Criado M, Lindstrom J M, Anderson C G, Dennert G
J Immunol. 1985 Dec;135(6):4245-51.
The channel-forming polyperforins P1 and P2 are thought to be formed from the contents of dense core vesicles of cytolytic effector cells. To test this hypothesis, granules from various cytotoxic effector cells were assayed for cytolytic activity on nucleated or unnucleated targets. The results show that in general, granules from cytolytic effector cells are cytolytic, whereas granules from noncytotoxic cells are not. Cytotoxicity of granules is not specific, but there appears to be a preference in that nucleated targets are lysed better than are erythrocytes by granules from T killer or natural killer cells. Granules from CTLL-2, however, preferentially lyse erythrocyte targets. This cell line has been in culture for a long period of time and has lost its cytotoxicity. We tested whether granules from CTLL-2 caused formation of transmembrane pores in erythrocyte target membranes. We found that granule- and complement-induced lesions have similar pore sizes. They are big enough to allow the total release of alpha-bungarotoxin, an 8000 Mr polypeptide with dimensions of 4 X 2.5 nm. Larger molecules are released partially or not at all. Under acidic conditions (pH 5.4) granules do not permeabilize target membranes. This may suggest a pH-dependent control mechanism in the formation, insertion, or function of polyperforin channels, in addition to a previously recognized Ca2+-dependent mechanism. Permeabilization of lipid vesicles by granules was studied to explore what the molecular requirements for channel insertion into membranes may be. Release of alpha-bungarotoxin induced by granules was observed in liposomes made of soybean lipid with or without cholesterol, suggesting that no membrane component other than lipid is required for the insertion of polyperforins, and that the action of polyperforins does not require other mechanisms in the target cell. When pure lecithin from soybean and egg, or synthetic phosphatidylcholines were used, slower release or no release of macromolecules was observed. We suggest that some kind of lipid specificity is required for perforin action. This may be related to the hydrophobic region of the lipid bilayer rather than to the polar portion, because different lecithins with varying fatty acid composition gave similar results.
形成通道的多穿孔素P1和P2被认为是由溶细胞效应细胞的致密核心囊泡内容物形成的。为了验证这一假设，对来自各种细胞毒性效应细胞的颗粒进行了检测，以观察其对有核或无核靶标的溶细胞活性。结果表明，一般来说，溶细胞效应细胞的颗粒具有溶细胞活性，而非细胞毒性细胞的颗粒则没有。颗粒的细胞毒性不具有特异性，但似乎存在一种偏好，即T杀伤细胞或自然杀伤细胞的颗粒对有核靶标的裂解效果比对红细胞的更好。然而，CTLL-2细胞系的颗粒优先裂解红细胞靶标。该细胞系已培养很长时间并失去了细胞毒性。我们测试了CTLL-2细胞系的颗粒是否会在红细胞靶膜上形成跨膜孔。我们发现颗粒诱导的损伤和补体诱导的损伤具有相似的孔径。这些孔径大到足以使8000道尔顿、尺寸为4×2.5纳米的α-银环蛇毒素完全释放。更大的分子则部分释放或根本不释放。在酸性条件下（pH 5.4），颗粒不会使靶膜通透。这可能表明，除了先前认识到的钙依赖性机制外，多穿孔素通道的形成、插入或功能还存在pH依赖性控制机制。研究了颗粒对脂质囊泡的通透作用，以探索通道插入膜的分子要求可能是什么。在含有或不含有胆固醇的大豆脂质制成的脂质体中观察到了颗粒诱导的α-银环蛇毒素释放，这表明多穿孔素的插入除了脂质外不需要其他膜成分，并且多穿孔素的作用在靶细胞中不需要其他机制。当使用来自大豆和鸡蛋的纯卵磷脂或合成磷脂酰胆碱时，观察到大分子释放较慢或不释放。我们认为穿孔素的作用需要某种脂质特异性。这可能与脂质双层的疏水区域有关，而不是与极性部分有关，因为具有不同脂肪酸组成的不同卵磷脂给出了相似的结果。
