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. 2010 Dec;162(3):543–549. doi: 10.1111/j.1365-2249.2010.04265.x

Efficacy of intravenous immunoglobulin (IVIG) affinity-purified anti-desmoglein anti-idiotypic antibodies in the treatment of an experimental model of pemphigus vulgaris

D Mimouni *,†,‡, M Blank *,‡, A S Payne §, G J Anhalt , C Avivi **, I Barshack ‡,**, M David †,‡, Y Shoenfeld *,‡,1
PMCID: PMC3026558  PMID: 20964642

Abstract

Pemphigus vulgaris is a rare life-threatening autoimmune bullous disease caused by immunoglobulin G (IgG) autoantibodies directed against desmogleins 1 and 3. Previously, we showed that intravenous immunoglobulin (IVIG) ameliorates anti-desmoglein-induced experimental pemphigus vulgaris in newborn naive mice. The aim of this study was to examine the efficacy of anti-anti-desmoglein-specific IVIG in a similar model. Pemphigus-vulgaris-specific IVIG (PV-sIVIG) was affinity-purified from IVIG on a column of single-chain variable fragment (scFv) anti-desmogleins 1 and 3. The anti-idiotypic activity of PV-sIVIG was confirmed by enzyme-linked immunosorbent assay, inhibition assay. After induction of pemphigus by injection of anti-desmogleins 1 and 3 scFv to newborn mice, the animals were treated with PV-sIVIG, IVIG (low or high dose) or IgG from a healthy donor (n = 10 each). The skin was examined 24–48 h later, and samples of affected areas were analysed by histology and immunofluorescence. In vitro study showed that PV-sIVIG significantly inhibited anti-desmogleins 1 and 3 scFv binding to recombinant desmoglein-3 in a dose-dependent manner. Specificity was confirmed by inhibition assay. In vivo analysis revealed cutaneous lesions of pemphigus vulgaris in mice injected with normal IgG (nine of 10 mice) or low-dose IVIG (nine of 10 mice), but not in mice treated with PV-sIVIG (none of 10) or high-dose IVIG (none of 10). On immunopathological study, PV-sIVIG and regular IVIG prevented the formation of acantholysis and deposition of IgG in intercellular spaces. In conclusion, the PV-sIVIG preparation is more effective than native IVIG in inhibiting anti-desmoglein-induced pemphigus vulgaris in mice and might serve as a future therapy in patients with the clinical disease.

Keywords: autoantibodies, autoimmunity, desmoglein, IVIG, pemphigus

Introduction

Pemphigus is a group of organ-specific autoimmune mucocutaneous disorders with an established immunological basis. Its clinical hallmark is the presence of intraepithelial blisters and erosions on the skin and the mucous membranes. Immunohistological studies of pemphigus lesions have shown that immunoglobulin G (IgG) autoantibodies directed against the adhesion molecules desmoglein 1 and desmoglein 3 in the affected epithelium cause cell-to-cell detachment of epidermal and mucosal epithelial cells (acantholysis) [1–3]. The goal of therapy is to eliminate these pathogenic autoantibodies [4]. However, at present there are no available selective inhibitors of desmoglein autoantibodies, and therapy is therefore based upon antibody removal and non-specific immunosuppression.

Left untreated, pemphigus vulgaris (PV) has a natural history of relentless progression, with 50% mortality at 2 years and almost 100% at 5 years [5]. Since the 1950s, the survival of patients with PV improved remarkably with the introduction of corticosteroids and cytotoxic drugs, which have powerful anti-inflammatory and immunomodulatory effects. However, their use is limited severely by immunosuppression, myelosuppression and numerous side effects.

Intravenous immunoglobulin (IVIG), a blood product prepared from donor serum, is used as replacement therapy in immunodeficient conditions [6,7]. Recent studies have revealed an extremely wide spectrum of IVIG antibody activity. Not only does IVIG recognize a large number of antigens of bacteria, viruses and other infectious agents, it also exhibits anti-idiotypic specificity [8,9]. Commercial IVIG preparations contain multiple anti-idiotypic antibodies, such as anti-factor VIII antibodies [10], anti-DNA autoantibodies [11–13], anti-intrinsic factor antibodies [13], anti-thyroglobulin (Tg) autoantibodies [13], anti-neutrophil cytoplasmic antibodies [14], anti-microsomal antibodies [15], anti-neuroblastoma antibodies [16], anti-phospholipid antibodies [17], anti-platelet antibodies [18], anti-Sm idiotype (ID-434) [19] and anti-GM1 antibody [20]. Therefore, in the last decade, IVIG has been used increasingly as an immunomodulatory agent in the treatment of autoimmune and systemic inflammatory diseases, including systemic lupus erythematosus, dermatomyositis and polymyositis, multiple sclerosis, myasthenia gravis, Guillain–Barré syndrome and anti-phospholipid syndrome [21,22].

Anti-idiotypic antibodies are effective in the treatment or prevention of disease manifestations because they inhibit the binding of the pathogenic autoantibodies to their corresponding antigen, as shown both in vitro[12,13,23,24] and in vivo[17,19,25]. An in vitro study of systemic lupus erythematosus suggested that the value of anti-idiotypic antibodies may also be attributable to their inhibitory effect on the spontaneous secretion of anti-desmoglein by peripheral B lymphocytes [26]. In addition, IVIG may act via the idiotypic network, causing soluble circulating immune complexes to aggregate and become insoluble and, consequently, removable by the reticuloendothelial system.

Our previous study demonstrated the efficacy of IVIG in the prevention of blister formation in an experimental model of PV [27]. Recently, our positive findings were confirmed in a large double-blind placebo-controlled clinical trial [28].

The amount of specific anti-idiotypes in commercial IVIG preparations is extremely low. Therefore, we speculated that the use of isolated anti-idiotypes against pathogenic autoantibodies could yield even better results with a fraction of the amount of IgG, with a lower rate of adverse reactions. To test this theory, we developed a modulated anti-idiotypic preparation using concentrated specific natural polyclonal anti-desmoglein anti-idiotypic antibodies from commercial IVIG.

The aim of the present study was to evaluate the effect of treatment with IVIG affinity-purified anti-desmoglein anti-idiotypic antibodies on the immunological and clinical findings in a mouse model of PV.

Methods

Antibodies

Desmogleins 1 and 3 single-chain variable fragment (scFv) was produced in the Top10F' strain of Escherichia coli (Invitrogen, Carlsbad, CA, USA) and purified by nickel chelation affinity chromatography, as described previously [29].

Rabbit anti-desmogleins 1 and 3 were derived from rabbits immunized with anti-desmogleins 1 and 3 scFv and used as a source of anti-idiotypic antibodies. The rabbit anti-sera were first cleared of an irrelevant human scFv, AM3-13, along with excess haemagglutinin (HA) peptide (Sigma, St Louis, MO, USA) coupled to Affigel-15 matrix (Bio-Rad, Hercules, CA, USA), as described previously [30]. The result was evaluated by testing for depletion of anti-HA activity by enzyme-linked immunosorbent assay (ELISA). To produce an affinity column comprising normal human IgG, 10 mg of human IgG (Enco Ltd, Petah Tiqwa, Israel) was coupled to 1 ml of Affigel 10 matrix (Bio-Rad), according to the manufacturer's instructions. The anti-HA- and anti-AM3-13-depleted rabbit anti-sera were incubated with the human IgG affinity column. The flow-through fractions comprising the cleared anti-sera were concentrated by Centricon YM-10 ultrafiltration (Millipore, Billerica, MA, USA).

Preparation of PV-specific IVIG (PV-sIVIG) anti-idiotypic antibodies

A column of desmogleins 1 and 3 scFv was constructed employing 500 µg of desmogleins 1 and 3 scFv coupled to 500 µl Affigel-15 matrix (Bio-Rad), according to the manufacturer's instructions. IVIG (100 mg) was loaded overnight at 4°C. The bound anti-anti-desmogleins 1 and 3-specific IVIG (PV-sIVIG) was eluted with 2 M of glycin-HCl (pH 2·5) and dialysed against phosphate-buffered saline (PBS) (pH 7·4).

Preparation of F(ab)2 and Fc IVIG

F(ab)2 or Fc fragments were prepared according to a standard method [31]. IVIG was dialysed against 100 mM of Na-acetate buffer, pH 4·0, and digested with pepsin [2% weight-for-weight (W/W); Sigma] or papain (2% W/W; Sigma) at 37°C for 18 h. Any remaining traces of undigested IgG and Fc fragments were removed by binding to a protein-A column (Pharmacia Biotech, Norden AB, Sollentuna, Sweden). The efficiency of the digestion was confirmed by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

Inhibition of anti-desmogleins 1 and 3 scFv binding to desmoglein 3 by IVIG

To define 50% anti-desmogleins 1 and 3 antibody binding to desmoglein 3, we used commercial plates coated with desmoglein 3 (MESACUP Desmoglein test ‘Dsg3’; MBL Medical & Biological Laboratories, Nagoya, Japan). The plates were blocked for 1 h at 37°C in blocking buffer [0·1 M NaHCO3, pH 8·6, 5 mg/ml bovine serum albumin (BSA)] and then incubated with anti-desmogleins 1 and 3 at different concentrations for 2 h at room temperature. The binding was probed with rabbit anti-desmogleins 1 and 3 followed by anti-rabbit-IgG conjugated to horseradish peroxidase (Dako, Carpinteria, CA, USA) and appropriate substrate {ABTS [2,20-Azino-di(3-ethylbenzthiazoline-sulphonate]; Sigma}. Anti-desmoglein 3 at 50% binding was incubated with either PV-sIVIG, whole-molecule IVIG or fragments of IVIG, F(ab)2 and Fc at different concentrations. The percentage inhibition was calculated as follows:

graphic file with name cei0162-0543-mu1.jpg

In vivo PV-sIVIG treatment of anti-desmogleins 1 and 3-induced PV in a mouse model

C57BL/6 pregnant mice (12–14 weeks old) were purchased from Harlan Laboratories (Jerusalem, Israel). PV was induced in the newborn mice by subcutaneous injection of anti-desmogleins 1 and 3 scFv, 20 µg/48 h. The mice were then divided into four treatment groups (n = 10 each): (i) PV-sIVIG (30 µg/mouse); (ii) low-dose IVIG (30 µg/mouse); (iii) high-dose IVIG (2 mg/mouse); and (iv) IgG from a healthy donor (2 mg/mouse) (controls). An additional control group of newborn mice in which PV was not induced were treated with PV-sIVIG (30 µg/mouse) for the purpose of eliminating the possibility of PV induced disease by the PV-sIVIG. Eight hours later, the newborn mice were inspected for the formation of blisters and erosions, and skin sections were cut from lesional and perilesional areas and fixed in 4% PBS-buffered formalin (Sigma).

Microscopic and immunofluorescence studies

The formalin-embedded skin samples were cut into four slices, deparaffinized and blocked with 3% BSA. Anti-rabbit IgG-fluorescein isothiocyanate was added to the slides for 2 h at room temperature, and the slides were washed and analysed by fluorescence microscopy.

Results

In vitro inhibitory activity of PV-sIVIG (Figs 1 and 2)

Fig. 1.

Fig. 1

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Enzyme-linked immunosorbent assay competition assay demonstrating the single-chain variable fragment binding neutralization activity of pemphigus vulgaris-specific intravenous immunoglobulin (PV-sIVIG), IVIG, its F(ab)2 (92% inhibition) and Fc fractions (7% inhibition, P < 0·001) to desmoglein 3.

Fig. 2.

Fig. 2

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Enzyme-linked immunosorbent assay competition assay demonstrating the single-chain variable fragment binding neutralization activity of pemphigus vulgaris-specific intravenous immunoglobulin (PV-sIVIG), low-dose intravenous immunoglobulin (IVIG) (98% compared to 9%, respectively, P < 0·001), normal dose IVIG (97% inhibition) and control immunoglobulin G (cIgG) (7% inhibition) to desmoglein 3.

In vitro analysis of the efficacy of treatment with IVIG fraction specific for anti-desmogleins 1 and 3 (PV-sIVIG) revealed significant inhibition of anti-desmogleins 1 and 3 scFv binding to desmoglein 3. At a dose of 30 µg/ml, PV-sIVIG inhibited desmoglein 3 binding by 98 ± 8% compared to only 9 ± 3% for the same dose of IVIG (P < 0·001). The effective dose of PV-sIVIG was 66-fold lower than the effective dose of commercial IVIG. A high dose of IVIG (2 mg/ml) had the same effect as PV-sIVIG (P > 0·05). IgG from a healthy donor had no effect on anti-desmogleins 1 and 3 scFv binding to desmoglein 3. Moreover, the F(ab)2 fraction of PV-sIVIG inhibited anti-desmogleins 1 and 3 binding to desmoglein 3 by 92 ± 4%, whereas the Fc portion of the PV-sIVIG inhibited binding by only 7 ± 2% (P < 0·001).

Prevention of blistering and erosions by PV-sIVIG

Lesions first appeared 16–48 h after injection of low-dose IVIG and control IgG (positive findings in nine of 10 newborn mice tested) (Table 1). They consisted clinically of either discrete cutaneous vesicles or extensive sloughing of the skin with positive Nikolsky sign (Fig. 3a). No cutaneous lesions appeared in any of the newborn mice in the PV-sIVIG or normal-dose IVIG groups (Fig. 3b).

Table 1.

Incidence of blisters and erosions in newborn mice

Source of IgG injected to newborn mice Treatment of newborn mice No. of mice with cutaneous lesions
Anti-desmogleins 1 + 3 scFv PV-sIVIG 0/10
Anti-desmogleins 1 + 3 scFv IVIG (normal dose) 0/10
Anti-desmogleins 1 + 3 scFv IVIG (low dose) 9/10
Anti-desmogleins 1 + 3 scFv Control IgG 9/10
Control IgG PV-sIVIG 0/10
Control IgG IVIG (normal dose) 0/10
Control IgG IVIG (low dose) 0/10
Control IgG Control IgG 0/10
None PV-sIVIG 0/10
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IgG, immunoglobulin G; IVIG, intravenous immunoglobulin; PV-sIVIG, pemphigus vulgaris-specific IVIG; scFv, single-chain variable fragment.

Fig. 3.

Fig. 3

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Cutaneous lesions expressed by extensive sloughing (black arrow) of the skin (back) induced in neonatal mice by injection of single-chain variable fragment (a). No lesions in pemphigus vulgaris-specific intravenous immunoglobulin (b).

Immunopathology

Histological analysis of lesional skin from two mice revealed typical intraepidermal vesicles with remaining basal cell layer attached to the dermis (suprabasal detachment) and a few acantholytic keratinocytes in the detached area (Fig. 4).

Fig. 4.

Fig. 4

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Histological examination of the affected mice showing subepidermal clefting with acantholysis (×100). The black arrow points to the position where the intra-epidermal separation, typical of pemphigus, is beginning.

Direct immunofluorescence of samples of perilesional epidermis from two mice demonstrated autoantibody deposition in the intercellular spaces. The fluorescence was more pronounced in the lower part of the epidermis (Fig. 5).

Fig. 5.

Fig. 5

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Direct immunofluorescence of perilesional epidermis from a pemphigus-induced mouse injected with low-dose intravenous immunoglobulin (30 µg/mouse) demonstrated autoantibody deposition in the intercellular spaces. The fluorescence was more pronounced in the lower part of the epidermis (as pointed by the white arrow).

On analysis of skin from mice in the PV-sIVIG or normal-dose IVIG groups, there was no autoantibody deposition in the intercellular spaces or suprabasilar separation.

Discussion

This study offers strong immunopathological evidence that IVIG exerts anti-anti-desmoglein activity (anti-desmoglein anti-idiotypes) which is capable of neutralizing the binding of PV-IgG to desmogleins 1 and 3. Furthermore, our study shows that IVIG anti-idiotypic antibodies are useful agents in the prevention of blister formation in experimental PV.

Desmoglein 1 and desmoglein 3 are adhesion molecules expressed predominantly in the basal layers of the human epidermis, the site of suprabasilar acantholysis in PV [32]. Antibodies reactive with desmogleins 1 and 3 are considered to be highly specific serological markers for diagnosis. In the individual patient, antibody levels correlate with disease activity, showing a remarkable increase during exacerbations and a drop during remissions [33].

An important clue to the pathogenicity of desmoglein 3 antibodies was provided by the study of Anhalt et al. [1], wherein the passive transfer of IgG from patients with PV to newborn mice resulted in the development of suprabasilar acantholysis and intercellular deposition of IgG and C3, as demonstrated by immunofluorescence. In more recent studies, even monovalent Fab immunoglobulin fragments were found to be pathogenic in these mice [34,35]. Another study using the same experimental model showed that the blister formation was abolished when anti-desmoglein 3 IgG from the sera of patients was immunoadsorbed with recombinant desmoglein 3 [2].

It is important to emphasize that in PV it is the antibodies that cause the tissue injury, in the absence of any inflammatory mediators [1,36,37]. The exact pathogenetic mechanism underlying the blister formation is still not understood completely. A direct inhibitory effect of the antibodies on the cell-to-cell adhesion function of the desmogleins was supported by a remarkable experiment by Koch et al. [38], wherein the genetic deletion of desmoglein 3 in mice led to the development of suprabasilar blisters in the oral mucosa and skin, very similar to the phenotype of patients with PV. In another study, anti-desmoglein-3 antibodies appeared to interfere directly with desmoglein function within the desmosome, causing split desmosomes, without keratin retraction, in areas of acantholysis [39]. The anti-desmoglein antibodies might deplete the desmosomes of desmoglein directly or, alternatively, deplete the cell surface of desmoglein before it becomes incorporated into the desmosome, thereby decreasing the precursor pool [40–43]. In either case, it may be concluded that PV antibodies target desmoglein 3 for endocytosis and lysosomal degradation: adhesion on the cell surface is necessary to prevent the endocytosis of organizing desmosomes [44]. Various studies have suggested a role for signalling pathways, associated with either acantholysis or causal. For example, adding PV-IgG to keratinocytes caused phosphorylation of desmoglein 1, leading to its dissociation from plakoglobin [45], a part of some signalling pathways. Plakoglobin was found to be a necessary ingredient for PV-IgG to cause retraction of keratin filaments in culture, serving possibly as a marker of early acantholysis [46]. A study of PV-IgG-treatment-induced phosphorylation of heat shock protein 27 in cells implicated the p38 mitogen-activated protein kinase (MAPK) signalling pathway by showing that inhibiting their pathway prevented cytoskeletal reorganization, associated presumably with loss of cell adhesion [47]. In additional studies, blocking various signalling pathways prevented PV IVIG-induced acantholysis in neonatal mice [48]. However, which, if any, of these signalling mechanisms is necessary or sufficient for acantholysis, their exact involvement in causing acantholysis, or whether they are activated as a result of acantholysis, remains to be determined.

In order to reduce anti-desmoglein 3 autoantibody synthesis, only agents that are known to suppress antibody production, alter antibody action, inhibit antibody binding to antigen or encourage antibody catabolism have a rational basis for therapeutic use in PV. However, only a limited number of drugs have this effect, and none is restricted to desmoglein autoantibodies. Several uncontrolled clinical studies [49,50] and a recent well-designed double-blind placebo-controlled study [26] have demonstrated the efficacy of IVIG in patients with moderate to severe pemphigus disease. The influence of IVIG was correlated strongly with the clinical status and the reduction of desmogleins 1 and 3 titres [51,52]. This treatment is limited, however, by the low cost-efficiency ratio of IgG and the extremely problematic worldwide shortage in plasma.

We speculated that the manipulation of the idiotypic network by anti-idiotypic antibodies contained in IVIG [13,14,53] may be the main mechanism of action of the drug in the treatment of pemphigus, and that owing to the relatively low amount of specific anti-idiotypic antibodies in commercial IVIG preparations, isolating pathogenic autoantibodies of PV might be more effective. Our premise was based on earlier studies by Blank et al. [54–56], which showed that this approach was very effective in an experimental model of anti-phospholipid syndrome and systemic lupus erythematosus. Other groups reported greater benefit for IVIG specific to anti-acetylcholin receptor than native IVIG in the treatment of rats with myasthenia gravis [57]. Moreover, our earlier work showed that F(ab)2 fragments were as efficient as the native antibodies in treating experimental PV, whereas Fc fragments were ineffective [27].

In the present study, we prepared polyclonal anti-desmogleins 1 and 3 anti-idiotypic antibodies by affinity-purifying commercial IVIG on a column constructed of scFv against desmogleins 1 and 3, and then tested the efficacy of this preparation in the most frequently used animal model of pemphigus. Our preparation was able to suppress the autoantibody response (no intercellular IgG deposition, no acantholysis) and the development of blisters and erosions using a 66-fold lower IgG dose than commercial IVIG. The same low dose of IVIG had no effect. Theoretically, the configuration of IVIG anti-idiotypic antibodies may resemble the structure of the antigen itself and induce the disease. We ruled out this hypothesis by showing that injection of PV-sIVIG did not induce the disease.

In summary, the use of concentrated polyclonal anti-idiotypic antibodies to anti-desmogleins 1 and 3 autoantibodies is more effective than native IVIG in inhibitory anti-desmoglein-induced PV in mice. Further studies are needed to determine if these findings can be applied to increase both the efficacy and efficiency of the treatment of PV in the clinical setting.

Acknowledgments

This work was supported by a grant from Tel Aviv University.

Disclosure

Nothing to disclose.

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