Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions

Background. Activated protein C (APC) therapy reduces mortality in high-risk patients with severe sepsis. The effects of APC on inflammatory responses have also been reported. Neutrophils are key cells involved in early host defence mechanisms in sepsis. We hypothesized that APC may have effects on neutrophil function.

Methods. Neutrophils were isolated from 10 healthy volunteers and incubated in the presence of lipopolysaccharide (LPS) with and without a range of therapeutically relevant concentrations of recombinant human APC. Respiratory burst activity was determined using flow-activated cell sorting (FACS) analysis. Apoptosis was determined using Annexin-V staining and FACS analysis. Cytokine bead array was used to simultaneously measure three key cytokines in culture supernatants: interleukin (IL)-1 beta, -6, and -8. For chemotaxis, neutrophil migration through a 5 mu m membrane was measured in response to formyl-methyl-leucine-phenylalanine (FMLP) or IL-8 in the presence and absence of APC.

Results. Exposure to LPS resulted in significant increases in respiratory burst activity, IL-1 beta, -6, and -8 expression (all P < 0.0001) and decreased the number of apoptotic cells (P < 0.0001). The APC exposure resulted in a significant release of IL-6 (P=0.04) without affecting other cytokines. Respiratory burst and apoptosis were also unaffected by APC. Neutrophil chemotaxis in response to either FMLP or IL-8 was reduced by APC (P=0.005 and 0.007, respectively).

Conclusions. This pilot study showed that APC treatment of human neutrophils results in a decreased IL-6 expression and chemotaxis, without affecting other cytokines, apoptosis, or respiratory burst activity.