Dermal-Epidermal Junction Disruptors: Toxicodynamic Mechanisms

项目总结/摘要 真皮-表皮连接（DEJ）破坏剂是皮肤毒物，其导致表皮从真皮脱离， 导致皮肤起泡（水疱）的作用。原型DEJ破坏剂是氮芥（MEC）， 芥末;然而，MEC和其他DEJ干扰物引起水疱的毒力机制仍然存在 在科学文献中几乎没有描述。这里提出的工作的总体目标是调查 与DEJ破坏相关的毒理学机制。这将通过研究肥大细胞的作用来实现 衍生的介质（组胺和丝氨酸蛋白酶类胰蛋白酶），嗜中性粒细胞衍生的介质（髓过氧化物酶，MPO和 基质金属蛋白酶-9，MMP-9）和角质形成细胞特异性胶原（胶原IV，COL 4和胶原XVII，COL 17） 皮肤对MEC的反应。在该提案的目标1中，假设肥大细胞衍生的介质影响DEJ， 将对中断进行评估。为此，将小鼠耳皮肤局部暴露于生理盐水（未处理）、二甲基亚砜 (DMSO载体）或MEC。在暴露于盐水、媒介物或MEC之前20分钟，对另外的小鼠组进行处理 与抗组胺多塞平（5%乳膏局部给药）或类胰蛋白酶抑制剂亮抑酶肽（以10或100 mg的试验剂量）一起使用， 30 mg/kg，ip）。在2、8或24小时收获所有耳组织，并固定用于组织病理学分析或 均质化并通过ELISA测定组胺的存在，通过蛋白质印迹法测定类胰蛋白酶的存在。在目标2中， 根据该提议，我们将检验嗜中性粒细胞衍生的MMP-9有助于MEC破坏DEJ的假设。为了验证这一 假设，我们将分析来自野生型和Mmp 9-/-敲除的幼稚、载体和MEC处理的小鼠耳活检组织 在暴露后2、8或24小时获得的小鼠，使用光学显微镜检查DEJ破坏的存在，并使用免疫组织化学方法检查DEJ破坏的存在。 使用免疫组织化学（IHC）检测髓过氧化物酶（一种嗜中性粒细胞选择性标记物）的存在。在目标3中， 我们将检验MEC通过在表皮角质形成细胞水平上进行干预而促进起泡的假设 并影响参与维持DEJ的关键角质形成细胞表达的胶原蛋白的表达，即COL 4和 COL17.为了检验这一假设，我们将分析来自上述时间点和样品的小鼠耳活组织检查， 目的1通过对福尔马林固定的石蜡包埋组织进行免疫组化检测，探讨这两种基因在组织中的表达情况 蛋白质COL 4和COL 17在暴露于MEC后随时间变化。此外，目标3中的实验将 利用暴露于MEC 2、8或24小时的人3D皮肤培养物来研究对COL 4和COL 17蛋白的影响 在人体组织中的表达。总而言之，该提案旨在调查三个相关但独立的假设， 揭示了DEJ干扰物用于引起水泡的毒理学机制，并将发现新的靶点 以减少化学诱导的皮肤起泡。这里提出的工作将提供一个广泛的， 在皮肤肥大细胞水平上，MEC对损伤的“全组织”毒代动力学反应， 中性粒细胞和表皮角质形成细胞，并将通过3D人体皮肤研究进行补充。最重要的是， 参与该项目的学生将学习广泛的技能，包括实验设计，动物护理，动物 给药、组织学技术、IHC、蛋白质印迹以及如何使用ImageJ软件评估组织蛋白质表达。

Project Summary/Abstract Dermal-epidermal junction (DEJ) disruptors are cutaneous poisons that cause the epidermis to detach from the dermis, an effect that results in skin blistering (vesication). The prototype DEJ disruptor is mechlorethamine (MEC), a nitrogen mustard; however, the toxicodynamic mechanisms involved in vesication by MEC and other DEJ disruptors remains mostly undescribed in the scientific literature. The overall goal of the work proposed here is to investigate the toxicodynamic mechanisms associated with DEJ disruption. This will be achieved by studying the role of mast cell derived mediators (histamine and the serine protease tryptase), neutrophil-derived mediators (myeloperoxidase, MPO and matrix metalloproteinase-9, MMP-9) and keratinocyte-specific collagens (collagen IV, COL4 and collagen XVII, COL17) in the response of skin to MEC. In Aim 1 of the proposal, the hypothesis that mast cell-derived mediators influence DEJ disruption will be evaluated. To this end, mouse ear skin will be topically exposed to saline (naïve), dimethylsulfoxide (DMSO, vehicle) or MEC. Additional groups of mice will be treated 20 min prior to exposure to saline, vehicle or MEC with the antihistamine doxepin (5% cream administered topically) or the tryptase inhibitor leupeptin (at test doses of 10 or 30 mg/kg, ip). All ear tissues will be harvested at 2, 8 or 24 hr and either fixed for histopathologic analysis or homogenized and assayed for the presence of histamine via ELISA and tryptase via western blotting. In Aim 2 of the proposal, we will test the hypothesis that neutrophil-derived MMP-9 contributes to DEJ disruption by MEC. To test this hypothesis, we will analyze naïve, vehicle and MEC-treated mouse ear biopsies from wildtype and Mmp9 -/- knockout mice obtained at 2, 8 or 24 hr after exposure for the presence of DEJ disruption using light microscopy and for the presence of myeloperoxidase, a neutrophil-selective marker, using immunohistochemistry (IHC). In Aim 3 of the proposal, we will test the hypothesis that MEC promotes blistering by interfering at the level of epidermal keratinocytes and affecting the expression of key keratinocyte-expressed collagens involved in maintaining the DEJ, namely COL4 and COL17. To test this hypothesis, we will analyze mouse ear biopsies from the time points and samples described above for Aim 1 but here carry out IHC on the formalin-fixed paraffin embedded tissues to evaluate how the expression of these two proteins, COL4 and COL17, changes over time after following exposure to MEC. In addition, experiments in Aim 3 will utilize human 3D skin cultures exposed to MEC for 2, 8 or 24 hr to investigate effects on COL4 and COL17 protein expression in human tissues. All in all, the proposal seeks to investigate three related yet independent hypotheses that will shed new light on the toxicodynamic mechanisms used by DEJ disruptors to cause blisters and will uncover novel targets for the purpose of reducing chemically induced cutaneous blistering. The work proposed here will provide a broad, “whole tissue” toxicodynamic response to injury by MEC at the levels of the cutaneous mast cells, the infiltrating neutrophils and the epidermal keratinocytes and will be complemented by the 3D human skin study. Most importantly, students involved in this project will learn a wide range of skills including experimental design, animal care, animal dosing, histology techniques, IHC, western blotting and how to use ImageJ software to assess tissue protein expression.