Leukocyte modulation inhibits endotoxin-induced disruption of intracellular calcium homeostasis.

Abstract

Sepsis is associated with disruption of intracellular calcium homeostasis. The specific mechanisms responsible for these changes remain unclear. This study attempts to modify endotoxin-induced alterations in erythrocyte intracellular calcium dynamics through modulation of the activated leukocyte and its products. Paired anticoagulated whole-blood specimens were obtained from healthy adult volunteers (n = 30). Specimens were incubated with 2 micrograms/mL endotoxin [lipopolysaccharide (LPS)] or saline control in the presence and absence of the white blood cell. Studies were repeated in specimens pretreated with allopurinol, superoxide dismutase, and pentoxifylline (PTX). After incubation, erythrocytes were separated, washed, and loaded with the fluorescent calcium chelator, FURA-2. Free cytosolic calcium concentration was determined on 10(6) cells using fluorescent spectroscopy. Values were computer-calculated every 1.8 seconds for 1 minute, and mean results were used for analysis. Differences were evaluated by analysis of variance. The LPS resulted in a significant increase in intracellular calcium concentration (LPS 70.95 nM vs. control 44.04 nM). This increase was dependent on the presence of the white blood cell and could not be induced in its absence (control 30.15 --> LPS 32.78). Pretreatment inhibited these endotoxin-induced alterations: allopurinol, 50.49 nM; superoxide dismutase, 49.12 nM; and PTX, 40.23 nM (p < 0.01). Endotoxin induces a significant increase in intracellular calcium concentration. This alteration seems to be mediated by activated neutrophils and can be ameliorated by both leukocyte modulation (PTX) and free radical scavengers.