Early In Vitro Lipopolysaccharide-Induced Serum Protein Aggregation in Tolerant Serum

Abstract : In a rat LPS shock model, an intravenous injection of LPS (3 - 30 mg/kg body weight) corresponds to an estimated peak load of 0.13 1.3 mg LPS/mi serum. We tested the effects of such LPS concentrations in vitro. Rat serum was incubated with LPS (0.1 - 2.0 mg LPS/mi serum) at 370C. The exact amount of precipitate (ppt) depended on both LPS concentration and incubation time. By varying the incubation time (5 see - 60 min), with a set concentration of LPS (0.5 mg/mi), we found differences between normal and tolerant rat serum. For normal serum, the amount of ppt was the same at all incubation time points. With tolerant serum, the amount of ppt after 5 min incubation was twice the amount from normal serum. Much of this enhanced ppt faded by the 10 min incubation time point. Precipitated protein was freed of LPS by NaCI/EDTA extraction, and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions. Some protein from normal serum aggregates with LPS, as shown by SDS-PAGE bands at 18, 28, 68, 1 1 0, and 190 kDa. Two particular components, at 65 Kda (preceding rat albumin) and 43 kDa, distinguished tolerant from normal serum. These precipitated from tolerant serum within 5 sec of incubation, but required much longer incubation (2 - 3 min) to be precipitated from normal serum. Neither band was detected using non-reducing conditions. Unlike normal serum, tolerant serum interacts with LPS in vitro in a dynamic way, which may hold clues to the protective effects of tolerance. LPS, serum, aggregation, rats, tolerance, neutralization.