Noncleavable transmembrane mouse tumor necrosis factor-α (TNFα) mediates effects distinct from those of wild-type TNFα in vitro and in vivo
Noncleavable transmembrane mouse tumor necrosis factor-α (TNFα) mediates effects distinct from those of wild-type TNFα in vitro and in vivo
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DOI：
10.1074/jbc.274.53.38112
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发表时间：
1999-12-31
影响因子：
4.8
通讯作者：
Imboden, MA
中科院分区：
文献类型：
--
作者：
Mueller, C;Corazza, N;Imboden, MA
肿瘤坏死因子-α（TNF α）以两种生物活性形式存在，26-kDa跨膜形式和蛋白水解切割和分泌形式。我们通过突变相应的DNA序列依次灭活小鼠TNF α的所有三个已知切割位点。用非分泌型突变体TNF α转染的鼠T细胞杂交瘤以细胞接触依赖性方式有效地裂解L929靶细胞，并诱导小鼠内皮瘤细胞上血管细胞粘附分子-1的表达。随后将编码该突变体的基因组小鼠TNF α克隆作为转基因导入TNF α（-/-）α-光毒素-α（-/-）小鼠。在转基因中保留TNF基因座的富含3'AU的调控元件，以确保充分的基因调控。跨膜TNF α转基因小鼠完全免于内毒素休克，并且在用脂多糖刺激后血清中未检测到TNF α生物活性。然而，来自这些动物的活化的CD 4 T细胞以细胞接触依赖性方式裂解L929细胞。脂多糖给药后，跨膜TNF α转基因小鼠产生的白细胞介素-12水平显著高于野生型小鼠或TNF缺陷小鼠。这表明跨膜TNF α可能极大地影响体内细胞免疫应答的过程，并在体外和体内发挥与分泌的TNF α定量和定性不同的功能。
Tumor necrosis factor-alpha (TNF alpha) exists in two biologically active forms, a 26-kDa transmembrane form and a proteolytically cleaved and secreted form. We sequentially inactivated all three known cleavage sites of mouse TNF alpha by mutating the corresponding DNA sequences. A murine T cell hybridoma transfected with the nonsecretable mutant TNF alpha efficiently lysed L929 target cells in a cell contact-dependent manner and induced expression of vascular cell adhesion molecule-1 on mouse endothelioma cells. A genomic mouse TNF alpha clone encoding this mutant was subsequently introduced as a transgene into TNF alpha(-/-) lymphotoxin-alpha(-/-) mice. The 3' AU-rich regulatory elements of the TNF locus were maintained in the transgene to assure adequate gene regulation. Transmembrane TNF alpha transgenic mice were fully protected from endotoxic shock, and no TNF alpha bioactivity was detectable in the serum after stimulation with lipopolysaccharide. Activated CD4 T cells from these animals, however, lysed L929 cells in a cell contact-dependent way. After administration of Lipopolysaccharide, transmembrane TNF alpha transgenic mice produced significantly higher levels of interleukin-12 than wild-type mice or TNF-deficient mice. This indicates that transmembrane TNF alpha may greatly affect the course of a cellular immune responses in vivo and exerts quantitatively and qualitatively distinct functions from secreted TNF alpha in vitro and in vivo.