Sasu S, Cooper A L, Beasley D
Division of Nephrology, Department of Medicine, New England Medical Center Hospitals and Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Am J Physiol Heart Circ Physiol. 2001 Apr;280(4):H1615-23. doi: 10.1152/ajpheart.2001.280.4.H1615.
After injury to the blood vessel wall, vascular smooth muscle cells (SMC) synthesize interleukin (IL)-1 and inducible nitric oxide (NO) synthase (iNOS). The present study tested whether endogenous production of IL-1 alpha stimulates iNOS expression in vascular SMC, and assessed whether IL-1 alpha exerts autocrine effects on the cells producing IL-1 alpha or juxtacrine effects on cells that contact the IL-1 alpha producing cells. Rat aortic SMC were transiently transfected with expression plasmids encoding either IL-1 alpha precursor, which localizes to the plasma membrane, or mature IL-1 alpha, which remains cytosolic. iNOS mRNA levels, determined by RT-PCR, and production of nitrite, a stable oxidation product of NO, were markedly elevated in SMC overexpressing IL-1 alpha precursor, and modestly elevated in SMC overexpressing mature IL-1 alpha, relative to SMC transfected with vector alone. Exposure to exogenous IL-1 beta or TNF-alpha further stimulated iNOS gene expression in SMC producing IL-1 alpha; low levels of IL-1 beta (20 pg/ml) were effective in SMC transfected with IL-1 alpha precursor plasmid, whereas SMC transfected with mature IL-1 alpha plasmid or vector alone required higher concentrations of IL-1 beta (200 and 2,000 pg/ml, respectively). The increases in iNOS mRNA levels and NO production in SMC overexpressing IL-1 alpha precursor were prevented by exogenous IL-1 receptor antagonist, suggesting that these effects were mediated by the type I IL-1 receptor. Immunostaining studies indicated that IL-1 alpha precursor stimulates iNOS gene expression via cell-cell contact. Expression of iNOS was enhanced in cells that were in contact with a cell overexpressing IL-1 alpha precursor (identified by coexpression of green fluorescent protein), and in cells that were overexpressing IL-1 alpha themselves, but only when the cell contacted another cell. Together these results indicate that IL-1 alpha precursor acts by cell-cell contact as an autocrine and juxtacrine enhancer of iNOS gene expression, inducing moderate iNOS expression on its own, and markedly augmenting the responsiveness of rat aortic SMC to exogenous cytokines.
血管壁损伤后，血管平滑肌细胞（SMC）合成白细胞介素（IL）-1和诱导型一氧化氮（NO）合酶（iNOS）。本研究检测内源性IL-1α的产生是否刺激血管SMC中iNOS的表达，并评估IL-1α对产生IL-1α的细胞发挥自分泌作用，还是对与产生IL-1α的细胞接触的细胞发挥旁分泌作用。用编码定位于质膜的IL-1α前体或保留在细胞质中的成熟IL-1α的表达质粒瞬时转染大鼠主动脉SMC。相对于仅用载体转染的SMC，通过RT-PCR测定的iNOS mRNA水平以及NO的稳定氧化产物亚硝酸盐的产生在过表达IL-1α前体的SMC中显著升高，在过表达成熟IL-1α的SMC中适度升高。暴露于外源性IL-1β或TNF-α进一步刺激产生IL-1α的SMC中iNOS基因的表达；低水平的IL-1β（20 pg/ml）对用IL-1α前体质粒转染的SMC有效，而用成熟IL-1α质粒或仅用载体转染的SMC需要更高浓度的IL-1β（分别为200和2000 pg/ml）。外源性IL-1受体拮抗剂可阻止过表达IL-1α前体的SMC中iNOS mRNA水平和NO产生的增加，表明这些作用是由I型IL-1受体介导的。免疫染色研究表明，IL-1α前体通过细胞间接触刺激iNOS基因表达。与过表达IL-1α前体的细胞（通过绿色荧光蛋白的共表达鉴定）接触的细胞以及自身过表达IL-1α的细胞中iNOS的表达增强，但仅当细胞与另一个细胞接触时才增强。这些结果共同表明，IL-1α前体通过细胞间接触作为iNOS基因表达的自分泌和旁分泌增强剂，自身诱导适度的iNOS表达，并显著增强大鼠主动脉SMC对外源性细胞因子的反应性。
