Immune diagnosis of pure ocular mucous membrane pemphigoid: indirect immunofluorescence versus immunoblot*

ARTICLE

Auteur(s) : Marcell F Jonkman¹, Anton C De Groot¹, Tanja PAM Slegers², Marcel CJM De Jong¹, Hendri H Pas¹

¹Center for Blistering Diseases, Department of Dermatology, University Medical Center Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands
²Dept of Ophthalmology (currently Scheper Ziekenhuis, Emmen) University Medical Center Groningen, University of Groningen, the Netherlands

accepté le 7 Mai 2009

Mucous membrane pemphigoid (MMP) describes a heterogeneous group of chronic, inflammatory, mucous membrane-dominant, subepithelial blistering diseases, that manifest a varying constellation of oral, ocular, skin, genital, nasopharyngeal, esophageal, and laryngeal lesions [1]. Life-threatening airway obstruction and sight-threatening ocular scarring can occur in the patients affected by this condition [2, 3]. In 60% of patients with MMP, the conjunctivae are involved in the process, and here we denote this subset ocular mucous membrane pemphigoid (OMMP) [4, 5]. MMP may also be limited to the eyes and is then called pure ocular mucous membrane pemphigoid (pure OMMP) [6, 7] (synonym: pure ocular cicatricial pemphigoid [8]). In a series of 74 patients with MMP with ocular involvement, 13 patients (18%) had such pure OMMP [9]. An early manifestation of OMMP may be the presentation of noninfective corneal epithelial damage [10].

MMP is considered to be an autoimmune disease. Several epithelial basement membrane zone (BMZ) components have been recognized by autoantibodies in the serum of MMP patients with ocular involvement, including BP230, BP180, LAD-1, laminin-332, type VII collagen, α6-integrin, and β4-integrin subunit [9, 11, 12]. In studies of MMP patients with pure OMMP, either no serum antibodies directed against BMZ antigens could be detected [13] or only with conjunctiva as a substrate for indirect immunofluorescence [14].

Diagnosing pure OMMP is based on the clinical picture of progressive cicatrisation of the ocular mucous membrane and immunohistopathological investigation of the conjunctiva. With direct immunofluorescence (DIF) or immunoperoxidase (DIP) analysis of a freeze biopsy of the conjunctiva, linear depositions of the immunoglobulins IgG, IgA, of C3 or combinations thereof can be demonstrated alongside the epithelial basal membrane zone in patients with OMMP. DIP analysis has been reported to be more sensitive than DIF investigation [15, 16]. Only 80% of patients with OMMP have a positive conjunctival biopsy [9]. In the case of a negative biopsy, the demonstration in the serum of auto-antibodies against BMZ antigens would be helpful in establishing the diagnosis [9].

The aims of this study were: 1) to investigate whether the DIP method is – indeed – more sensitive than DIF in demonstrating IgG and/or IgA depositions along the epithelial basement membrane zone in a freeze biopsy of the conjunctiva in patients with pure OMMP; 2) to analyse whether circulating antibodies against basement membrane auto-antigens can be detected in DIF positive and DIF negative patients and which method is the most sensitive for this.

Materials and methods

A conjunctival biopsy was taken from the most seriously affected eye after topical anaesthesia with one drop of 0.2% oxybuprocaine hydrochloride. Using a microsurgical pair of forceps and scissors a tissue sample of 3 × 2 mm was cut from the upper part of the conjunctiva bulbi near to the limbus. We did not take the sample from the inflammatory zone close to the fornix conjunctivae, as this may enhance the risk of symblepharon and also the risk of false-negative immunofluorescence results.

The conjunctival biopsy was stretched and pinned to Perspex, placed in an aluminium cup and frozen immediately in liquid nitrogen. Blood was sampled for serum indirect immunofluorescence (IIF) and for immunoblot investigation. DIF microscopy was performed on 4 μm frozen conjunctival sections with fluorescein-labelled goat antibodies (Protos Immunoresearch, Burlingame CA) directed against human IgG, IgA, IgM, C3c and fibrin. For the first step of IP microscopy of the conjunctivae we used mouse-IgG antihuman IgG and monoclonal mouse-IgG antihuman IgA₁. As second step, peroxidase-conjugated rabbit anti-mouse IgG was employed.

IIF microscopy of the serum samples (1:20) was performed on monkey oesophagus and on 1M NaCl-split human skin as substrates with fluorescein-labelled goat antibodies against human IgG and IgA (DAKO, Glostruk, Denmark) as previously described [17]. IB was performed with patient serum (1:300) and an extract of human cultured keratinocytes using polyclonal mouse anti-human IgG and goat anti-human IgA [18]. The incubation with serum was performed at 37 °C as this greatly enhances sensitivity for immunblot of anti-BP180 antibodies [18].

Results

Discussion

The diagnosis of pure OMMP is based on the demonstration of linear depositions of IgG, IgA, C3, or combinations thereof, alongside the epithelial BMZ with DIF or DIP analysis of a freeze biopsy of the conjunctiva. In this series of 11 patients with chronic cicatrizing keratoconjunctivitis as the initial sign, the diagnosis of pure OMMP was established with DIF in 5 (45%). Power et al. reported that the use of an immunoperoxidase technique increases the sensitivity of conjunctival biopsies in the diagnosis of OMMP [16]. These authors observed an increase in the percentage of biopsies positive for OMMP from 52% using the DIF technique alone to 83% with the addition of the DIP technique. In a recent study from the group in Baltimore [9], however, the sensitivity of a single conjunctival biopsy using standard DIF techniques (80%) was similar to the sensitivity for using both DIF and DIP techniques (83%) as reported by Power et al. [16]. In our study, DIP investigation of the conjunctival biopsies did not score better results than the DIF investigation and had no additional diagnostic value.

The DIF of a single conjunctival biopsy establishes the diagnosis in approximately 80% of patients with OMMP [9]. In case of a negative DIF, the demonstration of circulating auto-antibodies against BMZ antigens may suggest an autoimmune cause and thus be of diagnostic value in patients with OMMP or pure OMMP. In the 74 patients with ocular MMP reported by Thorne et al., circulating auto-antibodies against BMZ antigens were detected in 23 (31%). It was not mentioned how often these auto-antibodies were found in the 13 patients with pure OMMP [9].

Oyama et al. [11] investigated the sera of 124 patients with MMP, 77% of whom had ocular involvement (it was not stated how many patients had pure OMMP). As assessed by an IIF study on salt-split skin, 102 sera (82%) were positive for IgG antibodies to BMZ, whereas 77 (62%) were positive for IgA antibodies. Among these positive samples, 73 sera (59%) had a dual IgG and IgA antibody response. By immunoblot analysis using human epidermal, dermal and amniotic proteins, the authors characterized IgG and IgA autoantibodies in the 124 MMP patients. The majority of MMP patients had detectable levels of IgG (93 of 124, 75%) and/or IgA autoantibodies (63 of 124, 51%) to full-length BP180 or its soluble ectodomains, 120-kDa LAD-1 or 97-kDa LABD97 antigens. A substantial number of MMP sera contained IgG autoantibodies to other BMZ antigens; 34 (27%) reacted with BP230, 26 (21%) with β₄ integrin and three (2%) with laminin-332 (figure 2) [11].

The number of OMMP patients with circulating anti-laminin-332 may, however, be larger if ELISA is used for detection. Bekou et al., using a newly developed ELISA, demonstrated that the majority (75%) of MMP sera contained anti-laminin-332 antibodies (specificity 84.3%) [22]. On the other hand, an ELISA for anti-laminin-332 may raise false-positive results as demonstrated in 13% of bullous pemphigoid sera by Lazarova et al. [23].

In studies of MMP patients with lesions limited to the eye (pure OMMP) either no serum antibodies directed against BMZ antigens could be detected [13], or only with conjunctiva as a substrate for indirect immunofluorescence [14]. In our study, IIF of serum detected circulating antibodies in just one of five patients (20%) with pure OMMP and only with salt split skin as substrate. On the other hand, serum IB analysis proved to be positive for IgG and/or IgA antibodies against BMZ autoantigens in 4 of the 5 (80%) patients with pure OMMP. Thus, the immunoblot used in this study appears to be more sensitive than IIF analysis in these patients.

Auto-antibodies against β4-integrin, which may be associated with non-pure OMMP [11, 24-26], were not detected, although monoclonal anti-β₄ integrin demonstrated that our substrate (extract of cultured human keratinocytes) clearly contained this protein.

In patients with OMMP and pure OMMP, treatment with dapsone or immunosuppressants such as oral corticosteroids, cyclophosphamide and mycophenolate mofetil may control the disease and prevent further progression [20, 21]. As chronic use of such drugs may cause serious complications [19], especially in the typically elderly patient with OMMP, it is imperative that the correct diagnosis is made and ascertained. For this, DIF investigation of a conjunctival biopsy is the most reliable diagnostic method. When the DIF is negative, the demonstration of antibodies against BMZ antigens by means of serum analysis may be helpful in establishing the diagnosis of OMMP. Our results suggest that for this, the use of the immunoblot is advisable, as it appears to be more sensitive than the IIF, at least in patients with DIF proven pure OMMP patients. Whether this is also the case in DIF negative patients cannot be deducted from this study and should further be investigated in patients with cicatrizing conjunctivitis with a negative DIF.

The number of patients in this study is small and the investigation was partly retrospective. Therefore, the results should be interpreted with some caution.

Acknowledgements

References

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* Most of the data presented have previously been reported in: Jonkman MF, Slegers T, Navadeh E, de Jong MCJM, Pas HH, van Rij G. Immunodiagnostiek van oculair slijnmvliespemfigoïd. Ned Tijdschr Derm Venereol 2004; 14: 398-402. Permission for publication here was obtained from the publisher and editor.