Chemical and Biologic Properties of A Protein-Rich Fraction of Bacterial Lipopolysaccharides

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Abstract
Butanol LPS preparations from three serotypes of Escherichia coli lose their ability to stimulate C3H/HeJ lymphocytes after the removal of a protein-rich fraction (LAP) by extraction with phenol. This loss of mitogenic activity for LPS nonresponder spleen cells is accompanied by an increase in equilibrium density of approximately 0.03 g/cm3 by each of the three lipopolysaccharides. The LAP preparations exhibited potent mitogenic activity for both LPS responder C3H/St and nonresponder C3H/HeJ lymphocytes. Each LAP produced an optimal response at approximately 10 µg/ml and each was toxic at a 10-fold higher concentration. Like LPS, LAP is mitogenic for congenitally athymic nude mice. Although LAP is isolated in association with LPS and shares certain of its biologic properties, the two B cell mitogens may be distinguished by several criteria. Of primary importance is the ability of LAP to stimulate LPS-nonresponder lymphocytes. In addition, these experiments demonstrate that the LAP-induced response by C3H/HeJ lymphocytes is not blocked by preincubation with high concentrations of LPS. The response to LAP by C3H/St spleen cells is not blocked by polymyxin B, an inhibitor of the lipid A-induced mitogenic response. Furthermore, such LAP activity is destroyed by mild alkaline hydrolysis under conditions that do not decrease LPS activity. Finally, we observed that the peak level of 3H-thymidine incorporation that results from stimulation with LPS is consistently lower than the level which is obtained with optimal LAP doses. The experiments presented here also demonstrate that isolated LAP and protein-free LPS can reassociate in vitro to generate an LPS-LAP complex with mitogenic activity for C3H/HeJ lymphocytes.

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