Shibata Y, Bjorkman D R, Schmidt M, Oghiso Y, Volkman A
Department of Pathology and Laboratory Medicine, East Carolina University School of Medicine, Greenville, NC.
Blood. 1994 Jun 1;83(11):3316-23.
Previously, we found that murine bone marrow-derived macrophages (MO) induced in vitro by MO-specific colony-stimulating factor (M-CSF) have little capacity to release prostaglandin E2 (PGE2) and other eicosanoids. This work focused on the functional and transcriptional expression of the key enzymes for the PGE2 synthesis in the MO. Nonadherent bone marrow cells were cultured with RPMI1640 plus 10% fetal bovine serum (FBS) further supplemented with either M-CSF or granulocyte-macrophage (GM)-CSF and interleukin-3 (IL-3). Cellular PGG/H synthase (cyclooxygenase) levels were quantified by cytometric analysis with antibodies specific for the two isozymes of PGG/H synthase (PGG/H synthases 1 and 2). The enzyme activity was monitored by adding exogenous arachidonic acid (AA) substrate to the bone marrow MO cultures and to the cell-free particulate fractions. The levels of PGE2 converted were quantitated by radioimmunoassay (RIA). mRNA levels of the enzymes were detected by Northern blot analysis hybridized with mouse PGG/H synthase cDNA probes, 2.7 kb (PGG/H synthase 1) and 4.2 kb (PGG/H synthase 2). In addition, cellular phospholipase A2 (PLA2) activities were detected with sn-2-14C-arachidonyl phosphatidylcholine as a substrate. Cells proliferating in the presence of GM-CSF and IL-3 for more than 4 days showed significant release of PGE2 (> 7 ng/10(6) cells) when stimulated by AA. These cells also expressed significant amounts of PGG/H synthase 1 protein, its mRNA (2.7 kb) and cellular PLA2. M-CSF-induced MO, in sharp contrast, expressed little PGG/H synthase protein, mRNA, cellular enzyme activity, or PGE2 release, despite comparable levels of cellular PLA2 activity. These data suggest that the capacity of differentiating marrow-derived MO to form PGE2 is growth factor-dependent.
此前，我们发现，由单核细胞特异性集落刺激因子（M-CSF）体外诱导产生的小鼠骨髓来源巨噬细胞（MO）释放前列腺素E2（PGE2）和其他类花生酸的能力较弱。这项工作聚焦于单核细胞中PGE2合成关键酶的功能和转录表达。将非贴壁骨髓细胞与RPMI1640加10%胎牛血清（FBS）一起培养，并进一步补充M-CSF或粒细胞-巨噬细胞（GM）-CSF和白细胞介素-3（IL-3）。通过使用针对PGG/H合酶（PGG/H合酶1和2）两种同工酶的特异性抗体进行细胞计数分析来定量细胞PGG/H合酶（环氧化酶）水平。通过向骨髓单核细胞培养物和无细胞颗粒部分中添加外源性花生四烯酸（AA）底物来监测酶活性。通过放射免疫测定（RIA）对转化的PGE2水平进行定量。用与小鼠PGG/H合酶cDNA探针杂交的Northern印迹分析检测酶的mRNA水平，分别为2.7 kb（PGG/H合酶1）和4.2 kb（PGG/H合酶2）。此外，以sn-2-14C-花生四烯酰磷脂酰胆碱为底物检测细胞磷脂酶A2（PLA2）活性。在GM-CSF和IL-3存在下增殖超过4天的细胞在受到AA刺激时显示出显著的PGE2释放（>7 ng/10(6)细胞）。这些细胞还表达了大量的PGG/H合酶1蛋白、其mRNA（2.7 kb）和细胞PLA2。与之形成鲜明对比的是，尽管细胞PLA2活性水平相当，但M-CSF诱导的单核细胞表达的PGG/H合酶蛋白、mRNA、细胞酶活性或PGE2释放都很少。这些数据表明，分化的骨髓来源单核细胞形成PGE2的能力是生长因子依赖性的。
