Identification of ocular cicatricial pemphigoid antibody binding site(s

PURPOSE. To identify specific site(s) on human ␤4 molecule to which sera from ocular cicatricial pemphigoid (OCP) patients bind and to determine its role in the process of blister formation. METHODS. Clone the fragments representing the extracellular and intracellular domain of ␤4 molecule from normal human conjunctival mRNA into an expression vector; map the region to which sera from OCP patients bind by Western blot analysis. Determine the role of the immunodominant region in pathogenesis by demonstrating the ability of the rabbit antibody to the immunodominant region to produce separation of basement membrane zone (BMZ) from the basal epithelial layer when incubated with normal human conjunctiva in an in vitro organ culture model. RESULTS. Majority of the OCP sera tested bound to the Cterminal end of the intracellular domain (IC3.0) of the human ␤4 integrin. Further subcloning of IC3.0 demonstrated that a smaller fragment extending from 1489 aa to 1572 aa (IC3.4) was responsible for this binding. This region may have multiple antibody binding sites. Antibody to human IC3.0 and IC3.4 produced in rabbit, resulted in BMZ separation, histologically identical with that observed when normal human conjunctiva was cultured with OCP sera in an human conjunctival organ culture model. CONCLUSIONS. These observations identify IC3.4 as the antibody binding site for sera of OCP patients and suggest a possible role for it in blister formation. Indirectly it highlights certain important aspects of the structural and functional dynamics of the biology of the hemidesmosomes and basement membranes. (Invest Ophthalmol Vis Sci. 2001;42:379 -385) M ucous membrane pemphigoid (MMP) is a multisystemic autoimmune inflammatory disease that affects mucous membrane derived from stratified squamous epithelium including the conjunctiva and occasionally the skin. 1,2 In some patients, the involvement of the conjunctiva is more prominent than other mucous membranes. Such a subset of MMP patients are referred to as having ocular cicatricial pemphigoid (OCP). Progressive subepithelial fibrosis follows the chronic conjunctivitis, resulting in severe dryness of the eye, ocular keratinization, and blindness secondary to corneal scarring. Sera of patients with subepithelial blistering diseases have demonstrable levels of circulating antibodies that bind to different antigens within the basement membrane zone (BMZ) of skin and mucosa. 10 There are several human autoimmune diseases in which the target autoantigens are identified as being intracellular in location. MATERIALS AND METHODS Sera The method section confirms adherence to the Declaration of Helsinki. Sera used in this study were obtained from 20 patients with active mucous membrane pemphigoid before beginning of systemic treatment. These patients had the pemphigoid disease process involving multiple mucosa but not the skin. Ocular involvement was the most prominent symptom, resulting in blindness in many of these patients. The clinical diagnosis of OCP was established by routine histology and confirmed by direct immunofluorescence of the conjunctiva. The presence of IgG and or complement was detected in the conjunctival BMZ. Sera of these OCP patients binds to the epidermal side of the salt split skin. Control sera were obtained from 20 healthy individuals, 5 patients each with confirmed bullous pemphigoid (BP) and pemphigus vulgaris (PV). Blood samples were collected after informed consent, and the study was approved by the institutional review board