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Keywords:

  • protein C;
  • TNF-α;
  • NF-κB;
  • sepsis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

Activated protein C (APC) protects against sepsis in animal models and inhibits the lipopolysacharide (LPS)-induced elaboration of proinflammatory cytokines from monocytes. The molecular mechanism responsible for this property is unknown. We assessed the effect of APC on LPS-induced tumour necrosis factor α (TNF-α) production and on the activation of the central proinflammatory transcription factor nuclear factor-κB (NF-κB) in a THP-1 cell line. Cells were preincubated with varying concentrations of APC (200 µg/ml, 100 µg/ml and 20 µg/ml) before addition of LPS (100 ng/ml and 10 µg/ml). APC inhibited LPS-induced production of TNF-α both in the presence and absence of fetal calf serum (FCS), although the effect was less marked with 10% FCS. APC also inhibited LPS-induced activation of NF-κB, with APC (200 µg/ml) abolishing the effect of LPS (100 ng/ml). The ability of APC to inhibit LPS-induced translocation of NF-κB is likely to be a significant event given the critical role of the latter in the host inflammatory response.

Protein C (PC) is a natural anticoagulant that plays an important role in coagulation homeostasis by proteolytic cleavage and inactivation of procoagulant factors factor Va and VIIIa ( Esmon, 1987). In addition to its well-defined role in coagulation, recent evidence has suggested that PC also has potent anti-inflammatory properties, namely activated protein C (APC) protects baboons from lethal doses of Escherichia coli endotoxin ( Taylor et al, 1987 ) and inhibits lipopolysacharide (LPS) induced proinflammatory cytokine production from monocytes ( Hancock et al, 1995 ; Murakami et al, 1997 ). Moreover, the apparent reduction in cytokine-mediated multiorgan failure associated with PC replacement therapy in patients with severe meningococcaemia further supports that this pathway plays an important role in negatively modulating the host inflammatory response ( Rivard et al, 1995; Smith et al, 1997 ; Rintala et al, 1998 ; Smith & White, 1999). The molecular mechanism responsible for the anti-inflammatory properties of APC is unknown. A potential molecular target for APC is nuclear factor-κB (NF-κB), which is a critical transcription factor in LPS-induced proinflammatory cytokine production ( Yao et al, 1997 ).

NF-κB functions as a heterodimer, usually consisting of p65 and p50 protein subunits ( Siebenlist et al, 1994 ). In unstimulated cells, NF-κB is localized in the cytoplasm by binding to I-κBα and I-κBβ and thus preventing nuclear translocation ( Baldwin, 1996). Activation of cells with LPS, cytokines, viruses, oxidants or activators of protein kinase C leads to the induction of several signal transduction pathways which result in the phosphorylation of I-κB kinase ( DiDonato et al, 1996 ). The subsequent phosphorylation of I-κB results in rapid proteasomal degradation with release of NF-κB, which in turn is translocated into the nucleus where it binds to specific sequences in the promoter regions of target genes responsible for the generation of a variety of proinflammatory molecules such as tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), IL-6, IL-8 and intercellular adhesion molecule 1 (ICAM-1) ( DiDonato et al, 1996 ; Barnes & Karin, 1997)

Increased nuclear translocation of NF-κB has been implicated in a wide range of disease states, including endotoxin-induced sepsis, malignancy and chronic inflammatory disorders such as rheumatoid arthritis, asthma and ulcerative colitis ( Barnes & Karin, 1997; Bohrer et al, 1997 ). We assessed the ability of APC to inhibit LPS-induced nuclear translocation of NF-κB in a THP-1 monocyte cell line.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

APC (lot PCA 162) was provided by Baxter Hyland Immuno, Vienna, Austria. PC was isolated from human plasma by immunoaffinity chromatography with anti-PC monoclonal antibody (mAb). APC was prepared by activation of immunopurified PC with human thrombin, followed by separation by ion exchange chromatography and was certified as sterile and pyrogen free by the manufacturer.

The human monocyte THP-1 cell line (European Collection of Cell Cultures, Salisbury, UK) was cultured at 37°C under 5% CO2 at a density of 2–9 × 105 per ml. The cell culture medium was RPMI-1640 (Gibco BRL) supplemented with 2 m m glutamine (Sigma), 10% fetal calf serum (FCS) (Gibco BRL), penicillin 50 units/ml and streptomycin 50 µl/ml (Gibco BRL). Cells were counted and > 90% viability was confirmed by ethidium bromide acridine orange (Sigma). The cells were then resuspended at a concentration of 1 × 106/ml in culture medium which contained FCS at varying concentrations (10%, 2% and 0%). A 2-ml cell suspension was transferred to a six-well plate and incubated with varying concentrations of APC (final concentrations of 200 µg/ml, 100 µg/ml and 20 µg/ml) 10 min before the addition of LPS (100 ng/ml or 10 µg/ml). The reaction was stopped after 4 h by the addition of 2 ml of ice-cold phosphate-buffered saline. After centrifugation at 1000 g for 1 min, the supernatant was collected and stored at −70°C for subsequent analysis. TNF-α was measured in the supernatant fraction using a commercially available enzyme-linked immunosorbent assay (ELISA) (R & D Systems). Nuclear extracts were prepared from the cells as previously described and the protein content was determined by Bradford reagent (Bio-Rad) ( Osborn et al, 1989 ).

Electrophoretic mobility shift assay (EMSA) An oligonucleotide (Promega) containing the κB binding site (underlined) 5′-AGTTGAG GGGACTTTCCCAGGC-3′ was annealed to a complimentary primer and end-labelled using the T4 polynucleotide kinase which exchanges a phosphate group at the 5′ end of the double-stranded DNA with the radiolabelled phosphate group [γ-32P]-dATP (Amersham). Binding reactions were performed in a 20-µl volume containing 4 µg of nuclear extract, 2 µg of poly-(dI–dC) as a non-specific competitor of DNA, 1 µl of labelled oligonucleotide (10 000 c.p.m.) and 2 µl of binding buffer (100 m m Tris, pH 7·5, 1 m NaCl, 50 m m DTT, 10 m m EDTA, 40% glycerol and 1 mg/ml nuclease-free bovine serum albumin). After a 30-min binding reaction at room temperature, the protein–DNA complexes were separated from the free DNA probe by electrophoresis and visualized on a 4% non-denaturing acrylamide gel run on 0·5× Tris-borate/EDTA buffer (89 m m tris-HCl, 89 m m boric acid, 2 m m EDTA). Gels were dried and visualized by autoradiography. Band intensities were quantified by densitometric analysis using a Pharmacia LKB Imagemaster DTS densitometer and diversity one software.

Statistical analysis All experiments were performed in triplicate. The percentage inhibition of NF-κB and TNF-α was evaluated for statistical analysis by the Mann–Whitney U-test. Analysis of the effect of FCS on LPS-induced TNF-α was determined by measurement of analysis of variance ( statview, Abacus Concepts, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

LPS at 10 µg/ml was a strong stimulator of TNF-α production in THP-1 cells even in the absence of serum, inducing a 50- to 60-fold increase in TNF-α compared with resting cells. The presence of 2% and 10% FCS further enhanced this response with over a 100-fold increase being evident at 10% FCS. LPS at 100 ng/ml also strongly stimulated TNF-α production, resulting in a 50-fold increase in the presence of 10% FCS ( Fig 1). APC resulted in significant inhibition of TNF-α production. The most marked inhibition was seen at an APC concentration of 200 µg/ml, which blocked the effect of 100 ng/ml LPS by 75·3 ± 2·9%. The inhibitory effects of APC were less marked at the higher concentration of LPS (10 µg/ml) and in the presence of FCS; however, APC 200 µg/ml resulted in at least a 50% reduction in TNF-α under all conditions tested ( Fig 1).

image

Figure 1. Effect of APC on LPS-induced TNF production in the THP-1 cell line. THP-1 cells (1 × 109/ml) were preincubated with APC at varying concentrations (200 µg/ml, 100 µg/ml and 20 µg/ml) before the addition of LPS 100 ng/ml and 10 µg/ml. The effect of FCS was assessed by varying the concentration of FCS (0%, 2% and 10%) in the cell culture medium immediately before the addition of LPS 10 µg/ml. The concentration of TNF-α in the supernatant was determined at 4 h by ELISA. Data are expressed as means ± 1SD of triplicate experiments. The percentage inhibition of TNF-α at different concentrations was analysed by the Mann–Whitney test, Asterisk, P < 0·05. FCS resulted in a dose-dependent increase in LPS-induced TNF-α production [P < 0·001 by analysis of variance ( anova)].

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We explored a possible mechanism for this inhibition by examining the effect on NF-κB, the key transcription factor involved in TNF-α gene expression. Treatment of THP-1 with 100 ng/ml LPS strongly activated NF-κB in THP-1 cells ( Fig 2A, compare lanes 3 and 1). Preincubation with 200 µg of APC completely blocked the effect of LPS (compare lanes 4 and 3) and partially reduced basal NF-κB (compare lanes 2 and 1). A more marginal effect was evident at 100 µg/ml of APC (lane 5) and no effect was seen with 20 µg/ml of APC. Inhibition of NF-κB was demonstrated both in the presence and absence of FCS (not shown).

image

Figure 2. APC inhibits nuclear translocation of NF-κB after stimulation with LPS 100 ng/ml. Nuclear extracts were isolated from THP-1 cells pretreated with APC 200 µg/ml, 100 µg/ml and 20 µg/ml for 10 min before the addition of LPS 100 ng/ml. At 4 h, nuclear extracts were prepared from the cells and exposed to a radiolabelled oligonucleotide that contained the consensus sequence for NF-κB. The NF-κB–oligonucleotide complexes were visualized by autoradiography after electrophoresis on a 4% non-denaturing acrylamide gel (A). Band intensities were then quantified by densitometric analysis. Band intensities were standardized against values obtained with resting cells, which were arbitrarily assigned a value of 1. Data are expressed as the means ± SD for triplicate experiments (B). Asterisk, P < 0·05, determined by the Mann–Whitney U-test. 1, resting; 2, APC 200 µg/ml; 3, LPS 100 ng/ml; 4, LPS 100 ng/ml + APC 200 µg/ml; 5, LPS 100 ng/ml + APC 100 µg/ml; 6, LPS 100 ng/ml + APC 20 µg/ml.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

The molecular basis for the anti-inflammatory properties of APC has not yet been fully elucidated. In vivo studies, using animal models of sepsis, have demonstrated that APC results in a significant reduction in TNF-α ( Esmon et al, 1991 ) and inhibition of the PC pathway by antibodies to PC or PS correspondingly increases TNF-α production ( Taylor et al, 1991; Taylor, 1994). In addition, APC has been shown in vitro to bind to monocytes and to inhibit the LPS-induced proinflammatory cytokine production ( Grey et al, 1994 ; Hancock et al, 1995 ; Murakami et al, 1997 ).

In this study, we have demonstrated that APC inhibited LPS-induced TNF-α production and NF-κB activation. The presence of FCS resulted in a dose-dependent increase in LPS-induced TNF-α production and a reduction in the inhibitory effects of APC. The increase in TNF-α associated with FCS is not surprising and probably reflects the presence of co-stimulatory molecules such as LPS binding proteins in the serum ( Fenton & Golenbock, 1998). This stronger response to LPS may be the reason why inhibition by APC was less marked. Alternatively, this effect may result from the presence of physiological inhibitors of APC such as α1-antitrypsin, α2-macroglobulin or protein C inhibitor at normal or increased concentrations in FCS ( Suzuki et al, 1989; Heeb et al, 1991 ). Likewise, the concentration of LPS influences the ability of any given concentration of APC to inhibit TNF-α. The effect of APC was less marked at the higher concentration of LPS (10 µg/ml). Thus, the protective effect of APC in sepsis may be influenced by the concentration of LPS, the circulating levels of APC/PC and the concentration of LPS co-stimulatory factors or protein C inhibitors in the serum.

The activation of monocytes by LPS leads to the initiation of proximal signalling events, which in turn result in the increased transcription and stability of TNF messenger RNA (mRNA) ( Zuckerman et al, 1991 ; Taylor, 1994; Yao et al, 1997 ). Increased transcription requires the concerted binding of NF-κB, Egr-1 and c-jun to the promoter region of the TNF-α gene ( Yao et al, 1997 ). The importance of NF-κB in enhancing transcription is supported by studies in which transfection with TNF promoter constructs lacking the κB3 binding site for NF-κB resulted in a significant reduction in LPS-induced transcription of a downstream reporter gene ( Yao et al, 1997 ).

The concentration of APC used in this study was higher than the concentration in the plasma of PC by about 50-fold. This requirement may be the result of the low affinity of receptors for APC on THP-1 cells. Alternatively, unknown factors may be responsible for increasing the concentration or activity of APC at the surface of the monocyte in vivo.

The ability of APC to downregulate LPS-induced NF-κB activation may be of considerable clinical significance. Although there is substantial experimental evidence to suggest that TNF-α plays a pivotal role in the pathophysiology of sepsis, preliminary clinical trials with anti-TNF-α monoclonal antibodies have failed to demonstrate an improvement in morbidity or mortality in patients with sepsis ( Abraham et al, 1995 ). This may be due to the considerable pleiotropy and redundancy among proinflammatory cytokines or the differential expression of individual cytokines at various stages in the evolution of the inflammatory response to endotoxin ( van Deuren et al, 1994 ). Therefore, inhibition of any single proinflammatory cytokine at an arbitrary time point in the evolution of sepsis may not sufficiently alter the host immune response to improve clinical outcome. Thus, the ability of APC to inhibit TNF-α may be insufficient to explain its protective effects in animal models of sepsis. Alternatively, the ability of APC to inhibit such a critical transcription factor as NF-κB would have a more profound effect on the host inflammatory response. NF-κB regulates a variety of genes responsible for the generation of proinflammatory cytokines, chemokines, enzymes that generate mediators of inflammation, immune receptors and adhesion molecules ( Barnes & Karin, 1997). Furthermore, the proinflammatory cytokines TNF-α and IL-1β cause activation of NF-κB, thereby providing a positive regulatory loop. Thus, NF-κB activation in response to LPS leads to a co-ordinated increase in a wide variety of proinflammatory proteins which in turn lead to amplification and perpetuation of the inflammatory response ( Barnes & Karin, 1997). The inhibition of NF-κB would interrupt this positive feedback loop and globally downregulate the production of a wide variety of proinflammatory molecules. It is this particular property of NF-κB that makes it such an attractive molecular target for novel anti-inflammatory therapies and offers an explanation for the protective effect of APC in animal models of sepsis and the apparent reduction in mortality associated with protein C replacement therapy in patients with severe meningococcaemia.

In summary, the ability of APC to inhibit LPS-induced nuclear translocation of NF-κB identifies a potentially important immunomodulatory pathway. This mechanism may be responsible at least in part for the protective effect of APC in sepsis and also identifies a molecular target for novel anti-inflammatory therapies. Further work is required to assess the effect of APC on endotoxin-induced NF-κB in vivo and to elucidate the molecular mechanism(s) responsible for the inhibition of NF-κB.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References

We thank to Immuno/Baxter for supplying the APC.

References

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. References
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