keratin was also recognized by the Cascas-42 serum by immunoblotting and autoradiography. These studies suggest that in murine epidermis, the 59 kD keratin is a transmembrane protein with an extracellular domain recognized by the IAP-Cascas-42 antibodies.