Effects of recombinant human activated protein C in human models of endotoxin administration

Abstract

Alterations in the generation of activated protein C (APC) as well as in the interactions of APC with the endothelial protein C receptor are present in severe sepsis and acute lung injury. Administration of recombinant human activated protein C (rhAPC) improves the survival of critically ill patients with sepsis, but the mechanisms by which rhAPC produces benefit are not well defined. Human models of systemic and pulmonary endotoxin exposure may provide important insights into the mechanisms of action of rhAPC in critical illness. In volunteers given systemic endotoxin, rhAPC had minimal effects on physiologic parameters, including blood pressure, markers of inflammation, and measures of sepsis-induced coagulopathy. In contrast, in the setting of pulmonary endotoxin exposure, rhAPC decreased neutrophil migration into the airspaces and also diminished neutrophil chemotaxis. Administration of rhAPC did not affect other parameters of neutrophil function, including kinase activation, production of proinflammatory cytokines, or apoptosis. Such results indicate that the effects of rhAPC in inhibiting the infiltration of neutrophils into the lungs and other inflammatory sites may contribute to its beneficial effects in sepsis.

Figure 1.

Recombinant human activated protein C (rhAPC) decreases endotoxin-induced neutrophil and total leukocyte accumulation in the airspaces. Total white cells (WBC) and neutrophils present in bronchoalveolar lavage (BAL) obtained 16 h after endobronchial LPS instillation in volunteers administered rhAPC (filled circles; n = 8) or placebo (open circles; n = 7). Solid bar represents the mean of log values for each group. (A) Total WBC recovered per milliliter of BAL fluid. Administration of rhAPC significantly reduced recovery of all leukocytes (p = 0.037 by Student's t test). (B) Total neutrophils recovered per milliliter of BAL fluid. Administration of rhAPC reduced recovery of neutrophils (p = 0.048 by Student's t test). Adapted by permission from Reference 26.

Figure 2.

rhAPC decreases neutrophil migration. (A) Effect of rhAPC on neutrophil chemotaxis in vitro. Peripheral blood neutrophils isolated from healthy volunteers were exposed to rhAPC (solid diamonds: 10⁻⁴ g/ml; asterisks: 10⁻⁶ g/ml; solid triangles: 10⁻⁸ g/ml; solid circles: 10⁻¹⁰ g/ml) or left untreated (open squares) for 20 min. The mean chemotaxis index is shown (n = 5) for migration using an interleukin-8 (1 nM) gradient over a 2-h period. Pretreatment of the neutrophils with rhAPC resulted in a significant reduction in migration for all concentrations tested when compared with untreated cells (10⁻⁴ g/ml, p < 0.0001; 10⁻⁶ g/ml, p < 0.0001; 10⁻⁸ g/ml, p < 0.0001; 10⁻¹⁰ g/ml, p = 0.01, by two-way analysis of variance [ANOVA]). (B) Administration of rhAPC results in significant decreases in neutrophil chemotaxis ex vivo. Neutrophils were isolated from BAL obtained 16 h after endobronchial LPS-instillation in volunteers treated with rhAPC (solid circles) or placebo (open circles). Mean index ± SEM (n = 3 for rhAPC-treated group, n = 4 for untreated group; p = 0.012 by two-way ANOVA). This research was originally published in Reference 26.