Localisation of bullous pemphigoid antigen 180 (BP180) in cultured human keratinocytes: functionally relevant...

The expression of BP180 has previously been demonstrated to be influenced by both calcium (Ca(2+)) concentration and binding of anti-BP180-antibodies in cultured keratinocytes of the skin squamous cell carcinoma line DJM-1. Here, BP180 expression was studied in cultured normal human epidermal keratinocytes by confocal laser scanning microscopy. We exploited an experimental system, in which BP180 was previously shown to mediate, upon incubation with anti-BP180 antibodies, a specific signal-transducing event that leads to the release of inflammatory mediators, such as IL-8 from cultured normal human epidermal keratinocytes (NHEK). We found that without addition of BP180-specific IgG, BP180 is predominantly expressed on the cell surface irrespective of the Ca(2+) concentration. In contrast, cell surface BP180 was greatly reduced in NHEK kept in high Ca(2+) medium after incubation with BP180-specific IgG for 12 h compared to low Ca(2+) medium. This effect was seen with antibodies to both N- and C-terminal fragments of the BP180 ectodomain, respectively. In addition, a slightly higher BP180 expression was found in NHEK cultured in low compared to high Ca(2+) medium by Western blotting. Interestingly, in contrast to NHEK kept under low Ca(2+ )conditions, in NHEK grown in high Ca(2+) medium, no elevated levels of IL-8 were released after treatment of cells with anti-BP180 IgG compared to normal IgG. Our data indicate that the Ca(2+)-modulated expression of BP180 is functionally relevant. This finding sheds further light on the complex pathomechanism in blister formation of BP180-related autoimmune blistering skin diseases.