Epitopes Targeted by Bullous Pemphigoid T Lymphocytes and Autoantibodies Map to the Same Sites on the Bullous Pemphigoid 180 Ectodomain

Bullous pemphigoid is a blistering skin disease characterized by autoantibodies directed against the NC16A domain of bullous pemphigoid 180 (collagen XVII), a transmembrane protein of epidermal basal cells. Passive transfer studies in mice have shown that antibodies that bind to this immunodominant region of bullous pemphigoid 180 are capable of inducing a skin disease that closely mimics bullous pemphigoid, supporting the hypothesis that epitopes within NC16A are involved in the pathogenesis of bullous pemphigoid. In this study, we examined the autoimmune T cell response in bullous pemphigoid patients. T cells from eight of 12 bullous pemphigoid patients, all of whom had circulating anti-bullous pemphigoid 180 autoantibodies, showed a specific proliferative response to recombinant forms of NC16A. T cell lines and clones developed from four of these patients recognize the same NC16A peptides as those targeted by autoantibodies from the corresponding individuals. These NC16A-responding T lymphocytes express alpha/beta T cell receptors and CD4 memory T cell surface markers and exhibited a Th1/Th2 mixed cytokine profile that may support the production of antibodies. This new information will aid in defining the key steps involved in the development of the autoimmune response in bullous pemphigoid

Tight Clustering of Extracellular BP180 Epitopes Recognized by Bullous Pemphigoid Autoantibodies

Bullous pemphigoid is a blistering skin disease associated with autoantibodies against the BP180 antigen, a transmembrane component of the hemidesmosome. Anti-BP180 antibodies have been demonstrated to be pathogenic in a passive transfer mouse model. One extracellular site on human BP180 (MCW-1) was previously shown to be recognized by 50–60% of bullous pemphigoid sera. To facilitate the identification of additional autoantibody-reactive epitopes, recombinant forms of the BP180 ectodomain were generated using both bacterial and mammalian expression systems. One recombinant protein, sec180e, that was expressed in COS-1 cells and that contained the entire BP180 ectodomain, provided us with a tool to detect conformational epitopes. Bullous pemphigoid sera immuno-adsorbed against the major noncollagenous NC16A domain no longer reacted with sec180e, indicating that autoantibody reactivity to the BP180 ectodomain is restricted to the NC16A region. Immunoblot analysis of bullous pemphigoid sera immunoadsorbed with a series of recombinant NC16A peptides revealed the presence of three novel autoantigenic sites that, along with the MCW-1 epitope, are clustered within the N-terminal 45 amino acid stretch of NC16A. All 15 bullous pemphigoid sera tested reacted with a recombinant protein containing this BP180 segment. No disease-associated epitopes were detectable within the remaining 28 amino acids of NC16A. Thus, bullous pemphigoid patient autoantibodies react with a set of epitopes on the BP180 ectodomain that are highly clustered. This autoantibody-reactive region on human BP180 shows overlap with the corresponding murine BP180 site that is targeted by antibodies that are pathogenic in the mouse model of bullous pemphigoid. These findings suggest new directions for the development of diagnostic and therapeutic tools for this disease

Two Sets of Interacting Collagens Form Functionally Distinct Substructures within a Caenorhabditis elegans Extracellular Matrix

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of ∼154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed ∼2 h apart during matrix synthesis

The autoantigen in anti-p200 pemphigoid is synthesized by keratinocytes and fibroblasts and is distinct from nidogen-2

Anti-p200 pemphigoid is a subepidermal immunobullous disorder associated with tissue-bound and circulating autoantibodies reactive with a 200 kDa protein on the dermal side of salt-split-skin. The autoantigen, named p200, is a non-collagenous glycoprotein located at the lamina lucida-lamina densa border of the epidermal basement membrane. However, its identity and cellular origin remain elusive. Here, we used biochemical and genetic approaches to characterize the autoantibody reactivity in three new patients with anti-p200 pemphigoid. We show that the target antigen p200 is synthesized by both keratinocytes and fibroblasts, is disulfide-bonded, and participates in calcium-dependent molecular interactions. Lack of collagen XVII (BP 180), collagen VII, or laminin 332 (laminin 5) from the dermal-epidermal junction does not destabilize p200. Colocalization within the basement membrane zone and an identical molecular weight suggested nidogen-2 as candidate autoantigen in anti-p200 pemphigoid, but biochemical analysis demonstrated that p200 is distinct from nidogen-2. In conclusion, the results define further the biochemical characteristics of p200 and demonstrate its in vitro-synthesis by keratinocytes and fibroblasts, thus providing a basis for identification and further characterization of this autoantigen