Keratinocytes as modulators of autoantibody-induced tissue injury

获得性大疱性表皮病是一种慢性皮肤黏膜自身免疫性皮肤水疱病。自身免疫反应针对VII型胶原（COL 7），其是真皮-表皮连接的组成部分。导致水疱形成的机制相对较好地表征。在特异性同种型和特异性糖酵解的抗COL 7抗体与COL 7结合后，Fc依赖性机制诱导促炎环境。这种环境的形成部分但不完全依赖于补体激活。我们已经收集了证据，细胞因子在调节这种促炎环境的贡献。详细地，实验EBA中细胞因子的功能表征表明，（i）MIP-1 a不有助于疾病表现，（ii）TNF-α的抑制具有最小的作用，（iii）需要IL-1和（iv）GM-CSF，以及（v）IL-6具有深刻的保护作用。总体而言，这导致中性粒细胞的CD 18依赖性迁移。嗜中性粒细胞与自身抗体的结合由特异性Fc γ受体介导。随后，中性粒细胞释放活性氧和蛋白水解酶，诱导水疱形成。显然不相关的工作表明，自身抗体，即抗BP 180抗体与角质形成细胞的结合诱导IL-6和IL-8的释放。鉴于，如果细胞因子释放也可以通过角质形成细胞与抗COL 7 IgG的孵育来触发，则这些角质形成细胞衍生的细胞因子可以介导EBA中促炎环境的产生。为了测试这一点，我们将HaCaT细胞与抗COL 7抗体孵育。在初步实验中，HaCaT细胞与抗COL 7抗体孵育诱导几种细胞因子的释放。来自抗COL 7抗体处理的HaCaT细胞的上清液诱导中性粒细胞迁移。与对照IgG孵育没有这种影响。由于几种鉴定的细胞因子的释放受NF-κ B活化控制，我们接下来测试了表皮中受损的NF-κ B信号传导是否对通过将抗COL 7 IgG转移到小鼠中的EBA诱导产生影响。为此，我们与Ingo Haase教授合作，他从表皮角质形成细胞（RelAepi）中产生了靶向缺失RelA的小鼠。在先导实验中，通过转移抗COL 7 IgG诱导实验EBA，RelAepi小鼠与野生型对照相比发展出显著更温和的EBA表型。基于这些观察结果，该项目将挑战以下假设：（i）抗COL 7 IgG诱导角质形成细胞释放NF-κ B依赖性和功能相关的细胞因子，（ii）抑制角质形成细胞NF-κ B活化会损害实验性EBA的诱导，（iii）阻断NF-κ B活化对EBA具有治疗作用。这将导致更好地理解原型，器官特异性自身免疫性疾病中自身抗体诱导的组织损伤的发病机制，重点关注自身免疫反应靶向的细胞。

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.