Activity of Macrophage Processed Endotoxin
The difference in relative biological activity between isolated chemically purified endotoxic lipopolysaccharide (LPS) and endotoxins generated in situ with intact bacteria used in models of pathogenic infection is an important consideration for interpretation of experimental studies of pathogenesis. We have addressed this problem by establishing an in vitro model in which endotoxins are generated in their normal state following phagocytosis of intact bacteria. These studies have been facilitated by labeling specifically the LPS component of E. coli with tritiated galactose by growing an expimerase deficient strain of this organism in the presence of 3H-galactose. Macrophages are allowed to phagocytose the organisms for several hours followed by removal of unphagocytosed E. coli. This procedure results in more than 90% of the macrophage associated labeled bacteria being internalized. After 48–72 h of subsequent incubation, the radiolabeled material is harvested from the culture supernatant and from macrophage lysates. A partially purified LPS is obtained by phenol-water extraction. As a control in these studies, E. coli organisms are incubated with culture medium and then subjected to phenol-water extraction. The extracted LPS is purified by isopycnic density gradient ultracentrifugation to obtain LPS free from the majority of non-LPS-associated components.
KeywordsMitogenic Activity Limulus Amebocyte Lysate Splenocyte Proliferation Murine Splenocyte Limulus Amebocyte Lysate Assay
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