Derigs H G, Burgess G S, Klingberg D, Nahreini T S, Mochizuki D Y, Williams D E, Boswell H S
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Leukemia. 1990 Jul;4(7):471-9.
We examined the role of augmented formation of intracellular cyclic AMP (cAMP) in the mediation of stromal cell growth factor production that occurs constitutively or upon cytokine stimulation. Clonal murine marrow adherent cell lines were stimulated under serum-free conditions by interleukin-1 (IL-1) or lipopolysaccharide (LPS) and one (+/+ -1.LDA11) was found to produce low quantities of granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF identity was confirmed by the ability of supernatants from stromal cells to promote proliferation of the factor-dependent cell line FDC-P1, neutralization of this activity by antiserum to GM-CSF, and by Northern blot analysis. However, optimal concentrations of IL-1 and tumor necrosis factor-alpha (TNF-alpha), in combination, led to synergistic (greater than 5-fold higher quantity) GM-CSF production compared with either stimulus alone in the +/+ -1. LDA11 cell line, capable of GM-CSF production after only single stimulation with IL-1 or LPS. In addition, synergistic stimulation by IL-1 and TNF-alpha led to equivalent high amounts of GM-CSF in another cell line incapable of GM-CSF production after induction with only IL-1 or LPS. Any of several means to raise intracellular cAMP levels, including addition of 8-bromo-cyclic AMP (8Br cAMP) (0.25-1mM), pertussis toxin (20-100 ng/ml), or addition of prostaglandin E1 (PGE1) (1 microM), failed to stimulate GM-CSF production alone and strongly inhibited GM-CSF production in stromal cells stimulated by IL-1, LPS, or the synergistic combination of IL-1 and TNF-alpha. In addition, PGE1 and pertussis intoxication were agonists of adenylate cyclase in membranes of marrow adherent cells, whereas IL-1 and LPS were not. The role for regulators of intracellular cAMP was specific because any of the cAMP agonists alone, or in the presence of cytokine stimulators of stromal cells, strongly enhanced IL-6 production, an event known to be cAMP-responsive. Thus, acute formation of intracellular cAMP is a negative regulator of stromal cell GM-CSF production mediated by cytokines, but positively regulates IL-6 production and may be an important determinant of cytokine-directed marrow microenvironmental function. These findings on the requirement for augmentation versus inhibition of cytokine-mediated production of hemopoietic growth factors might be applied to an analysis of marrow stromal cell heterogeneity.
我们研究了细胞内环状AMP（cAMP）生成增加在介导基质细胞生长因子产生过程中的作用，该过程可组成性发生或在细胞因子刺激后发生。在无血清条件下，用白细胞介素-1（IL-1）或脂多糖（LPS）刺激克隆的小鼠骨髓贴壁细胞系，发现其中一个细胞系（+ / + -1.LDA11）产生少量粒细胞巨噬细胞集落刺激因子（GM-CSF）。通过基质细胞上清液促进因子依赖性细胞系FDC-P1增殖的能力、抗GM-CSF抗血清对该活性的中和作用以及Northern印迹分析，证实了GM-CSF 的特性。然而，与单独使用任何一种刺激物相比，在+ / + -1.LDA11细胞系中，IL-1和肿瘤坏死因子-α（TNF-α）的最佳浓度联合使用可导致协同（高出5倍以上）产生GM-CSF，该细胞系仅在经IL-1或LPS单次刺激后就能产生GM-CSF。此外，在另一个仅经IL-1或LPS诱导后无法产生GM-CSF的细胞系中，IL-1和TNF-α的协同刺激也导致了等量的高产量GM-CSF。几种提高细胞内cAMP水平的方法，包括添加8-溴环AMP（8Br cAMP）（0.25 - 1mM）、百日咳毒素（20 - 100 ng/ml）或添加前列腺素E1（PGE1）（1 microM），单独使用时均未能刺激GM-CSF的产生，并且在由IL-1、LPS或IL-1与TNF-α的协同组合刺激的基质细胞中强烈抑制GM-CSF的产生。此外，PGE1和百日咳毒素中毒是骨髓贴壁细胞膜中腺苷酸环化酶的激动剂，而IL-1和LPS则不是。细胞内cAMP调节剂的作用具有特异性，因为任何一种cAMP激动剂单独使用或在存在基质细胞细胞因子刺激剂的情况下，都会强烈增强IL-6的产生，这是一个已知对cAMP有反应的事件。因此，细胞内cAMP的急性形成是细胞因子介导的基质细胞GM-CSF产生的负调节剂，但对IL-6的产生起正调节作用，并可能是细胞因子导向的骨髓微环境功能的重要决定因素。这些关于造血生长因子细胞因子介导产生的增强与抑制需求的发现，可能适用于骨髓基质细胞异质性的分析。
