Keratinocytes as modulators of autoantibody-induced tissue injury

角质形成细胞作为自身抗体诱导的组织损伤的调节剂

• 批准号: 239218327
• 负责人: Professor Dr. Ralf Joachim Ludwig
• 金额: --
• 依托单位: Lübecker Institut für Experimentelle Dermatologie (LIED)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/239218327?language=en
• 关键词: Keratinocytes; modulators; autoantibody; induced; tissue

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.

获得性大疱性表皮松解症(EBA)是一种慢性大疱性黏膜自身免疫性皮肤水疱病。自身免疫反应针对的是III型胶原(COL7)，它是真皮-表皮连接的组成部分。导致水泡形成的机制被相对较好地描述。在特定的同种类型和特定的糖酵解的抗COL7抗体与COL7结合后，Fc依赖的机制诱导了促炎环境。这种环境的形成部分但不完全依赖于补体的激活。我们已经收集了细胞因子在调节这种促炎环境中的作用的证据。对实验性EBA中细胞因子的功能研究表明，(1)MIP-1a不参与疾病的表现，(2)抑制肿瘤坏死因子-α的作用很小，(3)需要IL-1和(4)GM-CSF，(5)IL-6具有深刻的保护作用。总体而言，这导致了中性粒细胞依赖CD18的迁移。中性粒细胞与自身抗体的结合是由特异的Fc-γ受体介导的。随后，中性粒细胞释放活性氧和蛋白水解酶，导致水泡的形成。显然无关的工作表明，自身抗体，即抗BP180抗体与角质形成细胞结合可诱导IL-6和IL-8的释放。鉴于，如果角质形成细胞与抗COL7抗体孵育也能触发细胞因子释放，这些角质形成细胞衍生的细胞因子可以介导EBA促炎环境的产生。为了测试这一点，我们将HaCaT细胞与抗COL7抗体孵育。在初步实验中，HaCaT细胞与抗COL7抗体孵育后，诱导了几种细胞因子的释放。抗COL7抗体处理的HaCaT细胞上清液可诱导中性粒细胞迁移。而与对照免疫球蛋白孵育则无此作用。由于几种已识别的细胞因子的释放是由核因子-kB激活控制的，我们接下来测试了表皮中受损的核因子-kB信号是否通过将抗COL7抗体转移到小鼠体内而影响EBA的诱导。为此，我们启动了与Ingo Haase教授的合作，他培育了从表皮角质形成细胞(RelAepi)定向缺失relA的小鼠。在一项通过转移抗COL7抗体诱导实验性EBA的先导性实验中，RelAepi小鼠与野生型对照相比，发展出明显温和的EBA表型。基于这些观察，该项目将挑战以下假设：(1)抗COL7抗体诱导角质形成细胞释放依赖的和功能性相关的细胞因子；(2)抑制角质形成细胞的核因子-kB激活削弱实验性EBA的诱导；(3)阻断核因子-kB的激活对EBA有治疗作用。这将有助于更好地理解自身抗体诱导的组织损伤的发病机制，在一种典型的、器官特有的自身免疫性疾病中，重点关注自身免疫反应所针对的细胞。