ALPHA(2)-MACROGLOBULIN FUNCTIONS AS A CYTOKINE CARRIER TO INDUCE NITRIC-OXIDE SYNTHESIS AND CAUSE NITRIC OXIDE-DEPENDENT CYTOTOXICITY IN THE RAW-264.7 MACROPHAGE CELL-LINE
ALPHA(2)-MACROGLOBULIN FUNCTIONS AS A CYTOKINE CARRIER TO INDUCE NITRIC-OXIDE SYNTHESIS AND CAUSE NITRIC OXIDE-DEPENDENT CYTOTOXICITY IN THE RAW-264.7 MACROPHAGE CELL-LINE
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DOI：
10.1074/jbc.270.37.21919
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发表时间：
1995-09-15
影响因子：
4.8
通讯作者：
GONIAS, SL
中科院分区：
文献类型：
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作者：
LYSIAK, JJ;HUSSAINI, IM;GONIAS, SL
一氧化氮（NO）是巨噬细胞活动的重要介质。我们研究了 α(2)-巨球蛋白 (α(2)M) 对巨噬细胞 NO 合成的调节，α(2)-巨球蛋白是一种蛋白酶抑制剂和某些生长因子的载体，包括转化生长因子-β (TGF-β)。天然 alpha(2)M 和 alpha(2)M 受体识别的衍生物 alpha(2)M-甲胺 (alpha(2)M-MA) 可增加 RAW 264.7 鼠巨噬细胞系的亚硝酸盐生成。亚硝酸盐积累水平（NO 合成指标）与用干扰素-γ 观察到的水平相当。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑/琥珀酰脱氢酶测定或台盼蓝排除法测定，天然 α(2)M 和 α(2)M-MA 还增加了诱导型一氧化氮合酶 (iNOS) mRNA 水平，并显着减少了活细胞数量。在稍高的 α(2)M 浓度下，[H-3]胸苷掺入受到抑制。当加入 iNOS 竞争性抑制剂 N-G-单甲基-L-精氨酸时，所有这些活性几乎完全被抵消。通过原位切口平移，天然 alpha(2)M 和 alpha(2)M-MA 将具有可检测单链染色质切口的细胞百分比分别从 4% 增加到 12% 和 17%。这种变化表明细胞凋亡；然而，电子显微镜研究表明受损细胞的形态存在差异。为了确定 α(2)M 增加巨噬细胞 NO 合成的机制，我们研究了保留部分活性的蛋白水解 α(2)M 衍生物。与天然 alpha(2)M 和 alpha(2)M-MA 相比，保留生长因子结合活性的 600 kDa 衍生物可增加 RAW 264.7 细胞 NO 合成和 iNOS mRNA 水平。纯化的 18-kDa α(2)M 受体结合片段对 NO 合成或 iNOS 表达没有影响。因此，α(2)M 的生长因子载体活性而非其受体结合活性对于 NO 合成调节至关重要。 TGF-β 中和抗体模仿 alpha(2)M 的活性，增加 RAW 264.7 细胞 NO 合成并降低细胞活力。这些研究表明，α(2)M 可以调节巨噬细胞 NO 合成并深刻影响细胞功能，而无需进入细胞且不与特定的质膜受体结合。
Nitric oxide (NO) is an important mediator of macrophage activities. We studied the regulation of macrophage NO synthesis by alpha(2)-macroglobulin (alpha(2)M), a proteinase inhibitor and carrier of certain growth factors, including transforming growth factor-beta (TGF-beta). Native alpha(2)M and the alpha(2)M receptor-recognized derivative, alpha(2)M-methylamine (alpha(2)M-MA), increased nitrite generation by the RAW 264.7 murine macrophage cell line. The level of nitrite accumulation, which is an index of NO synthesis, was comparable to that observed with interferon-gamma. Native alpha(2)M and alpha(2)M-MA also increased inducible nitric oxide synthase (iNOS) mRNA levels and substantially reduced the number of viable cells, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium/succinyl dehydrogenase assay or trypan blue exclusion. At slightly higher alpha(2)M concentrations, [H-3]thymidine incorporation was inhibited. All of these activities were counteracted nearly completely when the iNOS competitive inhibitor N-G-monomethyl-L-arginine was included. By in situ nick translation, native alpha(2)M and alpha(2)M-MA increased the percentage of cells with detectable single strand chromatin nicks from 4 to 12 and 17%, respectively. This change suggested apoptosis; however, electron microscopy studies demonstrated variability in the morphology of injured cells. To determine the mechanism by which alpha(2)M increases macrophage NO synthesis, we studied proteolytic alpha(2)M derivatives that retain partial activity. A 600-kDa derivative that retains growth factor binding activity increased RAW 264.7 cell NO synthesis and iNOS mRNA levels comparable to native alpha(2)M and alpha(2)M-MA. The purified 18-kDa alpha(2)M receptor-binding fragment had no effect on NO synthesis or iNOS expression. Thus, the growth factor-carrier activity of alpha(2)M and not its receptor-binding activity is essential for NO synthesis regulation. A TGF-beta-neutralizing antibody mimicked the activity of alpha(2)M, increasing RAW 264.7 cell NO synthesis and decreasing cellular viability. These studies demonstrate that alpha(2)M can regulate macrophage NO synthesis and profoundly affect cellular function without gaining entry into the cell and without binding specific plasma membrane receptors.