NORMAL HUMAN EPIDERMAL-KERATINOCYTES EXPRESS IN-VITRO SPECIFIC MOLECULAR-FORMS OF (PRO)FILAGGRIN RECOGNIZED BY RHEUMATOID ARTHRITIS-ASSOCIATED ANTIFILAGGRIN AUTOANTIBODIES

Background: The so-called antikeratin antibodies and the antiperinucle ar factor are the most specific serological markers of rheumatoid arth ritis (RA). They were recently shown to be largely the same autoantibo dies and to recognize human epidermal filaggrins and profilaggrin-rela ted proteins of buccal epithelial cells (collectively referred to as ( pro)filaggrin). Materials and Methods: To further characterize the tar get antigens, we investigated their expression by normal human epiderm al keratinocytes cultured in differentiating conditions, using immunof luorescence and immunoblotting with RA sera and three different monocl onal antibodies to (pro)filaggrin. Results: On the cornified, stratifi ed epithelial sheets obtained in vitro, RA sera with anti(pro)filaggri n autoantibodies (AFA) produced granular staining of the stratum granu losum and diffuse staining of the stratum corneum. The antigens recogn ized by RA sera strictly colocalized with (pro)filaggrin in keratohyal in granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, inc luding a neutral/acidic isoform of filaggrin and several proteins with sizes and pi intermediates between this isoform and profilaggrin. The y also recognized urea-soluble high-molecular-weight profilaggrin-rela ted molecules. Conclusions: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro)filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are a bsent from epidermis. Moreover, these epitopes, which are present on t he keratohyalin granules of buccal epithelial cells but not on those o f epidermal cells, are present on the granules of the cultured keratin ocytes. This work completes the molecular characterization of the prot eins targeted by AFA.