The mitogenic effect of lipopolysaccharide: Metabolic processing of lipopolysaccharide by mouse lymphocytes

Abstract

Bacterial lipopolysaccharide (LPS), from S. enteritidis, consisted of two components, a heavy fraction with a molecular weight > 2 × 10 6 and a light fraction with a molecular weight < 40,000. Assuming the specific radioactivity to be the same for the two fractions, the ratio of heavy: light fractions was 4.6; both fractions were mitogenic to splenocytes of nude mice, but neither was active with lymphocytes from the genetically low responding strain C 3H/HeJ. When splenic lymphocytes from nude mice or from strains shown to be high responders to LPS were pulsed with labeled LPS for 1 hr the cell-bound label was progressively unloaded into the medium and recovered in the supernatant, reaching an asymptotic value of 70–80% unloading in about 6 hr. Chromatography of the unloaded material on agarose columns revealed that the ratio of heavy: light fractions declined progressively, reaching a value of 1.45 in 12 hr. Cells from high responders were incubated for 1 hr with purified heavypeak material, and the low molecular weight peak, unloaded from the cells after 12 hr, was purified by gel filtration and was concentrated by lyophilization; this fraction was found to be highly mitogenic when tested with splenic lymphocytes of the low LPS responder strain C 3H/HeJ. Thymic lymphocytes from high responder strains could not be stimulated by this fraction. Populations of thymocytes and splenocytes from the low responders bound and unloaded the LPS as did the high responders, but the ratio of heavy: light fractions remained constant during the unloading. Thus, only cells capable of showing a vigorous blastogenic response to LPS are able to process the molecule into a form which induces a high response from cells of the low responder strain. We hypothesize that an activated form of the low molecular weight species is in fact the active mitogen.