Mathias S, Perez R, Diffley P
Department of Biological Sciences, University of Notre Dame, IN 46556.
J Immunol. 1990 Nov 15;145(10):3450-5.
The purpose of this study was threefold: to determine if the variant surface coat glycoprotein (VSG) of Trypanosoma brucei rhodesiense induces IL-1 alpha; to study the kinetics of IL-1 alpha transcription, maturation and secretion; and to compare VSG to LPS in its ability to induce IL-1 alpha. VSG was added to cultures of the P388D1 murine macrophage cell line. RNA was dotted onto nitrocellulose and hybridized with a murine IL-1 alpha cDNA probe. Maximal production of IL-1 alpha mRNA occurred in a dose- and time-dependent manner, peaking at 25 micrograms/ml VSG, within 2 h. Induction of IL-1 alpha was not due to contaminants because 1) absorption of VSG with a mAb abrogated IL-1 alpha mRNA synthesis, 2) the addition of polymyxin B did not affect mRNA levels, and 3) cellular IL-1 alpha was detectable in VSG-treated splenocytes from endotoxin nonresponder C3H/HeJ mice. Murine splenic macrophages also had enhanced levels of IL-1 alpha mRNA after administration of VSG in vivo or during an acute infection. Antiserum generated against the synthetic peptide SGDDSKYPV (amino acids 177-185 from the murine IL-1 alpha sequence) was used to measure the levels of the 33-, 22-, and 14-kDa proteins in cell lysates and medium of VSG-stimulated P388D1 cells. The 22-kDa protein was the predominant cellular form until secretion started. Secretion of the 14-kDa form began abruptly 6 to 8 h after the addition of VSG. By 12 h, the 33-kDa precursor was the major cytoplasmic form. In comparative analyses, LPS-stimulated P388D1 cells produced more transcript, generated peak levels of 22-kDa protein 3 h earlier, and began to secrete the 14-kDa molecule 5 h earlier. The rate of IL-1 alpha accumulation in the medium was linear between 6 and 24 h after LPS treatment, but began to drop by 8 h in VSG-treated cells. Functional (comitogenic) IL-1 activity was also detected in media from VSG-treated splenic macrophages and P388D1 cells. Activity peaked at 50 micrograms/ml and was lost if 0.2% IL-1 antisera were added to the cultures.
确定罗德西亚布氏锥虫的变异表面糖蛋白（VSG）是否诱导白细胞介素-1α（IL-1α）；研究IL-1α转录、成熟和分泌的动力学；并比较VSG与脂多糖（LPS）诱导IL-1α的能力。将VSG添加到P388D1小鼠巨噬细胞系的培养物中。将RNA点样到硝酸纤维素膜上，并用小鼠IL-1α cDNA探针进行杂交。IL-1α mRNA的最大产量呈剂量和时间依赖性，在25微克/毫升VSG时于2小时内达到峰值。IL-1α的诱导不是由于污染物，因为1）用单克隆抗体吸收VSG消除了IL-1α mRNA的合成，2）添加多粘菌素B不影响mRNA水平，3）在内毒素无反应性C3H/HeJ小鼠经VSG处理的脾细胞中可检测到细胞IL-1α。在体内给予VSG后或急性感染期间，小鼠脾巨噬细胞中IL-1α mRNA水平也有所提高。针对合成肽SGDDSKYPV（来自小鼠IL-1α序列的氨基酸177 - 185）产生的抗血清用于测量VSG刺激的P388D1细胞的细胞裂解物和培养基中33 kDa、22 kDa和14 kDa蛋白的水平。在分泌开始前，22 kDa蛋白是主要的细胞形式。添加VSG后6至8小时，14 kDa形式的分泌突然开始。到12小时时，33 kDa前体是主要的细胞质形式。在比较分析中，LPS刺激的P388D1细胞产生更多的转录本，22 kDa蛋白的峰值水平提前3小时出现，14 kDa分子的分泌提前5小时开始。LPS处理后6至24小时，培养基中IL-1α的积累速率呈线性，但在VSG处理的细胞中8小时后开始下降。在VSG处理的脾巨噬细胞和P388D1细胞的培养基中也检测到了功能性（致有丝分裂）IL-1活性。活性在50微克/毫升时达到峰值，如果向培养物中添加0.2%的IL-1抗血清则活性丧失。
