Dept. Immunology, Instituto de Microbiologia Paulo de Góes, UFRJ, Rio de Janeiro, Brazil.
Postgraduate Programme in Pathology, Faculdade de Medicina, Hospital Universitário Antônio Pedro, UFF, Rio de Janeiro, Brazil.
Int Immunopharmacol. 2021 May;94:107440. doi: 10.1016/j.intimp.2021.107440. Epub 2021 Feb 12.
Subcutaneous implants of heat-coagulated egg white (egg white implants, EWI) induce intense local eosinophilia and prime for hyperreactivity following airway ovalbumin challenge. The roles of allergen sensitization, surgical trauma-induced glucocorticoids, and the 5-lipoxygenase (5-LO) pathway were hitherto unexplored in this model, in which quantitative recovery and large-scale purification of the eosinophils from the inflammatory site for functional and immunopharmacological studies are difficult to achieve.
We overcame this limitation by shifting the implantation site to the peritoneal cavity (EWIp), thereby enabling quantitative leukocyte retrieval.
By day 7 post-surgery, eosinophil counts reached ~ 30% of all leukocytes recovered. Eosinophilia was prevented by: a) induction of allergen-specific oral tolerance to ovalbumin, the main allergen in egg white; b) inactivation of the 5-lipoxygenase pathway; c) blockade of endogenous glucocorticoid signaling by pretreatment with metirapone plus mifepristone before surgery. Highly purified eosinophils (~99% pure) could be obtained from the peritoneal exudate of EWIp-carrier mice in 2 simple, antibody-free steps. Preparative-scale yields, suitable for most biochemical, pharmacological, and molecular applications, were routinely obtained, and could be further enhanced through addition of pre-or post-surgery immunization steps (active or adoptive). The recovered eosinophils were fully functional in vivo, as demonstrated by the transfer of purified eosinophils into eosinophil-deficient Δdbl-GATA-1-KO mice, which upon subsequent challenge with eotaxin-1 present secondary accumulation of neutrophils, but not of mononuclear phagocytes.
These findings document glucocorticoid-, allergen- and 5-lipoxygenase-dependent eosinophilia, which makes EWIp carriers an abundant source of pure, nontransgenic eosinophils for immunopharmacological studies.
皮下植入热凝结的蛋清（蛋清植入物，EWI）可诱导强烈的局部嗜酸性粒细胞增多，并在气道卵清蛋白挑战后引发超反应性。在该模型中，过敏原致敏、手术创伤诱导的糖皮质激素和 5-脂氧合酶（5-LO）途径的作用迄今尚未得到探索，在该模型中，从炎症部位定量回收和大规模纯化嗜酸性粒细胞进行功能和免疫药理学研究是困难的。
我们通过将植入部位转移到腹腔（EWIp）克服了这一限制，从而能够定量回收白细胞。
手术后第 7 天，嗜酸性粒细胞计数达到回收白细胞的30%。通过以下方法可预防嗜酸性粒细胞增多：a）诱导卵清蛋白（蛋清中的主要过敏原）的过敏原特异性口服耐受；b）抑制 5-脂氧合酶途径；c）手术前用美替拉酮加米非司酮预处理阻断内源性糖皮质激素信号。可通过 2 个简单、无抗体的步骤从 EWIp 载体小鼠的腹腔渗出液中获得（99%纯度）高纯度的嗜酸性粒细胞。可常规获得适合大多数生化、药理学和分子应用的制备规模产量，并且可通过添加手术前或手术后免疫接种步骤（主动或被动）进一步增强。回收的嗜酸性粒细胞在体内具有完全的功能，这可通过将纯化的嗜酸性粒细胞转移到嗜酸性粒细胞缺陷型Δdbl-GATA-1-KO 小鼠中得到证明，随后用嗜酸性粒细胞趋化因子-1 进行挑战导致中性粒细胞的继发性聚集，但单核吞噬细胞没有聚集。
这些发现证明了糖皮质激素、过敏原和 5-脂氧合酶依赖性嗜酸性粒细胞增多，这使得 EWIp 载体成为用于免疫药理学研究的纯非转基因嗜酸性粒细胞的丰富来源。
