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

H. F. Galley, N. E. El Sakka, N. R. Webster, D. A. Lowes, B. H. Cuthbertson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)815-819
Number of pages5
JournalBritish Journal of Anaesthesia
Volume100
Issue number6
Early online date19 Apr 2008
DOIs
Publication statusPublished - Jun 2008

Keywords

  • blood, coagulation
  • blood, neutrophils
  • complications, septicaemia
  • NF-Kappa-B
  • human polymorphonuclear leukocytes
  • plasma cytokine levels
  • necrosis-factor-alpha
  • severe sepsis
  • oxidative stress
  • expression
  • antocoagulant
  • inflammation
  • apoptosis

Cite this

Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions. / Galley, H. F.; El Sakka, N. E.; Webster, N. R.; Lowes, D. A.; Cuthbertson, B. H.

In: British Journal of Anaesthesia, Vol. 100, No. 6, 06.2008, p. 815-819.

Research output: Contribution to journalArticle

Galley, H. F. ; El Sakka, N. E. ; Webster, N. R. ; Lowes, D. A. ; Cuthbertson, B. H. / Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions. In: British Journal of Anaesthesia. 2008 ; Vol. 100, No. 6. pp. 815-819.
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abstract = "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.",
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T1 - Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions

AU - Galley, H. F.

AU - El Sakka, N. E.

AU - Webster, N. R.

AU - Lowes, D. A.

AU - Cuthbertson, B. H.

PY - 2008/6

Y1 - 2008/6

N2 - 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.

AB - 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.

KW - blood, coagulation

KW - blood, neutrophils

KW - complications, septicaemia

KW - NF-Kappa-B

KW - human polymorphonuclear leukocytes

KW - plasma cytokine levels

KW - necrosis-factor-alpha

KW - severe sepsis

KW - oxidative stress

KW - expression

KW - antocoagulant

KW - inflammation

KW - apoptosis

U2 - 10.1093/bja/aen079

DO - 10.1093/bja/aen079

M3 - Article

VL - 100

SP - 815

EP - 819

JO - British Journal of Anaesthesia

JF - British Journal of Anaesthesia

SN - 0007-0912

IS - 6

ER -