Department of Immunology, Jagiellonian University School of Medicine, Cracow, Poland.
Inflamm Res. 2010 Apr;59(4):277-89. doi: 10.1007/s00011-009-0096-5. Epub 2009 Sep 26.
Denatured proteins are deposited in damaged tissues, around implanted biomaterials, during natural aging as well as in a heterogeneous group of amyloid diseases, such as Alzheimer's disease. There is evidence that tissue damage observed in amyloidosis is mediated mainly by factors released from activated macrophages, such as superoxide and nitric oxide (NO), as opposed to direct interaction between amyloid fibrils and nonimmune cells.
Mouse resident peritoneal macrophages were stimulated in serum-free medium with different preparations of nonamyloidogenic proteins: alcohol dehydrogenase (AD), bovine serum albumin (BSA) or fibrinogen (FG). Intra- and extracellular superoxide production was measured by, respectively, nitro blue tetrazolium (NBT) reduction and lucigenin-enhanced chemiluminescence. Levels of nitrite (reflecting NO release) were measured in culture supernatants.
Aggregates of denatured, nonamyloidogenic proteins, but not their native or denatured but not aggregated counterparts, stimulated superoxide and/or NO production in macrophages. The NO production was mediated by beta(1) and beta(2) integrins, with a possible contribution of receptor for advanced glycation end products (RAGE). It was catalyzed by inducible NO synthase (iNOS), enhanced synergistically by interferon-gamma (IFN-gamma), and inhibited by covalently modified proteins-components of advanced glycation end products. Although intracellular superoxide production was stimulated significantly by denatured BSA and AD, but not by FG, both denatured BSA and FG strongly enhanced zymosan-stimulated extracellular release of reactive oxygen species.
Our results point at similarities in macrophage responses to denatured nonamyloidogenic proteins and to amyloid fibrils. Thus, the tissue injury observed in amyloidosis may result from overstimulation of mechanisms that, under physiological conditions, enable macrophages to recognize and remove denatured proteins.
变性蛋白在组织损伤、植入生物材料周围、自然老化以及阿尔茨海默病等各种淀粉样变性疾病中沉积。有证据表明，淀粉样变性中观察到的组织损伤主要是由激活的巨噬细胞释放的因子介导的，如超氧阴离子和一氧化氮（NO），而不是淀粉样纤维与非免疫细胞的直接相互作用。
在无血清培养基中，用不同的非淀粉样蛋白制剂刺激小鼠常驻腹膜巨噬细胞：醇脱氢酶（AD）、牛血清白蛋白（BSA）或纤维蛋白原（FG）。通过分别用硝基蓝四唑（NBT）还原和荧光素增强化学发光法测量细胞内和细胞外超氧阴离子的产生。在培养上清液中测量亚硝酸盐（反映 NO 释放）的水平。
变性的、非淀粉样的蛋白聚集物，但不是它们的天然或变性但未聚集的对应物，刺激巨噬细胞产生超氧阴离子和/或 NO。NO 的产生是由β1 和β2 整合素介导的，可能有晚期糖基化终产物受体（RAGE）的贡献。它由诱导型一氧化氮合酶（iNOS）催化，干扰素-γ（IFN-γ）增强协同作用，并被共价修饰蛋白-晚期糖基化终产物的成分抑制。虽然变性的 BSA 和 AD 显著刺激了细胞内超氧阴离子的产生，但 FG 却没有，变性的 BSA 和 FG 都强烈增强了酵母聚糖刺激的细胞外活性氧的释放。
我们的结果表明，巨噬细胞对变性的非淀粉样蛋白和淀粉样纤维的反应存在相似性。因此，淀粉样变性中观察到的组织损伤可能是由于过度刺激了使巨噬细胞能够识别和清除变性蛋白的机制。
