Epidermolysis bullosa acquisita (EBA) is a chronic mucocutaneous autoimmune skin blistering disease. The autoimmune response is directed towards type VII collagen (COL7), which is an integral part of the dermal-epidermal junction. Mechanisms leading to blister formation are relatively well characterized. After binding of specific isotypes and specifically glycolysated anti-COL7 antibodies to COL7, Fc-dependent mechanisms induce a pro-inflammatory milieu. Formation of this milieu is partly, but not completely dependent on complement activation. We have gathered evidence for a contribution of cytokines in modulating this pro-inflammatory milieu. In detail, functional characterization of cytokines in experimental EBA showed, that (i) MIP-1a does not contribute to disease manifestation, (ii) inhibition of TNF-a has minimal effects, (iii) IL-1 and (iv) GM-CSF are required, and (v) IL-6 has profound protective effects. Overall, this leads to the CD18-dependent migration of neutrophils. Engagement of neutrophils with autoantibodies is mediated by specific Fc gamma receptors. Subsequently, neutrophils release reactive oxygen species and proteolytic enzymes, which induce blister formation. Apparently unrelated work demonstrated, that binding of autoantibodies, i.e. anti-BP180 antibodies to keratinocytes induce release of IL-6 and IL-8. Given, if cytokine release can also be triggered by incubation of keratinocytes with anti-COL7 IgG, these keratinocyte-derived cytokines could mediate generation of the pro-inflammatory milieu in EBA. To test this, we incubated HaCaT cells with anti-COL7 antibodies. In preliminary experiments, incubation of HaCaT cells with anti-COL7 antibodies induced release of several cytokines. Supernatants from anti-COL7 antibody treated HaCaT cells induced neutrophil migration. Incubation with control IgG had no such effects. As release of several of the identified cytokines is controlled by NF-kB activation, we next tested, if impaired NF-kB signaling in the epidermis has an impact on EBA induction by transfer of anti-COL7 IgG into mice. For this purpose, we initiated a cooperation with Prof. Ingo Haase, who generated mice with a targeted deletion of RelA from epidermal keratinocytes (RelAepi). In a pilot experiment, induction of experimental EBA by transfer of anti-COL7 IgG, RelAepi mice developed a significantly milder EBA phenotype compared to wild type controls. Based on these observations, the project will challenge the following hypothesis: (i) Anti-COL7 IgG induces NF-kB-dependent, and functionally relevant cytokine release from keratinocytes, (ii) Inhibition of keratinocyte NF-kB activation impairs induction of experimental EBA, and (iii) Blockade of NF-kB activation has therapeutic effects in EBA. This will lead to a better understanding of the pathogenesis of autoantibody-induced tissue damage in a prototypical, organ specific autoimmune disease, focusing on the cells, which are targeted by the autoimmune response.