Department of Medical Pharmacology, School of Medicine, Acibadem Mehmet Ali Aydinlar University, 34752 Istanbul, Turkey.
Translational Lung Research Center Heidelberg (TLRC-H), Part of the German Center for Lung Research (DZL), 69120 Heidelberg, Germany.
Int J Mol Sci. 2022 Jul 27;23(15):8315. doi: 10.3390/ijms23158315.
Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS; 1 µg/mL) and exposed to normoxia and hypoxia (1.5% O). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISC); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.
炎症和缺氧会损害肺泡屏障的紧密性，抑制钠和液体的重吸收，并导致水肿。我们测试了刺激的肺泡巨噬细胞是否会影响肺泡钠转运，以及缺氧是否会加重炎症的影响，并测试了涉及的信号通路。原代大鼠肺泡 II 型细胞 (rA2) 与大鼠肺泡巨噬细胞 (NR8383) 共培养，或在用脂多糖 (LPS; 1 µg/mL) 刺激后用 NR8383 条件培养基处理，并暴露于常氧和缺氧 (1.5% O) 下。LPS 引起巨噬细胞中 TNFα 和 IL-6 mRNA 的快速、短暂增加，并在巨噬细胞和 rA2 细胞中持续增加诱导型一氧化氮合酶 (NOS2) mRNA，导致亚硝酸盐水平升高和 TNF-α 和 IL-6 分泌到培养基中。在常氧下，24 小时 LPS 处理的 NR8383 降低了共培养物的跨上皮电阻 (TEER)，以及阿米洛利敏感的短路电流 (ISC)；然而，Na/K-ATPase 活性不受影响。LPS 刺激的 NR8383 的条件培养基对 rA2 也有抑制作用，但在透析去除小分子和亚硝酸盐后，抑制作用不那么明显。LPS 刺激的巨噬细胞对 TEER 和 Na 转运的影响在共培养物和 rA2 单核培养物中应用 iNOS 抑制剂 L-NMMA 完全阻止。LPS 刺激的 NR8383 与缺氧联合完全消除了 TEER 和 ISCΔamil。这些结果表明，LPS 刺激肺泡巨噬细胞通过 NO 依赖机制损害肺泡上皮细胞钠转运，其中部分 NO 是由 LPS 刺激的肺泡巨噬细胞信号诱导的 rA2 产生的。
