Bullous pemphigoid autoantibodies reactive with intracellular basal keratinocyte antigens: studies of subclass distribution and complement activation.

Using immunofluorescence (IF) and monoclonal antibodies (MoAbs) to IgG subclasses, terminal complement components, and S-protein/vitronectin, we have extended recent observations concerning reactivity of bullous pemphigoid autoantibodies with intracellular antigens located on the polar tips of basal human keratinocytes (HuK). Using three purified bullous pemphigoid IgG fractions, autoantibody reactivity with these intracellular antigens was present in all four IgG subclasses. When skin sections were used as substrate, an identical IgG subclass distribution of autoantibodies for each bullous pemphigoid IgG fraction was observed, but reactive with the basement membrane zone. All three bullous pemphigoid IgG preparations contained IgG subclass autoantibodies capable of complement fixation. Each IgG fraction resulted in fixation of all of the terminal complement components (C5, C6, C7, C8, and C9) and assembly of the membrane attack complex (MAC) on the polar tips of basal HuK. S-protein/vitronectin was not bound in a similar fashion. Normal IgG fractions yielded consistently negative reactions. Thus, bullous pemphigoid autoantibodies, fixed to polar tips of basal HuK, are found in all four IgG subclasses and will activate complement resulting in generation of MAC.

An electrophoretic method for selection of conditions for production of electrophoretically uniform protein colloidal gold complexes.

An electrophoretic method was developed to determine the conditions required for production of electrophoretically uniform protein-colloidal gold complexes from monodisperse colloidal gold (Au) and electrophoretically uniform protein. The method is based on the electrophoretic migration of protein-Au complexes in agarose. The results demonstrate that two variables, the pH of adsorption and the quantity of protein added, can be manipulated to vary the electrophoretic mobility of the resulting protein-Au complexes. Thus, agarose gel electrophoresis can be used to select the pH of adsorption and the quantity of protein required to produce electrophoretically uniform protein-Au complexes. This new electrophoretic mobility test can be used in place of or in addition to the classical procedure of Zsigmondy and its many variations, both visual and spectrophotometric. The procedure described is also useful for electrophoretic comparison of small quantities of various protein-Au samples.

Pemphigus immunoglobulin G subclass autoantibodies: studies of reactivity with cultured human keratinocytes.

The subclass distribution of pemphigus vulgaris (PV) autoantibodies has been investigated further. Cultured human keratinocytes (HuKs) were incubated with immunoglobulin G (IgG) fractions prepared from the serum samples of five patients with PV and one patient with pemphigus foliaceus (PF). The binding of IgG subclasses to HuK was detected by the use of indirect immunofluorescence done with monoclonal antibodies that were specific for each of the four human IgG subclasses. IgG1 and IgG4 binding to keratinocytes was detected in all of the pemphigus IgG preparations. In each instance, the titer of bound IgG1 was equal to or greater than the IgG4 titer. IgG3 binding was detected in only one PV IgG, and IgG2 pemphigus antibody activity was not detected. Differences in the immunofluorescence patterns between the five PV and one PF IgG and between the five PV and a second PF serum were noted. PV antigens were detected as a speckled pattern on HuKs, with accentuation in the intercellular spaces. By comparison, the pattern of PF antigen expression varied significantly depending on whether anti-IgG1 or anti-IgG4 antisera were used. Anti-IgG1 bound in a coarse granular pattern without accentuation in the intercellular spaces, but anti-IgG4 bound in a pattern nearly identical to that observed with PV IgG and its subclasses.

Deposition of the membrane attack complex of complement in pemphigus vulgaris and pemphigus foliaceus skin.

The present study was performed to determine whether complement activation in pemphigus vulgaris (PV) and pemphigus foliaceus (PF) results in the assembly of the terminal complement sequence or membrane attack complex (MAC) in skin lesions. Biopsy specimens of skin lesions from five patients with PV and three patients with PF contained C5, C7, C9, and the MAC related neoantigen (C5b-9 neoantigen) in intercellular substance areas (ICS), as well as IgG and the early complement components Clq, C4, and C3. The presence of these late complement components and the C5b-9 neoantigens in ICS sites of the skin lesions is indicative of complement activation by the pemphigus antibody, with subsequent assembly of the MAC. The binding of IgG and early complement components to ICS was observed in both non-lesional (normal appearing) skin and in skin lesions. However, no MAC could be detected in the normal appearing skin of our pemphigus patients. It was also noted that the MAC could be generated in vitro on cryostat sectioned normal human skin by pemphigus antibody in the presence of complement. Results of these studies suggest that complement activation may be related to membrane damage of epidermal cells in both PV and PF.

Complement fixation by pemphigus antibody. V. Assembly of the membrane attack complex on cultured human keratinocytes.

Previous studies have shown that pemphigus vulgaris (PV) IgG will fix early complement components (C1q, C4, and C3) to cultured murine epidermal cell surfaces and that PV IgG and complement alter epidermal cell membrane integrity. The present study was undertaken to determine if assembly of terminal complement components (C5, C6, C7, C8, and C9) and expression of C5b-9 neoantigens occur when PV IgG interacts with human keratinocyte (HuK) cell surface antigens in the presence of a source of complement. Monoclonal antibodies specific for C5, C6, C7, C8, C9, and C5b-9 neoantigens were screened for reactivity to the individual complement components in an assembled complex of human C5b-9 on rabbit red blood cell ghosts. Monoclonal antibodies (tissue culture supernatants) that bound to antigenic determinants accessible in the C5b-9 complex were selected for this study using immunofluorescence methods. HuK treated with PV IgG fixed C5, C6, C7, C8, C9, and C5b-9 neoantigens in a characteristic speckled pattern, while normal IgG did not. Heat inactivation or EDTA treatment of the complement source, or substitution of C2-depleted serum abolished C5, C6, C7, C8, C9, and C5b-9 neoantigen staining. PV IgG and complement also resulted in significant cytotoxicity to cell membranes as assessed using an ethidium bromide-fluorescein diacetate assay. These results suggest that PV IgG will activate the membrane attack complex of the complement system on HuK cell surfaces, resulting in cytotoxicity to cell membranes, further implicating complement in the pathogenesis of pemphigus.

Expression of pemphigus vulgaris antigen in cultured human keratinocytes: effect of inhibitors, tunicamycin, and lectins.

We have investigated expression of pemphigus vulgaris antigen(s) in cultured human keratinocytes induced by the addition of extracellular calcium. Cycloheximide (10(-4) M) inhibited pemphigus antigen expression and stratification but actinomycin D (2 micrograms/ml) had no effect. Tunicamycin, which inhibits dolichol pyrophosphate-mediated glycosylation of asparaginyl residues specifically, was used to study the role of glycosylation. When calcium switching was carried out in the presence of tunicamycin, human keratinocytes did not stratify, and the expression of pemphigus antigen was partially inhibited and limited to cell-cell contact areas. Analysis of biosynthetically labeled proteins showed that the synthesis of high-molecular-weight proteins was markedly reduced in the tunicamycin-treated cells. A reciprocal blocking test demonstrated that concanavalin A and wheat germ agglutinin receptor share an epitope with pemphigus vulgaris antigen(s). These results suggest that Ca++, newly synthesized protein, and N-asparaginyl glycosylation are required for normal pemphigus antigen expression and epidermal stratification in vitro. Pemphigus vulgaris antigen may have a highly glycosylated, high-molecular-weight protein chain with carbohydrates playing an important role in epidermal cell morphology, adhesion, and stability of cell surface antigens.

The effect of three variables on adsorption of rabbit IgG to colloidal gold.

The purpose of this investigation was to (a) begin to evaluate the coagulation curve as a means of selecting the minimal quantity of IgG required to stabilize colloidal gold (Au); (b) determine the effect of the quantity of IgG added, the pH of adsorption, and the isoelectric point (pI) range of the IgG on the quantity of IgG bound and the stability of the IgG-Au complex with respect to desorption; and (c) discuss these results with respect to current theory on the effect of pH on adsorption of IgG to surfaces. No absolute minimal value required to prevent coagulation could be determined despite the high reproducibility of the values obtained; approximate values were selected. Each variable had an effect on the quantity of IgG bound: as the quantity of IgG added increased, the quantity bound increased; as the pH of adsorption became more alkaline, the quantity bound decreased; and as the pI range of the IgG became more alkaline, the quantity bound increased. IgG-Au complexes with a variable number of bound IgG molecules, depending on the three variables selected, can be produced. Production of IgG-Au composed of uniform numbers of IgG is discussed. A modification of the current theory on the effect of pH on adsorption of IgG is proposed.

Complement fixation by Brazilian Pemphigus foliaceus autoantibodies.

This study was undertaken to determine if Brazilian Pemphigus foliaceus (BPF) autoantibodies will fix complement as do P. vulgaris (PV) autoantibodies. When sections of human skin were examined following indirect immunofluorescence (IF) staining, BPF autoantibodies reacted with the upper layers of epidermis, while PV autoantibodies were reactive with the lower layers. When tissue culture epidermal cells were used for indirect IF, BPF autoantibodies were not detected after plating until 24 h, while PV autoantibodies reacted within 18 h of plating. Using complement IF staining methods, BPF autoantibodies were found to fix C1q, C4 and C3 to whole skin sections in an intercellular pattern and to the surface of cultured keratinocytes. Although reactive with different epidermal cell surface antigens, autoantibodies in BPF will fix complement in a fashion similar to autoantibodies in PV.

Complement fixation by pemphigus antibody. IV. Enhanced epidermal cell detachment in the absence of human plasminogen.

It has been reportedly previously that complement enhances pemphigus vulgaris antibody-induced epidermal cell detachment. The present studies were designed to eliminate the possibility of the human plasminogen-plasmin system contributing to the cell detachment observed in previous complement-mediated studies. To assay for the presence of protease contamination in the reagents, a simple, sensitive, caseinolytic-fluorometric assay was used. Confluent murine or human epidermal cell cultures were incubated with pemphigus IgG with and without complement and plasminogen. Detached epidermal cells, as determined by light microscopy and cell-counter analysis, were enumerated at 48 hours. Incubating pemphigus IgG with complement resulted in marked cell detachment, compared with minimal cell detachment when pemphigus IgG was incubated with plasminogen. Culture media were collected after 48 hours and assayed for activation of plasminogen. It was determined that the added plasminogen had not been activated, particularly in experiments where significant detachment was observed. The plasminogen could still be activated by streptokinase and urokinase. These studies suggest that epidermal cell detachment in pemphigus appears to be mediated at least in part by complement activation.

The particulate (speckled-like thread) nuclear staining pattern: species and cellular distribution of Ro/SSA antigen.

Anti-Ro/SSA antibodies are associated with a particulate (speckled-like thread) nuclear immunofluorescence (IF) staining pattern when human spleen imprints are employed as substrate. The present study was undertaken to determine if these antibodies would yield a similar IF pattern with splenic imprints from other species, and with other human cell lines. All splenic imprints (mammalian, avian, amphibian and fish origin) demonstrated a particulate nuclear pattern when treated with anti-Ro/SSA antibody positive serum samples. Cytospin preparations of a variety of peripheral blood cells also demonstrated this particulate pattern when treated with anti-Ro/SSA antibodies. Similar findings were noted when cytospin preparations of cultured keratinocyte cell lines were used as substrate. The antigen, associated with the particulate IF staining pattern when treated with anti-Ro/SSA antibody positive serum samples, is expressed in a variety of tissues and species.

The application of glucose oxidase-labeled antibodies for the detection of proteins on nitrocellulose.

We have evaluated the sensitivity of immunostaining with glucose oxidase for the detection of monomeric human serum albumin (HSA) and monomeric human immunoglobulin G (IgG). A modification of a histochemical procedure was utilized by replacing phenazine methosulfate (PMS) with 1-methoxyphenazine methosulfate (mPMS) and by replacing Tris-HCl with Tris-citrate to improve the solubility of the tetrazolium compounds tested. mPMS is less sensitive to light, and may be stored for long periods in solution; it is now used routinely by histochemists in place of PMS in dehydrogenase cytochemistry. pH values of 6.3-8.3 were tested, with the reaction at pH 8.3 providing a slight increase in sensitivity. The reaction rate increased markedly as the pH became more alkaline. The minimum quantity of HSA detected was 3 ng applied directly to nitrocellulose and 10 ng when blotted. Human IgG was routinely detected at 250 pg and occasionally at 100 pg when dotted on the nitrocellulose.

Complement fixation by pemphigus antibody. III. Altered epidermal cell membrane integrity mediated by pemphigus antibody and complement.

The present study investigates the effects of pemphigus IgG and complement upon cell viability and/or membrane integrity using trypan blue exclusion, ethidium bromide (EB) staining, and fluorescein diacetate (FDA) conversion by living cells. Forty-eight-hour cultivated epidermal monolayers of neonatal BALB/c mice were incubated in media containing 1 mg/ml purified pemphigus IgG for 48 h in either the presence or absence of complement (absorbed AB sera). Adherent and detached cells were examined by both phase and fluorescence microscopy. Results from trypan blue exclusion showed that pemphigus IgG plus complement produced a modest decrease in exclusion of the dye compared to pemphigus IgG without complement. When FDA/EB comparisons were made, however, the differences were more substantial. When complement plus pemphigus IgG was added to cultures, the number of FDA-positive adherent cells decreased significantly and the number of EB-positive detached cells increased significantly. The effects of complement were inhibited by the use of heat-inactivated AB sera or by C1q depletion of AB sera. No significant effect on the cells was observed in the presence or absence of complement when pemphigus F(ab')2 fragments or when normal IgG was used. Plasminogen depletion of the complement source did not interfere with complement and pemphigus IgG effects as judged by the FDA/EB assay. These studies suggest that pemphigus antibody in the presence of complement alters cell membrane integrity and supports the contention that complement may play a significant role in the mechanism of acantholysis.

Complement fixation by pemphigus antibody. II. Complement enhanced detachment of epidermal cells.

We have previously demonstrated that pemphigus antibodies will fix complement to organ and tissue cultured epidermal cells in vitro. In the present study, we sought to determine the role complement plays in the detachment of cultured murine epidermal cells by pemphigus antibody. Forty-eight hour cultivated epidermal monolayers from neonatal BALB/c mice were treated with purified IgG fractions of pemphigus sera in the presence or absence of complement. In the absence of complement, pemphigus IgG produced slight cell detachment when compared to an identical amount of normal IgG. When cells were maintained in media with pemphigus IgG plus complement for 48 h, the cell detachment was significantly higher than that obtained with pemphigus IgG alone, or with normal IgG plus complement. Heat inactivation or Clq depletion of the complement source resulted in complete inhibition of cell detachment. When pemphigus F(ab')2 plus complement was used instead of whole pemphigus IgG plus complement, the cell detachment rate was again lowered significantly. Both pemphigus IgG and the complement source were depleted of plasminogen by passage over a lysine-Sepharose 4B column, and tested for their effects on cell detachment depleted of plasminogen. Depletion of plasminogen failed to inhibit cell detachment induced by pemphigus IgG and complement. These results suggest that complement activated by pemphigus antibody binding to the epidermal cell surface induces epidermal cell detachment and would argue against the plasminogen-plasmin system as the sole cause of cell detachment in pemphigus.

Anti-Ro/SSA antibodies. Association with a particulate (large speckledlike thread) immunofluorescent nuclear staining pattern.

Twenty-one patients with clinical and histopathologic evidence of subacute cutaneous lupus erythematosus and one patient with Sjögren's syndrome and vasculitis had anti-Ro/SSA (Sjögren's syndrome A) antibodies as demonstrated by double immunodiffusion assay using a saline extract of human spleen as the source of antigen. These serum samples were negative when tested for other nuclear antigens, including native DNA, Sm, and RNP. When tested for antinuclear antibodies (ANAs) by immunofluorescence, only ten of 22 serum samples were ANA positive on mouse liver substrate, while 18 of 22 had a positive ANA when HEp-2 tumor cells were utilized. With imprints of human spleen as test substrate, all 22 serum samples yielded a positive ANA result and a particulate (large speckledlike thread) staining pattern. Absorption of two of these serum samples with human spleen extract containing Ro/SSA antigen inhibited both the particulate staining pattern using human spleen imprints and the anti-Ro/SSA precipitin line by double immunodiffusion. These studies suggest that anti-Ro/SSA antibodies and antibodies producing the particulate nuclear staining pattern on human spleen imprints are either one and the same or closely paralleling antibody systems.

Analysis of rabbit lung lavage immunoglobulins during the course of pulmonary inflammation induced with aerosolized antigen.

Lung lavage fluids (LLF) from rabbits with pigeon dropping extract (PDE)-induced granulomatous pulmonary inflammation were studied for protein and immunoglobulin (Ig) G and A levels. It was found that the protein levels of the lung fluids of rabbits increased to a maximum after 2-3 weeks of aerosol treatment with PDE during which time inflammation of the lung increases. This is followed by a gradual decrease in protein content as the inflammation wanes and the lung returns to normal. These variations primarily reflect changes in IgG and IgA levels. IgG and IgA levels follow different courses. IgA reaches a maximum in the first week of inflammation and then gradually decreases. In contrast, IgG reaches a maximum level (2-3 weeks) and stays at an elevated level throughout the 12 week period of aerosol treatment with PDE. Antibodies to PDE in these two classes of immunoglobulins do not entirely reflect the immunoglobulin class levels. IgA antibody levels reach a maximum after extended aerosol challenge while IgG antibody reaches a maximum early and then declines to background levels. The specificity of the non-PDE antibody IgG is unknown at present. The distribution of IgA subclass producing cells in the lung is different than in the gut. In the lung the major subclass is g while in the gut it is f. The distribution of subclasses of IgA in the LLF, however, does not appear to reflect the cellular distribution. The reason for this is not clear.

Adaptation of the Ngo-Lenhoff peroxidase assay for solid phase ELISA.

A modification of the Ngo-Lenhoff peroxidase assay has been developed. By this method, peroxidase is detectable at 5 fmoles of peroxidase per ml. The assay is easy to perform and the reagents are inexpensive. We have modified the buffer by the addition of citric acid and sodium azide to reduce background color development and to inhibit the activity of catalase, respectively. Production of the indamine dye by the peroxidase reaction is rapid but it may be stopped by lowering the pH to 3.0. The pH change of 7.0 to 3.0 is accompanied by a color change from purple-blue to blue and a shift in the maximum absorbance from 590 nm to 595 nm. The sensitivity of the assay was unaffected by these modifications.

The effect of IgG and IgG fragments on the absorption of inhaled antigens across the air-blood barrier of isolated perfused rabbit lungs.

The absorption of inhaled soluble protein antigens across the alveolocapillary membrane can be inhibited by passive immunization in isolated rabbit lungs. The present study was carried out to determine the immunoglobulin class and structural features (Fc-receptor binding, divalent antigen binding) required to effect the inhibition. Isolated rabbit lungs from unimmunized rabbits were perfused with autologous blood to which whole antiserum, IgG or IgG fragments specific for either ovalbumin (OA) or human serum albumin (HSA) was added. The lungs were insufflated with an aerosol containing 125I-OA and 131I-HSA and blood samples were analysed for 125I and 131I in trichloracetic acid (TCA)-precipitable and TCA-soluble forms for 4 hr after insufflation. Whole antiserum and the IgG immunoglobulin fraction of the whole antiserum were equally effective in inhibiting the antigen absorption, indicating that the IGG antibody is sufficient for the effect. The F(ab')2 and Fab' fragments of the IgG molecule were as effective as native IgG, indicating that the antigen-binding site is the only structural requirement and that Fc-receptor and divalent antigen binding are not required.

Passive gold agglutination. An alternative to passive hemagglutination.

A method is described for coating colloidal gold with protein for use in passive agglutination. Coating of the gold is quick, reproducible and the sterile filtered product is stable over a long period of storage at 4 degrees C. Colloidal gold coated with each of the 4 subclasses of human IgG was tested with rabbit, monkey and goat antisera and found to be compatible with each. The visual properties of coated gold particles resemble blood cells and appear to be equally sensitive.

The detection of human B lymphocytes by both light and electron microscopy utilizing colloidal gold labeled anti-immunoglobulin.

Colloidal gold was used to label rabbit anti-goat IgG for the indirect detection of goat anti-human IgD and IgM. Utilizing this technique, surface immunoglobulin was visualized on human blood B lymphocytes at both the light and electron microscopic levels of resolution. By this technique, percentages of lymphocytes were found to closely correlate with those of the standard immunofluorescent procedure. The ease of the procedure along with the clarity of the marker in both light and electron microscopy suggest wide applicability in clinical and research studies of lymphocyte membrane markers.

Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat anti-human immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application.

A method is described for the adsorption of selected macromolecules to colloidal gold which is then used as an electron dense marker for the indirect detection of specific cell surface molecules. Membrane bound concanavalin A, which binds specific sugars on horseradish peroxidase, and wheat germ agglutinin, which binds specific sugars on ovomucoid are detected indirectly with gold labeled horseradish peroxidase and ovomucoid, respectively. Goat anti-human IgM on blood lymphocytes is detected with gold labeled rabbit anti-goat IgG. In the preparation of colloidal gold labeled proteins, the problems of flocculation of colloidal gold by proteins and nonadsorption of proteins to colloidal gold, are solved through a combination of concentration of protein and pH variable adsorption isotherms, which allows one to determine the conditions for adsorption of proteins to colloidal gold. Adsorption is pH dependent, the pH conditions correlating with the isoelectric point(s) of the major protein fraction(s); adsorption is influenced by interfacial tension, solubility and by the electrical charge on the molecules. Colloidal gold is inexpensive and preparation of a useful label is rapid, reproducible and the results easily quantitated from electron micrographs.