ARTICLE
INTRODUCTION
The molecular sequelae of events leading to the adverse
and sometimes lethal outcome of sepsis have not been fully elucidated
[1]. Cytokines are undoubtedly involved in these processes [2-4] with
the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF), interleukin-1,
macrophage migration inhibitory factor (MIF), and others playing a pivotal
role [4-8]. Less is known, however, about the role of the anti-inflammatory
cytokines and, also, the interplay between the various cytokines and the
regulatory loops involved is still poorly defined. Cytokines have been
considered promising clinical targets in the treatment of septic shock,
but to date, anti-cytokine-based therapeutic strategies such as the use
of anti-TNF antibodies, soluble TNF receptors, or interleukin-1 receptor
antagonists have failed to show a clear clinical benefit [9].
Activated protein C (APC), mostly known because of its
powerful anti-coagulant properties [10, 11], has recently been shown to
exhibit anti-inflammatory and probable anti-sepsis activities, and APC
or its precursor protein C have been implicated as therapeutical agents
for the treatment of meningococcaemia [12-14].
Protein C is a plasma serine protease proenzyme that is
produced in the liver and by endothelial cells and that is secreted into
the circulation following various stimuli [10]. Cleavage by thrombin of
a dodecapeptide from the N-terminal end of the protein C heavy chain results
in the generation of APC. APC acts to promote conversion of factors Va
and VIIIa to factors Vi and VIIIi, respectively, by proteolysis, thereby
inhibiting the blood clotting process [10, 15, 16].
Several lines of evidence have implicated protein C/APC
to be an endogeneous inhibitor of the inflammatory septic cascade. Acquired
severe protein C deficiency is a poor prognostic marker in severely infected
septic patients [17, 18]. In meningococcal septic shock in children, clinical
severity has been directly correlated with the reduction in circulating
levels of protein C [17, 19], and protein C replacement in this condition
not only led to normalization of plasma protein C levels and other parameters
of haemostasis, but has also resulted in significant improvement of organ
dysfunction, such as adult respiratory distress syndrome (ARDS), and improvement
of skin lesions [13, 20, 21]. Recently, protein C replacement therapy
has been reported to markedly improve survival rates in patients with
meningococcus-induced purpura fulminans [14]. In models of sepsis, preloading
baboons with APC prevented the coagulopathic response and lethal effects
of E. coli injections [22]. These and other studies have demonstrated
that the anticoagulant and fibrinolytic properties of protein C/APC most
likely contribute to its anti-inflammatory effects.
Evidence that APC has a direct anti-inflammatory action
is less clear-cut. Infusions of APC have been shown to suppress the peak
blood levels of TNF in rats challenged with high doses of lipopolysaccharide
(LPS) [23]. In vitro studies suggest that APC directly inhibits
TNF production from LPS-, interferon-gamma, and phorbol ester-stimulated
monocytes [24]; and although these observations have remained controversial
[25], a non-coagulation-related, direct anti-inflammatory action of APC
has also been argued for by investigations into the relationship between
coagulation and sepsis. For example, human APC is only a weak anticoagulant
in rats [26, 27], yet it exhibits anti-sepsis effects in these animals.
Inhibitors of coagulation do not show anti-sepsis effects comparable to
APC itself [28].
MIF was previously known as a classical T cell cytokine
that acts to inhibit the migration of monocytes/macrophages [29, 30],
but it has recently been shown to be an abundant cytokine with a broad
spectrum of proinflammatory properties, and to be a critical mediator
of Gram-negative and Gram-positive bacterial shock [5, 7, 8, 31, 32].
MIF is unique among cytokines in that it constitutes a physiological,
counter-regulatory system of the immunosuppressive and anti-inflammatory
actions of glucocorticoids [31, 33] and it exhibits both cytokine and
catalytic activities [34, 35]. Of note, MIF is found in the alveolar spaces
of patients with ARDS, it augments pro-inflammatory cytokine secretion
in ARDS, and it overrides the anti-inflammatory effects of glucocorticoids
in ARDS lungs [33, 36]. ARDS is known as one of the detrimental downstream
events following initiation of the septic cascade and has been closely
attributed to the previous activation of the extrinsic pathway of coagulation
leading to the formation of microthrombi and fibrin deposition.
In this study, we sought to establish the direct interference
of APC with cytokine-mediated toxicity. Because TNF is the pivotal mediator
of septic shock and to clarify the existing but controversial results
available, we first investigated directly the effect of APC on TNF production
by monocytes in vitro. To begin to examine whether APC action interferes
with other cytokine mediators that promote the septic cascade, we analyzed
potential direct effects of APC on MIF.
MATERIALS AND METHODS
Materials
APC (lot PCA 162) was kindly provided by Baxter Hyland
Immuno (Vienna, Austria) and was reconstituted as recommended by the supplier.
The APC provided was generated from protein C as follows: protein C was
isolated from human plasma by immunoaffinity chromatography and APC was
subsequently prepared by activation of protein C with human thrombin,
followed by purification by ion exchange chromatography. Protein C and
APC were certified as sterile and pyrogen-free by the manufacturer. Structural
integrity and native folding of APC was confirmed by far-UV CD spectropolarimetry.
Activity of APC preparations was tested by the Immunochrom®
PC assay essentially as described by the manufacturer. Lipopolysaccharide
(LPS) was of the type O111:B4 and was from Sigma-Aldrich GmbH (Deisenhofen,
Germany). Monoclonal anti-human TNF antibody was from R&D Systems
(Wiesbaden, Germany) and neutralizing polyclonal anti-MIF antibody was
prepared from rabbit serum as described [5]. Cell culture reagents were
from Life Technologies (Eggenstein, Germany) and miscellaneous chemicals
from Sigma-Aldrich GmbH or Life Technologies and were of the highest analytical
grade available.
Cell culture
THP-1 monocytic cells were obtained from the German collection
for microbiology and cell lines (DSMZ, Braunschweig, Germany) and were
cultured at a density of 0.1-0.5 x 106 cells/ml in RPMI 1640
containing 10% fetal calf serum (FCS), 2 mM L-glutamine, and penicillin-streptomycin
in 5% CO2 at 37° C. For experiments, cells were washed
twice in the same medium without FCS and were plated in 6- or 24-well
cell culture plates (Greiner Labortechnik, Frickenhausen, Germany) at
a density of 1 x
106 cells/ml in serum-free medium. Cells were rested for 3
hours. LPS stimulations were performed for 4-23 hours as indicated. APC
was always added 30 min before the stimulant.
ELISA
At the end of the incubations, reactions were transferred
to 1.5 ml centrifuge tubes, centrifuged gently at 400 g for 5 min, and
culture supernatants transferred to fresh tubes. Supernatants were then
either analyzed directly or stored at 80° C for up to 3 months.
Antibodies for the human TNF and human MIF enzyme-linked immunosorbent
assays (ELISA) were from R&D Systems and ELISA were performed following
the manufacturer's instructions. To confirm that APC proteolytic activity
did not affect test protein stability and detectability, TNF ELISA standards
were preincubated with 50 mug/ml APC and standard curves determined in
the presence versus absence of APC. Identical TNF standard values
were obtained for both curves. ELISA data are expressed as the mean ±
SD of the indicated number of experiments.
RESULTS
Dependence of APC activity on
serum concentration
Various preparations of APC have been used in previous
studies and their quality and status of biological activity have not always
been clear. Thus, we initially confirmed that the APC preparation we used
was natively folded and biologically active. APC exhibited full enzymatic
activity as measured by the amidolytic activity assay (data not shown),
but failed to have any inhibitory effect on TNF production in vitro,
when tested on peripheral blood monocytes or various monocytic cell lines
using standard conditions (data not shown). Detailed analysis of assay
parameters then showed that APC inhibition of TNF production was abolished
in the presence of FCS at concentrations as low as 1%, while its enzymatic
activity was unaffected under such conditions (data not shown). Serum-mediated
inhibition of the TNF-inhibitory activity of APC, i.e. as observed
in the presence of 10% FCS, could only be measurably overcome when the
concentration of APC was increased to beyond physiological values of >=
200 mug/ml, which are unsuitable to be handled in routine in vitro
settings.
Inhibition by APC of LPS-induced
TNF secretion
When incubations were performed under serum-free conditions,
APC potently inhibited TNF production in THP-1 monocytes in a dose- and
time-dependent fashion (Figure 1).
For example, APC at a concentration of 50 mug/ml inhibited TNF release
by more than 60%. Varying stimulatory LPS concentrations from 0.1-10 mug/ml
had no significant influence on the APC effect (data not shown). Together,
these data confirmed previous results by Hancock and coworkers [24] suggesting
that APC can act to directly inhibit TNF production in monocytes and implying
that conflicting results regarding this activity may have come from different
concentrations or properties of the serum supplement used during the incubations.
MIF secretion
by THP-1 monocytes
Although TNF is a pivotal cytokine mediator of septic shock,
the demonstration that APC has a direct anti-inflammatory action should
be sustained by the observation of the down-regulation of other pro-inflammatory
mediators of sepsis. Hence, we investigated whether APC could also block
LPS-stimulated production of MIF, another critical mediator of septic
shock that is distinct from TNF and other cytokines in that it may not
act by signalling through classical receptor-mediated pathways [38-40],
and in that it does not appear to follow classical secretion pathways
[41]. THP-1 monocytes were stimulated with LPS as established for TNF,
and production of MIF was measured by ELISA. Secretion of MIF increased
over time, but unlike secretion of TNF, it plateaued at approximately
20 hours (Figure 2). Of note,
resting monocytes also secreted MIF in a time-dependent manner. This latter
observation was not due to serum starvation, stress, or non-specific release
following cell death, as confirmed by lactate dehydrogenase (LDH) assay
performed in the culture supernatants of the analyzed cells (data not
shown). As with TNF, the secretion of MIF was found to gradually increase
as the concentration of LPS increased and was specific up to at least
10 mug/ml of LPS. MIF concentrations measured after a 10 hours stimulation
period ranged from 7.7 ± 1.8 ng/ml (resting conditions) to 44.9 ±
7.8 ng/ml (10 mug/ml LPS).
Inhibition by APC of LPS-induced
MIF secretion
As for TNF, APC at a concentration of 50 mug/ml, markedly
inhibited secretion of MIF, when THP-1 cells were stimulated with a standard
concentration of LPS (10 mug/ml) for 23 hours (Figure
3). Inhibition by APC was also evident, when cells were stimulated
for 4 hours only (data not shown) or with 100 ng/ml LPS (Figure
3). Under the latter conditions, inhibition by APC was complete, i.e.
MIF secretion was reduced to levels measured for resting control cells.
Direct comparison of the APC effect on TNF versus MIF release showed
that secretion of MIF was inhibited to a somewhat higher degree (68 ±
5% versus 43 ± 7%).
We then examined whether APC inhibition of MIF secretion
was dependent on TNF. LPS-induced release of MIF was not affected by neutralizing
anti-TNF antibodies that led to a complete reduction in TNF levels and,
under the conditions used, anti-MIF antibodies did not affect secretion
of TNF either (data not shown).
DISCUSSION
Evidence for a direct anti-inflammatory effect of APC and
the physiological significance of such observations had been controversial
[23-28]. Our results clearly confirm previous findings by Grey et al.
that had demonstrated a selective inhibitory effect of APC on the responses
of activated monocytes [24]. How-ever, our studies also imply that serum-based
inhibitors of APC anti-inflammatory activity may exist that may have interfered
with APC activity in earlier experiments. Using established experimental
APC concentrations, no inhibitory effect of APC on TNF secretion in monocytes
was observed when 1-10% FCS was included in the culture media. Serum inhibition
of APC activity could only be overcompensated when extremely high APC
concentrations were applied. Although our data do not allow for any quantitations
of serum inhibitor/APC ratios or affinities due to the in vitro
conditions used, i.e. fetal calf rather than human serum was used,
they should encourage further investigations into the molecular nature
of the proposed serum inhibitor of APC. The observed serum inhibitory
effect corresponds well with the fact that low PC levels are a predictor
of meningococcal septic shock.
The observation that APC also markedly inhibited the secretion
of MIF, another key cytokine mediator of septic shock [5, 7, 8], further
confirmed the significance of the anti-inflammatory activity of APC. In
fact, anti-MIF antibodies have recently been shown to potently block bacterial
septic shock in an E. coli pe-ritonitis and cecal ligation and
puncture (CLP) model and anti-MIF antibodies are currently considered
unique among potential anti-cytokine antibody-based therapeutic strategies
in that they confer protection against septic shock even when administered
several hours post-challenge [8].
MIF has been suggested to promote ARDS [33], while protein
C replacement therapy has been found to result in improvement of ARDS
lung conditions [13, 20, 21]. Thus, inhibition of MIF secretion by APC
offers an intruiging molecular pathway that could be part of the yet to
be defined regulatory mechanism underlying anti-inflammatory APC effects
in disease.
We found that LPS-induced secretion of MIF was fully independent
on the presence of TNF and vice versa. This indicated that inhibition
by APC of MIF and TNF release were either independent events or that inhibition
was due to interference with a mutually utilized upstream signalling event
(Scheme 1). The latter argument would be consistent with the notion that
TNF and MIF, although showing several similar and overlapping pro-inflammatory
activities, belong to different protein families with different mechanisms
of production, secretion, and target cell action (summarized in: [39]).
Preliminary data from ongoing investigations on potential
effects of APC on cellular signalling pathways that are initiated following
cellular activation by LPS would suggest that inhibition of pro-inflammatory
cytokine release by APC is due to interference with the NFkappaB/IkappaB
transcription factor pathway. Of note, NFkappaB-mediated activation has
been demonstrated to be involved in the production of TNF and a NFkappaB
element has also been identified in the 5'-upstream regulatory region
of the mouse MIF gene [42].
CONCLUSION
In summary, we have shown that APC can act to directly down-regulate
the secretion of two critical and mechanistically distinct cytokines mediators
of septic shock, indicating that inhibition by APC could represent a more
general anti-inflammatory principle.
Acknowledgements. We thank H. P. Schwarz for supplying
protein C, APC, and Immunochrom kits. We thank E. Wagner and D. Finkelmeier
for assistance with the cell culture and ELISA and H. Brunner, T. Calandra,
and O. Flieger for helpful discussions.
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