Isolation of a lipid a bound polypeptide responsible for LPS- initiated mitogenesis of C3H/HeJ spleen cells

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Abstract
Spleen cells from C3H/HeJ mice are responsive to some preparations of [Escherichia coli] LPS [lipopolysaccharide] but not to others; the method of extraction plays a critical role in determining activity. In particular, preparations of LPS prepared by extraction with aqueous butanol have potent mitogenic activity. The mitogenic activity of such positive preparations of LPS coisolates with the LPS during gel filtration chromatography and subsequent equilibrium banding on CsCl. Lipid A isolated from positive preparations of LPS also stimulates C3H/HeJ spleen cells. Treatment of positive preparations of LPS with hot phenol renders such preparations nonmitogenic for C3H/HeJ spleens, yet activity for other strains is only moderately decreased. These experiments 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. These experiments explore in greater detail the role of lipid A in the stimulation of C3H/HeJ spleen cells and attempt to distinguish between the 2 potential modes of action of phenol on LPS, i.e., the chemical alteration of the lipid A or the removal of a tightly bound contaminant by phenol treatment. Mitogenic activity of positive preparations of LPS is apparently associated with a phenol soluble polypeptide of .apprx. 10,000 MW. After partial purification this polypeptide initiates a significant mitogenic response at concentrations as low as 10 .mu.g/ml. The C3H/HeJ mouse strain is apparently a true nonresponder to the stimulatory effects of the lipid A region of LPS.