Vincent V A, Tilders F J, Van Dam A M
Research Institute Neurosciences Vrije Universiteit, Faculty of Medicine, Department of Pharmacology, Amsterdam, The Netherlands.
Glia. 1997 Mar;19(3):190-8. doi: 10.1002/(sici)1098-1136(199703)19:3<190::aid-glia2>3.0.co;2-3.
In mixed glial cell cultures from cerebral cortices of newborn rats, endotoxin induces inducible nitric oxide (iNOS), nitric oxide (NO), and interleukin-1 beta (IL-1 beta) production in microglial cells. Earlier we demonstrated that endotoxin induced iNOS but not IL-1 beta expression in microglial cells is inhibited by the presence of astroglial cells. In the present paper we describe studies on the mechanism by which astroglial cells exert selective suppressive action on iNOS expression by microglial cells. Expression of iNOS and IL-1 beta was studied by single or double label immunocytochemical techniques and cell identification was performed with GSA-I-B4-isolectin and an antibody against GFAP. Production of IL-1 beta and NO was determined by measurement of IL-1 beta and nitrite concentrations in cell lysates and the culture medium, respectively. TGF beta, a cytokine known to inhibit NO production by endotoxin challenged macrophages, was measured in culture medium of mixed glial cell cultures using a bioassay. Microglial, astroglial, and mixed glial cell cultures produced similar concentrations of TGF beta. The potential effect of TGF beta was studied by using immunoneutralizing antibodies against TGF beta 1 and TGF beta 2 on the induction of iNOS in microglial cells in the presence of astroglial cells. Incubation of the mixed glial cell culture with these TGF beta antibodies (3 micrograms/ml) markedly increased endotoxin-induced NO production and iNOS expression in microglial cells, whereas the production of IL-1 beta was not affected. The antibodies against TGF beta 1 and TGF beta 2 marginally increased NO production in pure microglial cell cultures, nonetheless in cultures of purified microglial cells recombinant TGF beta 1 and TGF beta 2 together with endotoxin inhibited NO production. We conclude that the presence of astroglial cells is essential for the inhibitory effect of TGF beta on NO production by microglial cells (possibly) by activation of TGF beta or by increasing the sensitivity of microglial cells for TGF beta.
在新生大鼠大脑皮质的混合胶质细胞培养物中，内毒素可诱导小胶质细胞产生诱导型一氧化氮合酶（iNOS）、一氧化氮（NO）和白细胞介素-1β（IL-1β）。此前我们证明，内毒素诱导的小胶质细胞中iNOS而非IL-1β的表达受到星形胶质细胞存在的抑制。在本文中，我们描述了关于星形胶质细胞对小胶质细胞iNOS表达发挥选择性抑制作用机制的研究。通过单标记或双标记免疫细胞化学技术研究iNOS和IL-1β的表达，并用GSA-I-B4异凝集素和抗GFAP抗体进行细胞鉴定。分别通过测量细胞裂解物和培养基中IL-1β和亚硝酸盐浓度来测定IL-1β和NO的产生。使用生物测定法在混合胶质细胞培养物的培养基中测量转化生长因子β（TGFβ），TGFβ是一种已知可抑制内毒素刺激的巨噬细胞产生NO的细胞因子。小胶质细胞、星形胶质细胞和混合胶质细胞培养物产生的TGFβ浓度相似。通过使用抗TGFβ1和TGFβ2的免疫中和抗体研究TGFβ在星形胶质细胞存在下对小胶质细胞中iNOS诱导的潜在作用。用这些TGFβ抗体（3微克/毫升）孵育混合胶质细胞培养物，可显著增加内毒素诱导的小胶质细胞中NO的产生和iNOS的表达，而IL-1β的产生不受影响。抗TGFβ1和TGFβ2抗体在纯小胶质细胞培养物中略微增加了NO的产生，尽管如此，在纯化的小胶质细胞培养物中，重组TGFβ1和TGFβ2与内毒素一起抑制了NO的产生。我们得出结论，星形胶质细胞的存在对于TGFβ对小胶质细胞产生NO的抑制作用至关重要，（可能）是通过激活TGFβ或通过增加小胶质细胞对TGFβ的敏感性来实现。
