BJA Advance Access originally published online on April 19, 2008
British Journal of Anaesthesia 2008 100(6):815-819; doi:10.1093/bja/aen079
Activated protein C inhibits chemotaxis and interleukin-6 release by human neutrophils without affecting other neutrophil functions
1 Academic Unit of Anaesthesia and Intensive Care
2 Health Services Research Unit, School of Medicine, University of Aberdeen, Aberdeen, UK
3 Department of Clinical Pathology, University of Alexandria, Alexandria, Egpyt
* Corresponding author. E-mail: h.f.galley{at}abdn.ac.uk
Accepted for publication February 29, 2008.
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Abstract |
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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)-1β, -6, and -8. For chemotaxis, neutrophil migration through a 5 µm 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-1β, -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.
Keywords: blood, coagulation; blood, neutrophils; complications, septicaemia
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Introduction |
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Sepsis is the most common cause of death in critically ill patients, accounting for
20% of deaths of intensive care unit admissions and associated with an overall mortality rate of 30%.1 Progression to severe sepsis and septic shock carries mortality rates of 50 and 80%, respectively. The only approved specific treatment for sepsis is the recently introduced activated protein C (APC). The use of recombinant human APC [drotrecogin alfa (activated), marketed under the brand name Xigris] has been shown to reduce the mortality rate in patients with severe sepsis (Prowess Study).2 In addition to its anti-coagulant actions, APC has been found to have anti-inflammatory activity, which may contribute to its clinical effectiveness. However, the extent of such effects is not completely known. The neutrophil is the first line of cellular host defence. It is a pivotal effector cell which is able to respond to mediators and recruit additional neutrophils to the sites of inflammation. The directed migration of neutrophils in response to activating factors is essential to their margination from the circulation. The respiratory burst is essential for the cytotoxic arsenal of these cells and neutrophils also have an important role in autocrine and paracrine regulation of immune cells through release of cytokines. The limitation of neutrophil activation is controlled by apoptosis. Neutrophil accumulation and activation have been implicated in the development of multiple organ failure in sepsis.3
In vitro, although APC has been shown to modulate aspects of cell function in a variety of cell types, the effects on neutrophil function are less clear. We determined the effect of APC upon aspects of human neutrophil function, including chemotaxis, respiratory burst, cytokine expression, and apoptosis.
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Methods |
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After Local Research Ethics Committee approval and informed consent, neutrophils were isolated from heparinized venous blood from 10 healthy volunteers [four males, six females, median (range) age 33 (20–50) yr] using single density gradient centrifugation as described previously.4 For chemotaxis studies, cells were placed in a microchemotaxis chamber in which a 5 µm pore-sized cellulose nitrate filter separated the upper chamber from the lower chamber. Migration in response to 1 µM FMLP and 1 nM IL-8 with and without pre-treatment with APC (100 ng ml–1) was determined in triplicate. After allowing 30 min for migration, filters were washed and stained with giemsa, then fixed and counted manually (3 fields well–1) by a single observer blinded to cell treatment. For other investigations, neutrophils were incubated overnight at 37°C in a humid atmosphere of 5% carbon dioxide/95% air in the presence of 2 µg ml–1 lipopolysaccharide (LPS) or phosphate-buffered saline (PBS) as control, 60 min after the addition of 0, 0.2, 2, 20, or 200 ng ml–1 recombinant human APC. The therapeutic concentration of APC during treatment in patients with severe sepsis is
50 ng ml–1.5 To assess membrane changes, one of the earliest features of apoptosis, Annexin-V and propidium iodide staining was used, as we have described previously6 and the samples were analysed by fluorescence-activated cell sorting (FACS) analysis. Annexin-V has a high affinity for phosphatidylserine, which undergoes translocation from the inner to the outer part of the plasma membrane early in apoptosis. As necrotic cells also expose phosphatidylserine as a result of loss of membrane integrity, propidium iodide was used as an exclusion dye to discriminate apoptotic from necrotic cells. The FACS analysis was performed using FACSCalibur (Becton Dickinson, San Jose, CA, USA) with an argon laser exciting at 488 nm; green fluorescence of fluorescein was detected at 530 nm, and red fluorescence of propidium iodide was detected at 610 nm. Typically, 10 000 events per sample were collected in list mode, stored, and analysed by CellQuest Pro (Becton Dickinson). Apoptotic neutrophils were calculated as the percentage of gated cells showing positive uptake of annexin-V and negative uptake of propidium iodide.
The generation of reactive oxygen intermediates was assayed using the modified Smith and Weidemann's method.7 Briefly, neutrophils were incubated with 200 nM phorbol myristate acetate (PMA) or PBS as a control for 10 min at 37°C, which stimulates NAPDH oxidase indirectly through the activation of protein kinase C, leading to the release of superoxide anion, which decomposes to hydrogen peroxide. Dehydrorhodamine-1,2,3 is a non-fluorescent probe which, when oxidized by hydrogen peroxide, forms the fluorescent compound rhodamine-1,2,3. For FACS analysis, neutrophils were gated using forward and side scatter and the green fluorescence of rhodamine was detected at 530 nm, using cells with PBS and without PMA as negative controls. A minimum of 10 000 events was recorded.
Cytokine accumulation in cell supernatants was measured using a cytometric bead assay after overnight incubation with LPS (Becton Dickinson). Briefly, four bead populations with distinct fluorescent properties are provided, coated with antibodies for tumour necrosis factor-
(TNF-), IL-1β, IL-6, and IL-8. Culture supernatants or recombinant cytokine standards were mixed with the beads and incubated for 3 h at room temperature. The FACS analysis was performed and the mean fluorescent intensity was plotted against standard cytokine concentrations using a specialized assay software. The within-assay precision (coefficient of variation) was 2–8% and between-assay precision was 4–10%.
Data were not normally distributed and so are presented as median (range) and were analysed using Friedman analysis of variance with Wilcoxon signed-rank post hoc test.
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Chemotaxis
The migration of neutrophils through a 5 µm filter was significantly increased when cells were incubated with either FMLP or IL-8 compared with saline control (data not shown). When cells were exposed to APC, the numbers migrating through the filters were significantly reduced compared with those exposed to FMLP or IL-8 alone (P=0.005 and 0.007, respectively, Fig. 1).
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Respiratory burst
The percentage of cells showing respiratory burst activity increased from 28.8 (23.0–49.9)% in control cells to 57.0 (34.0–74.2)% (P=0.0001) in cells exposed to LPS. However, there was no significant effect of incubation with APC (Fig. 2A).
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Apoptosis
Neutrophils are committed to apoptosis and in the absence of activation signals,
50% of cells will be in the early stages of apoptosis after 16 h culture.6 8 In the presence of LPS, apoptosis is inhibited. As expected, we found a significant decrease in apoptotic cells identified by Annexin-V when neutrophils were cultured with LPS compared with PBS controls [38.6 (31.3–50.1)% compared with 59.7 (50.1–75.0)%, P<0.0001]. When exposed to APC, there was no significant change in the number of apoptotic cells (Fig. 2B).
Cytokines
There was very marked inter-individual variation in responses to LPS in terms of all cytokines measured. Concentration of IL-1β increased from 29.4 (18.7–1957.3) to 61.7 (33.1–3862.6) pg ml–1 (P<0.0001) upon exposure to LPS compared with control, but there was no difference found when cells were treated with APC (data not shown). The concentration of IL-6 also increased markedly, from 84.9 (7.9–4167.2) to 200.7 (18.1–4268.5) pg ml–1 (P<0.0001), when cells were exposed to LPS, and exposure of cells to APC caused a concentration-dependent decrease in IL-6 release with a median decrease of 50% at the highest dose of APC (P=0.04, Fig. 3). The concentration of IL-8 was also significantly higher in supernatants from cells stimulated with LPS compared with control 0.8 (0.1–2.8) ng ml–1 compared with 2.8 (0.2–3.4) ng ml–1 (P<0.001), but APC had no significant effect upon the concentrations of IL-8 (data not shown).
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Discussion |
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We found that APC caused a concentration-dependent inhibition of IL-6 release from LPS-stimulated human neutrophils, without affecting other key cytokines. Migration of neutrophils towards chemoattractants was also inhibited by APC, but there was no effect on respiratory burst or apoptosis rates.
Sepsis has an incidence of 750 000 cases per year in the United States, with an overall mortality rate of nearly 30%.1 As populations age, the incidence of sepsis is projected to increase and the mortality rate of sepsis increases steadily with age. Development of several novel therapeutic strategies initially showed promise in the laboratory setting, yet has no survival benefit in human trials.9 Recombinant human APC treatment, however, was shown to decrease 28 day mortality in patients with severe sepsis.2 The subgroup analysis showed that APC was less effective in patients with lower APACHE II scores and so APC was approved only for use in patients with severe sepsis and a high predicted mortality.10 A follow-up study was terminated early because of an increase in serious bleeding complications in patients treated with APC who had a low risk of death, defined as having an APACHE II score <25.11
Protein C is a vitamin K-dependent serine protease zymogen which is converted to APC by the thrombin–thrombomodulin–endothelial protein C receptor (EPCR) complex. APC is one of the most important physiological anticoagulants. Various reports of in vitro modulation of apoptosis and aspects of immune function by APC have been reported.12 Neutrophils, monocytes, and endothelial cells express EPCR12 13 and therefore have the potential for functional modulation by APC. A better understanding of the modes of action of APC outside its role on coagulation is relevant for widening the therapeutic potential of APC. Therefore, we investigated the effects of APC on various neutrophil functions.
Neutrophils provide the first line for host defence mechanisms in sepsis.3 In response to chemotaxins, they migrate to the sites of injury where they are essential for clearing micro-organisms. However, they also have the potential to cause host tissue damage and may be the first step in the development of lung dysfunction in sepsis. Neutrophils produce and respond to chemotaxins including IL-8 and also produce a range of pro-inflammatory cytokines and reactive oxygen species. They can cause tissue damage through the so-called respiratory burst, producing reactive oxygen species and hydrogen peroxide. The enzyme myeloperoxidase is released from granules in the cells, and catalyses the production of hypochlorous acid. Patients with sepsis have evidence of oxidative stress and poor antioxidant defences.14–16 Unstimulated neutrophils spontaneously become apoptotic, but their persistence in tissues may prolong and exacerbate host injury after an inflammatory stimulus.3 6 8 We have shown that neutrophil apoptosis is delayed in patients with sepsis and organ failure.6
In the present study, we measured the effects of APC on several aspects of neutrophil function in vitro in cells isolated from healthy volunteers which were treated with an inflammatory stimulant. We found that the respiratory burst of neutrophils in response to PMA was not affected by APC, in agreement with Sturn and colleagues.13 Early spontaneous apoptosis of neutrophils was not affected by APC exposure. This concurs with a previous report of no effect of APC on neutrophil apoptosis.13 However, other reports showed that APC did inhibit camptothecin- or staurosporine-induced apoptosis in monocyte and endothelial cell lines, respectively,17 18 suggesting cell type differences. We also found that migration of neutrophils towards either FMLP or IL-8 was inhibited by APC. In human volunteers given systemic endotoxin, APC had minimal effects on markers of inflammation.19 In contrast, when endotoxin was instilled directly into the lungs of volunteers, i.v. APC administration decreased ex vivo neutrophil chemotaxis, and reduced neutrophil accumulation in the lungs without affecting IL-6, IL-8, or apoptosis,19 in agreement with our study. These results may suggest that the effect of APC in inhibiting the infiltration of neutrophils into the lungs and other inflammatory sites may contribute to its beneficial effects in sepsis. Interestingly, neither neutrophil nor monocyte phagocytosis has been shown to be modulated by APC.13 17
We found that although IL-1β release was significantly higher in cells after treatment with LPS, exposure to APC had no modulatory effect, in agreement with a previous study using the monocyte cell line, THP-1.17
We also found that neutrophil IL-6 expression was markedly inhibited by APC exposure. This confirms a previous in vitro study which showed that APC exposure reduced IL-6, also measured using a cytometric bead assay, released from THP-1 cells.17 Our findings also confirm the lower plasma IL-6 concentrations in patients treated with APC in the PROWESS study.2 However, a study using isolated human monocytes failed to show any effect of APC on low-dose LPS-induced IL-6,20 and a recent study reported no effect of APC on IL-6 in pigs treated with LPS.21 IL-6 has important roles in the host response mechanisms to sepsis. It causes B-cell differentiation to antibody forming cells, T-cell activation, and proliferation and formation of adhesion molecules. IL-6 contributes to the acute phase and inflammatory responses but also down-regulates the production of TNF- and IL-1β. High IL-6 concentrations have been shown to contribute to both morbidity and mortality in sepsis.22
In the present study, the release of IL-8 from neutrophils was not affected by APC. In THP-1 cells, LPS-induced IL-8 was decreased by APC in vitro.17 However, in a study of isolated human monocytes, APC had no effect on IL-820 and in endothelial cells APC was reported to up-regulate both IL-6 and IL-8.23 Thus, there are clear cell-specific differences.
The mechanism of the effect of APC on IL-6 has been suggested to be at the transcriptional level; in vitro studies using monocyte cell lines have described inhibition of translocation of the transcription factor nuclear factor kappa B (NF
B) into the nucleus when cells were exposed to APC.24 25 IL-6 is, in part, regulated by NFB, depending on cell type and stimulus. The potential effect of APC on other transcription factors has not been studied. Any effect on NFB is likely to have profound implications for patients with sepsis as others and we have shown marked activation of NFB in neutrophils from patients with severe sepsis, associated with mortality.26–29
In summary, we showed that at therapeutically relevant concentrations, APC suppressed the release of IL-6 from stimulated human neutrophils and inhibited chemotaxis, without affecting the respiratory burst, apoptosis, and expression of other key cytokines. These effects are likely to be cell-type specific but may have implications for treatment of patients with APC.
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Financial support was obtained from Eli Lilly, Basingstoke, UK.
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Declaration of interest. Dr Cuthbertson received funding from Eli Lilly for this research. Prof. Webster has received honoraria for lectures from Eli Lilly. 
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