Introduction

Mucous membrane pemphigoid (MMP) is a rare and chronic autoimmune blistering disease that involves multiple mucous membranes and the skin (Fleming and Korman, 2000). MMP heals with scarring, resulting in significant sequelae. Blindness occurs in approximately 25% of the patients, despite aggressive immunosuppressive therapy (Fleming and Korman, 2000). Involvement of the larynx can cause sudden asphyxiation and death (Fleming and Korman, 2000). Scarring in the vaginal, anal, and penile areas compromises the quality of life, and daily living (Fleming and Korman, 2000).

MMP is characterized by the presence of antibodies to proteins in the basement membrane zone of epithelial tissues. A subset with antibodies to laminin 5 is referred to as anti-epiligrin cicatricial pemphigoid (AECP) (Domloge-Hultsch et al., 1992). Some patients may have disease limited to the conjunctiva or may have disease that predominantly affects the conjunctiva, in addition to involvement of other mucosae. They are referred to as having ocular cicatricial pemphigoid (OCP) (Foster, 1986). Patients with OCP and generalized MMP have been found to have antibodies to a portion of the intracytoplasmic component of human 4 integrin (Chan et al., 1999). It has been demonstrated that the titer of these antibodies to 4 integrin correlate with disease activity and decrease as the disease improves, and becomes negative, when the patients go into remission (Letko et al., 2000). In another subset of MMP, the disease is limited to the oral cavity only. Long-term follow-up reveals that other mucosae or the skin are not involved. Such patients are referred to as having oral pemphigoid (OP) (Mobini et al., 1998). Patients with OP produce an antibody to human 6 integrin subunit (Bhol et al., 2001). Long-term follow-up studies indicate that the extent and severity of the oral disease correlates with the titer of antibody and as patients improve and go into a clinical remission, the antibody decreases and eventually disappears (Sami et al., 2002).

Some authors have reported that sera of MMP patients have antibodies to bullous pemphigoid (BP) antigens. However, on long-term follow-up, it was observed that antibodies to bullous pemphigoid antigen 1 (BPAg1) and bullous pemphigoid antigen 2 (BPAg2) do not correlate with disease activity or severity and their presence is erratic and unpredictable (Yeh et al., 2004). This would suggest that, although occasionally present, they are probably not pathogenic and could be produced in response to damage to basal epithelial cells. Hence, we did not include them in this study.

The purpose of this study was to determine if patients with generalized MMP or OCP have antibodies to 4 integrin subunit only or to 6 integrin subunit also. In addition, the purpose of the study was to determine if the patients with OP have antibodies only to human integrin 6 or antibodies to human integrin 4 subunit also.

Results

Immunoblot analysis of MMP and OP Sera

Sera from all 15 patients with MMP and OCP showed binding with a 205 kDa protein (Figure 1). The binding of the sera was identical to that of UMA9 antibody. Thus, MMP and OCP sera identified 4 integrin subunit as the target molecule. Binding of MMP and OCP sera to any other protein was not observed. Binding to 6 integrin was not detected in any of the 15 MMP sera.

Figure 1.

IB analysis of binding of MMP and OCP sera using BGL as substrate. Lanes 1–10 show the binding of sera of five MMP and five OCP patients to BGL absorbed with NHS and BP sera. Note the presence of binding to 205 kDa protein. Lane 11 demonstrates binding of UMA9 mAb to 250 kDa protein in BGL. Disease control includes binding of PV sera to desmoglein 3 observed as 130 kDa protein, and lanes 13–15 are three NHS, showing no binding.

Full figure and legend (31K)

Sera of 15 patients with OP demonstrated binding to a 120 kDa protein (Figure 2). This binding was identical to binding by GoH3 antibody. Hence, the sera from 15 patients with OP recognized 6 integrin subunit. Binding to any other protein was not observed. Binding to 4 integrin subunit was not observed.

Figure 2.

IB assay using BGL as substrate, demonstrating the binding of sera from patients with OP. Lanes 1–10 show the binding of 10 sera from patients with active OP. Note binding to 120 kDa protein. Lane 11 demonstrates the binding of mAb GoH3 to BGL absorbed with NHS and BP sera. Note binding to 120 kDa protein as 6 integrin subunit. Disease control includes binding of PV sera to desmoglein 3 observed as 130 kDa protein. Lanes 13–15 are three NHS, showing no binding.

Full figure and legend (21K)

The binding pattern of sera of OP and MMP patients to bovine gingivae lysate (BGL) absorbed with only normal human serum (NHS) and BP sera were identical. MMP, OP, and all sera did not bind to laminin, whereas GB3 did.

Bullous pemphigoid

Sera from all 15 patients, including BP patients, when reacted with BGL which had been absorbed with only NHS, demonstrated binding to a 230 kDa protein (BPAg1). Binding to a 180 kDa protein (BPAg2) was observed in 12 of the 15 BP sera (80%) (Figure 3).

Figure 3.

IB assay using BGL as substrate demonstrates binding of the sera from patients with BP. Lanes 1–10 show the binding of sera of untreated BP patients to BGL absorbed with NHS. Note the binding to BPAg1 (230 kDa) protein and BPAg2 (180 kDA) protein. In lane 11, PV serum bound to desmoglein 3 and in lane 12 PF serum bound to 160 kDa protein. Lanes 13–15 are three NHS reacted with BGL, showing no binding.

Full figure and legend (38K)

Controls

Sera from patients with pemphigus vulgaris bound to a 130 kDa (desmoglein 3) protein and a 160 kDa (desmoglein 1) protein, with pemphigus foliaceus bound to 160 kDa protein (desmoglein 1), and with AECP bound to a 165, 150, and 140 kDa protein (three subunits of laminin 5). The 15 NHS did not show any binding to any proteins.

Indirect immunofluorescence studies

The results of the indirect immunofluorescence, using salt-split normal human skin as substrate, are presented in Table 1. In all patients, the antibodies bound to the epidermal side of salt-split skin, tested at a titer of 1:10. Binding to the dermal side was not observed. The frequency of the Ig class was different in the three groups. OCP had a higher frequency of IgA antibodies. Non-ocular MMP and OP had predominantly IgG antibodies. Control BP sera bound to epidermal side and sera of an epidermolysis bullosa acquisita patient bound to the dermal side of split. NHS did not show any binding.

Table 1 - Indirect immunofluorescence assay using 1 M NaCl normal human split skin as substrate.

Full table

Antigen-binding specificity of subsets

When the lysates were absorbed with NHS and BP sera, it first reacted with (i) anti-4 antibody (UMA9) and (ii) MMP. Then, it reacted with OP sera and GoH3 mAbs. Binding to a 120 kDa protein, identified as 6 integrin, was observed (Figure 4).

Figure 4.

Blocking experiments using BGL to demonstrate the binding specificity of sera from patients with OP and mucous membrane or OCP. In left panel A, lane 1 demonstrates the binding of MMP sera and in lane 2 binding of UMA9 mAb to BGL absorbed with NHS and BP sera. Note 205 kDa protein identified as human 4 integrin subunit. In lanes 3 and 4, the BGL is first absorbed with MMP sera and UMA9 mAb, then reacted with OP sera in lane 3 and mAb GoH3 in lane 4. Note 120 kDa protein, identified as 6 integrin. In right panel B, in lane 5, BGL absorbed NHS and BP sera reacted with OP sera and in lane 6 reacted with mAb GoH3. Note binding to a 120 kDa protein. In lanes 7 and 8, BGL absorbed with OP sera and GoH3 reacted with MMP sera in lane 7, and UMA9 mAb in lane 8. Note binding to a 205 kDa protein, identified as human 4 integrin subunit.

Full figure and legend (45K)

When the lysates absorbed with NHS and BP, sera were reacted first with (i) mAb to human 6 integrin (GoH3) and (ii) OP sera. Then, they were reacted with MMP and OCP sera, and UMA9. Binding to a 205 kDa protein, identified as 4 integrin, was observed (Figure 4).

Blocking and cross-absorption studies

When the BGL was first absorbed with MMP/OCP sera and then reacted with UMA9 antibodies, MMP/OCP, OP sera, and GoH3 antibodies, the following was observed. A 205 kDa protein was not identified, indicating that the 4 protein had bound to MMP/OCP sera, and 4 integrin subunit was blocked and therefore not available to UMA9 antibodies. In the reverse experiment, the BGL strips were first absorbed with UMA9 antibodies. And then reacted with MMP and OCP sera. A 205 kDa protein binding was not observed. These experiments demonstrate that MMP/OCP sera both bind to 4 integrin subunit and block reactivity by cross-absorption. However, the strips reacted with GoH3 and OP antibodies, and this was demonstrated by binding to a 120 kDa protein. This indicates that while 4 integrin was blocked, 6 integrin is still available for binding to OP sera and GoH3 antibodies.

When the BGL is first absorbed with OP sera and then reacted to GoH3 antibodies, no binding to a 120 kDa band is observed, thus indicating that OP sera bound to the 120 kDa protein, which is 6 integrin. In the reverse experiment, when the BGL was first absorbed with GoH3 antibodies, and then reacted with OP sera, no binding to a 120 kDa protein was seen. These experiments demonstrated that OP sera and GoH3 can bind to 6 integrin and block reactivity by cross-absorption in the same substrate. However, in both experiments, binding to a 205 kDa protein was seen on strips reacted with MMP/OCP sera and UMA9 antibody. This indicates that 4 integrin is unaffected by these cross-absorption studies and is available for binding to MMP/OCP sera and UMA9 antibody. Positive controls demonstrated BP sera binding to 180 kDa protein (BPAg2) and 230 kDa protein (BPAg1). No binding was seen with NHS.

Longitudinal immunoblot analysis of sera from subsets of MMP

The immunoblot (IB) analysis of sera from MMP and OCP patients evaluated at 4-month intervals for 4 years demonstrated that their sera contained antibodies to only 4 integrin subunit. Antibodies to 6 integrin were never detected. A representative IB analysis of one MMP and one OCP patient is presented in Figure 5. The IB analysis of sera from OP patients evaluated at 4 months interval, for a 4-year period demonstrated binding to 6 integrin only. Binding to 4 integrin subunit was not observed. A representative patients' sera analysis is present in Figure 5. Once the binding was no longer detected, the patient was considered to be in a serological remission. Hence, data on time intervals beyond the induction of such a remission, although performed, are not presented in the figures. The titer of the antibody in the sera decreases with time, because of systemic therapy and eventually disappears.

Figure 5.

Longitudinal IB analysis of sera from subsets of MMP. (a) Sera from a representative patient with MMP was followed for 36 months. The IB analysis of sera from the patient was evaluated at 4-month intervals. Binding to a 205 kDa protein (4 integrin) was observed. The titer of the antibody at each time interval has been provided. The titer of the antibody in the sera decreases with time, becoming undetectable at the 28th month after the initiation of therapy. UMA9 antibody and NHS were used as controls. (b) Sera from a representative patient with OCP was followed for 36 months. The IB analysis of sera from the patient was evaluated at 4-month intervals. Binding to a 205 kDa protein (4 integrin) was observed. The titer of the antibody at each time interval has been provided. The titer of the antibody in the sera decreases with time, becoming undetectable at the 32nd month after the initiation of therapy. UMA9 antibody and NHS were used as controls. (c) Sera from a representative patient with OP was followed for 36 months. The IB analysis of sera from the patient was evaluated at 4-month intervals. Binding to a 120 kDa protein (6 integrin) was observed. The titer of the antibody at each time interval has been provided. The titer of the antibody in the sera decreases with time becoming undetectable at the 28th month after the initiation of therapy. GoH3 antibody and NHS were used as controls.

Full figure and legend (73K)

Discussion

Using an IB assay, we demonstrate that sera obtained from 15 untreated patients with mucous membrane or ocular pemphigoid bound only to 4 integrin subunit. They do not contain antibodies to the 6 integrin or to laminin. Sera of untreated patients with OP had antibodies that recognized only 6 integrin and did not bind to the human 4 integrin subunit or laminin. The specificity of this binding was further confirmed in the blocking and cross-absorption studies. The observations in the study clearly indicate that the two components 6 and 4 of integrin heterodimer, while in close proximity, act as autoantigens independently. Long-term follow-up of minimum of 4 years also demonstrates that during the clinical course patients with MMP and OCP produce autoantibodies to only human 4 integrin subunit, whereas patients with OP produce autoantibodies to only human 6 integrin subunit. Switching or crossover in autoantibody profile was not observed in the patients in this report. The importance of these observations lie in the fact that the clinical subsets of patients with MMP can be classified on the basis of their antibody profiles determined by their specific binding to the target antigens.

The features on indirect immunofluorescence studies using salt-split normal human skin are identical to those observed by other investigators (Bagan et al., 2005). The presence of antibodies to BPAg1 and BPAg2 in the sera of patients with MMP have been demonstrated to be transitory, does not correlate to disease activity or severity, and may be a secondary phenomenon, with no defined or specific role in the pathogenesis of the disease process (Yeh et al., 2004). The subset of patients referred to as having AECP produce antibodies to laminin 5, have clinical profile completely indistinguishable from patients with MMP, with antibodies to 4. Based on the available data, it appears that the sera of patients with antibodies to epiligrin or laminin 5 do not recognize antibodies to human 4 or 6 integrin subunits.

This division of subsets of MMP, based on serologies, has implications on therapy. Patients with MMP and OCP often require systemic immunosuppressive therapy (Miserocchi et al., 2002). Patients with OP often respond topical therapy and intralesional corticosteroid injection (Mobini et al., 1998). Patients with AECP have a poor prognosis and do not respond very favorably to conventional immunosuppressive therapy (Egan et al., 2003).

The benefit of identifying subsets of MMP is of importance because it correlates with differences in prognosis. Of patients with antibodies to laminin, those who have AECP have a statistically high risk of developing solid tumors (Egan et al., 2001). In sharp contrast, patients who have antibodies to either 6 or 4 do not have a high incidence of malignancy. On the contrary, they are observed to have a lower than normal or expected frequency of incidence of cancer (Letko et al., 2004). As laminin may be present in the periphery of the tumor, antibodies to it could play a role in the breakdown of the capsule or other barriers surrounding the tumor and may facilitate tumor growth as well as metastasis. The epitope to which the antibodies in AECP bind are present in the , , and domains of laminin 5 (Lazarova et al., 2001). The epitope in the human 4 integrin subunit to which MMP and OCP antibodies bind is present in the cytoplasm of the basal epithelial cells (Kumari et al., 2001). The epitope to which the antibodies bind in patients with OP is in the extracellular domain of 6 integrin subunit (Rashid et al., 2006). The role of the autoantibodies to the 4 and 6 integrin subunits, if any, in preventing or protecting from malignancy, has not been described or studied. It is possible that as the three target antigens have different locations, the binding of the antibody to the antigen could generate different signals, which could affect or influence different cellular and extracellular pathways.

Patients with MMP, OCP, and OP who are non-responsive to systemic corticosteroids and immunosuppressive agents have been treated with intravenous Ig (Ahmed and Dhal, 2003). A defined protocol has been used. The patients that have demonstrated good clinical response have gone into remission and remained in remission when followed over a prolonged period of time. This indicates that the autoantibodies to 6 and 4 can be influenced by intravenous Ig therapy and their production can be possibly eliminated (Letko et al., 2000; Sami et al., 2002). It is possible that the intravenous Ig could influence the regulation of autoantibody production through the idiotypic–anti-idiotypic network (Lacroix-Desmazes et al., 2002).

This study highlights the importance of identifying the clinical subsets of MMP. Once the diagnosis of MMP is made, the antigen specificity of the antibody should be studied. The antibody profile of the patient could have a significant influence on the prognosis and clinical outcome. This unique experiment in nature provides us a spectrum of clinical profiles that correlates with autoantibody profiles, without overlaps or crossovers. The association with malignancies provides an opportunity to study the influence and effect of autoantibodies to adhesion molecules on malignancy. This will enhance our knowledge and understanding of the pathogenesis of these diseases, the biology of the epithelium, its reaction to the underlying submucosal, and the role of the basement membrane in both adherence and communications.

Materials and Methods

Patients

Fifteen patients with MMP, with involvement of at least two mucosal surfaces, with or without the skin, were included in the study. In the non-ocular MMP patients, the clinical profile consisted of erosive lesions in the oral cavity, noticeably desquamative gingivitis. Scarring in the nasal, laryngeal, and esophageal mucosa was observed. Eight patients had predominantly ocular involvement in addition to involvement of other mucosae. The patients were most concerned about the ocular involvement, because of conjunctival scarring. These patients are referred to as OCP. Whereas the clinical diagnosis of MMP was based on clinical presentation, it was established by a subepithelial vesicle with a mixed infiltrate on routine histology in all patients. The diagnosis was confirmed by the deposition of IgG and/or complement at the basement membrane zone on direct immunofluorescence of at least one mucosal perilesional biopsy. All of the patients were tested for antibody binding on salt-split skin, as a substrate. All 15 sera bound to the epidermal side of the salt-split skin. All patients with OCP had IgA antibodies, whereas only two of the non-ocular MMP and one of the OP patients had IgA antibodies.

Fifteen patients with OP studied. The disease was limited to the oral cavity only. During a long-term follow-up, involvement of other mucosal sites was not observed. The gingiva, palate, and buccal mucosa were the most frequently involved sites. The histology, immunopathology, and serological features of OP were similar to MMP patients.

The patients with MMP, OCP, and OP were clinically followed for a minimum of 4 years. All the sera on the patients were obtained before systemic treatment was initiated. Subsequently, the sera were tested at every 4-month intervals, using IB assay. None of the patients had developed a malignancy during this follow-up period or had a malignancy before developing pemphigoid. The MMP, OCP, and OP patients reported in this study have not been reported in any earlier study.

Fifteen sera from patients with BP were also studied. The diagnosis in these patients was based on the presence of large tense bullae in an acral distribution. The histology, immunopathology, and serological features of BP were similar to MMP patients. None of the patients with BP had any mucosal involvement. The controls included sera of two patients with pemphigus vulgaris, two patients with pemphigus foliaceus, one patient with AECP, and 15 normal healthy human volunteers. Positive controls included UMA9 mAb to 4 integrin subunit (Ancell Corp., Bayport, MN), and GoH3 mAb to integrin 6 (R&D systems, Minneapolis, MN). The study was conducted according to the Declaration of Helsinki Principles. It was approved by the Institution Review Board. The patients provided their informed consent to participate in the study.

Indirect immunofluorescence studies

As monkey esophagus usually is not an ideal substrate to test MMP/OCP sera, the sera were tested on normal human salt-split skin. The sera were tested at a dilution of 1:10.

IB analysis

BGL was used as substrate. Recently, we have demonstrated that the information obtained by using BGL is similar to that obtained by using normal human epidermis or whole skin, as lysate in IB analysis, when identifying antigens in autoimmune mucocutaneous blistering diseases (Engineer et al., 2000). Bovine jaws were obtained from a local slaughterhouse. The entire mucosa was scraped off the jaw. The tissue so obtained was treated with tissue homogenizer. Then the lysate was prepared by a protocol similar to that for human skin or bovine tongue. The details have been described previously (Bhol et al., 2001).

A unique feature of this IB assay included absorption with the test lysate with NHS and sera from patients with BP. This additional step was as follows. Protein A sepharose beads were washed and equilibrated in 50 mM phosphate buffer (pH 8.0). After equilibration, the beads were suspended in NHS and mixed overnight at 4°C by rotation. The unbound serum proteins were washed by 50 mM phosphate buffer (pH 8.0), 100 mM NaCl, and 0.05% Tween. The beads were then packed into a column. Total protein concentration of the BGL sample loaded onto the column was 10 mg. This BGL preparation was passed through the column with the protein A sepharose beads coated with NHS. Thus, proteins present in BGL that were recognized by NHS were absorbed and eliminated.

The process was repeated twice to ensure removal of as many proteins from BGL that could be recognized by NHS. Half of this lysate was used as such for IB analysis in studies in this report. Half of the remaining lysate was depleted of antigens associated with BP by the following technique.

Protein A sepharose columns bound with serum from patients with BP were prepared as described above. The BGL proteins eluted from protein A sepharose column bound to NHS were then passed through this protein A sepharose column, which was immobilized with serum from BP patient, as described above. Using indirect immunofluorescence assay, it was determined that these sera had high titers of anti-basement membrane zone antibodies. By IB, it was confirmed that the anti-basement membrane zone antibodies were against both BPAg1 and BPAg2. This additional step facilitated the removal of BP antigens from the BGL to be used in the IB analysis. Thus, two batches of the BGL were finally prepared for the study. One in which the BP antigens were present, and the other in which the BP antigens had been removed.

The BGL was run on SDS-PAGE, and the proteins transferred to a nitrocellulose membrane as described earlier (Bhol et al., 2001).

Sera from the 15 patients with MMP and 15 patients with OP were reacted with BGL, absorbed with both normal human sera and sera of BP patients. The sera from 15 patients with BP, 15 normal, and four pemphigus patients were reacted with BGL, which had been absorbed only with the normal human sera.

Antigen-binding specificity of subsets

BGL was first absorbed with MMP and OCP sera. The control BGL strip was reacted with MMP and OCP sera, and UMA9 mAb only. Then, the same nitrocellulose strips were reacted with sera from OP patients and GoH3 antibodies.

BGL was first absorbed with OP sera and then GoH3 antibodies. The same nitrocellulose strips were reacted with sera from MMP, OCP patients and antibodies to 4 integrin (UMA9). Control strip of BGL was reacted with BP sera or GoH3 mAb only.

Blocking and cross-absorption studies

In the reverse experiment, the BGL strips were first reacted with UMA9 (mAb to 4 integrin) and then reacted with MMP and OCP sera, which were known to have high titers of antibodies to 4 integrin. The control BGL strip was reacted with UMA9 antibodies, MMP/OCP sera, OP sera, and GoH3 antibodies.

In the reverse experiment, the BGL was first absorbed with GoH3. These strips were then absorbed with OP sera (with known high titers to 6 integrin), MMP/OCP sera, and UMA9 antibodies.

Positive controls in both sets of experiments were two sera from patients with acute untreated BP, which were reacted with BGL not absorbed with BP sera. Negative control was NHS.

Longitudinal IB analysis of sera from subsets of MMP

Using the above-described IB assay, sera collected at 4-month intervals were evaluated for the presence of antibodies to 4 integrin subunit and 6 integrin. In the 30 patients in this study, UMA9 and GoH3 antibodies were used as controls.

Conflict of Interest

The authors state no conflict of interest.

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