Morrison D C, Betz S J, Jacobs D M
J Exp Med. 1976 Sep 1;144(3):840-6. doi: 10.1084/jem.144.3.840.
The experiments by Sultzer and Nilsson (1), and later by Watson and Riblet (2), established that spleen cells from the C3H/HeJ strain of mouse were refractory to the mitogenic effects of bacterial lipopolysaccharides (LPS). More recently, however, experiments from our laboratory (3) demonstrated that spleen cells from C3H/HeJ mice were in fact responsive to some preparations of LPS but not to others, and that the method of extraction played a critical role in determining activity. In particular, preparations of LPS prepared by extraction with aqueous butanol had potent mitogenic activity. Our data showed that the mitogenic activity of such positive preparations of LPS coisolated with the LPS during gel filtration chromatography and subsequent equilibrium banding on CsCl. In addition, lipid A isolated from positive preparations of LPS was also capable of stimulating C3H/HeJ spleen cells. Taken together, these experiments provided rather convincing data that it was the LPS (in particular the lipid A) itself, or some contaminant very tightly bound to the lipid A, which was responsible for its biological activity. We further demonstrated that treatment of positive preparations of LPS with hot phenol rendered such preparations nonmitogenic for C3H/HeJ spleens, yet activity for other strains was only moderately decreased. These experiments would suggest either that the phenol treatment chemically alters the lipid A region of the LPS molecule or that such treatment removes the putative tightly bound contaminant responsible for C3H/HeJ mitogenesis. In the experiments reported here, we have explored in greater detail the role of lipid A in the stimulation of C3H/HeJ spleen cells. For these experiments we have utilized our earlier observations that the antibiotic polymyxin B forms a highly stable molecular complex with the lipid A region of LPS (4), and that such polymyxin B-LPS complexes are unable to mitogenically stimulate B lymphocytes (5). In addition, we have attempted to distinguish between the two potential modes of action of phenol on LPS, namely, the chemical alteration of the lipid A or the removal of a tightly bound contaminant by phenol treatment. The results of the experiments we report here support the interpretation that mitogenic activity of positive preparations of LPS is associated with a low mol wt phenol soluble polypeptide of approximately 10,000 mol wt. After partial purification, this polypeptide intitiates a significant mitogenic response at concentrations as low as 10 mug/ml. We conclude that the C3H/HeJ strain of mouse is a true nonresponder to the stimulatory effects of the lipid A region of LPS.
苏尔策和尼尔森（1）以及后来沃森和里布利特（2）所做的实验表明，C3H/HeJ品系小鼠的脾细胞对细菌脂多糖（LPS）的促有丝分裂作用具有抗性。然而，最近我们实验室的实验（3）表明，C3H/HeJ小鼠的脾细胞实际上对某些LPS制剂有反应，而对其他制剂则无反应，并且提取方法在决定活性方面起着关键作用。特别是，用水性丁醇提取制备的LPS制剂具有很强的促有丝分裂活性。我们的数据显示，在凝胶过滤色谱及随后在CsCl上进行平衡区带分离过程中，此类LPS阳性制剂的促有丝分裂活性与LPS共分离。此外，从LPS阳性制剂中分离出的脂多糖A也能够刺激C3H/HeJ脾细胞。综合来看，这些实验提供了相当有说服力的数据，表明正是LPS（特别是脂多糖A）本身，或者是与脂多糖A紧密结合的某种污染物，导致了其生物活性。我们进一步证明，用热酚处理LPS阳性制剂会使其对C3H/HeJ脾细胞失去促有丝分裂活性，但对其他品系的活性仅略有降低。这些实验表明，要么酚处理在化学上改变了LPS分子的脂多糖A区域，要么这种处理去除了导致C3H/HeJ有丝分裂的假定紧密结合污染物。在本文报道的实验中，我们更详细地探讨了脂多糖A在刺激C3H/HeJ脾细胞中的作用。对于这些实验，我们利用了我们早期的观察结果，即抗生素多粘菌素B与LPS的脂多糖A区域形成高度稳定的分子复合物（4），并且这种多粘菌素B - LPS复合物不能促有丝分裂地刺激B淋巴细胞（5）。此外，我们试图区分酚对LPS的两种潜在作用方式，即脂多糖A的化学改变或通过酚处理去除紧密结合的污染物。我们在此报告的实验结果支持这样的解释，即LPS阳性制剂的促有丝分裂活性与一种分子量约为10,000的低分子量酚溶性多肽有关。经过部分纯化后，这种多肽在浓度低至10微克/毫升时就能引发显著的促有丝分裂反应。我们得出结论，C3H/HeJ品系小鼠对LPS脂多糖A区域的刺激作用是真正的无反应者。
