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.

大疱性表皮松解症（EBA）是一种慢性皮肤粘膜自身免疫性皮肤水疱病。自身免疫反应针对 VII 型胶原蛋白 (COL7)，它是真皮表皮连接的组成部分。导致水泡形成的机制已得到相对较好的表征。特定同种型和特异性糖酵解抗 COL7 抗体与 COL7 结合后，Fc 依赖性机制会诱导促炎环境。这种环境的形成部分但不完全依赖于补体激活。我们已经收集了细胞因子在调节这种促炎环境中的贡献的证据。详细而言，实验 EBA 中细胞因子的功能特征表明，(i) MIP-1a 不会导致疾病表现，(ii) TNF-a 的抑制作用最小，(iii) IL-1 和 (iv) GM- CSF 是必需的，并且 (v) IL-6 具有深远的保护作用。总体而言，这导致中性粒细胞依赖 CD18 迁移。中性粒细胞与自身抗体的结合是由特定的 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，从而诱导水疱形成。显然不相关的工作表明，自身抗体（即抗 BP180 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将角质形成细胞与抗 COL7 IgG 一起孵育来触发，那么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 COL7 抗体一起孵育。在初步实验中，HaCaT 细胞与抗 COL7 抗体一起孵育，诱导多种细胞因子的释放。抗 COL7 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 一起孵育则没有这种效果。由于几种已确定的细胞因子的释放是由 NF-kB 激活控制的，因此我们接下来测试了表皮中受损的 NF-kB 信号传导是否会通过将抗 COL7 IgG 转移到小鼠体内来影响 EBA 诱导。为此，我们发起了与 Ingo Haase 教授的合作，他培育了从表皮角质形成细胞中定向删除 RelA 的小鼠 (RelAepi)。在一项初步实验中，通过转移抗 COL7 IgG 诱导实验性 EBA，与野生型对照相比，RelAepi 小鼠表现出明显更温和的 EBA 表型。基于这些观察结果，该项目将挑战以下假设：(i) 抗 COL7 IgG 诱导角质形成细胞释放 NF-kB 依赖性且功能相关的细胞因子，(ii) 抑制角质形成细胞 NF-kB 激活会损害实验性 EBA 的诱导，(iii) 阻断 NF-kB 激活对 EBA 具有治疗作用。这将有助于更好地了解典型的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所针对的细胞。