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*
Central Laboratory of the Netherlands Red Cross Blood Transfusion Services and Laboratory for Experimental and Clinical Immunology, Academic Medical Centre, University of Amsterdam, The Netherlands;
Behringwerke AG, Marburg, Germany;
Oklahoma Medical Research Foundation, Oklahoma City, OK 73104; and
§
Department of Internal Medicine, Free University Hospital, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-granules, the exposure of negatively
charged phospholipids, assembly of the prothrombinase complex, and
release of vesicles that express prothrombinase activity (24, 25, 26).
Hence, activation of complement and contact systems may contribute to
the coagulant and inflammatory sequelae of sepsis through several
mechanisms. Activation of both the complement and contact system is regulated by C1-esterase inhibitor (C1-inh).3 C1-inh, which belongs to the superfamily of serine-proteinase inhibitors, is the only known inhibitor of C1r and C1s, components of the classical pathway of complement (27), as well as the major inhibitor of factor XII and prekallikrein of the contact system (28, 29). Although C1-inh is an acute phase protein, antigenic levels of C1-inh tend to be normal in patients with fatal septic shock, while levels of proteolytically inactivated C1-inh are increased, suggestive of an increased turnover and a relative deficiency of biologically active C1-inh during sepsis (30).
We have previously demonstrated that C1-inh substitution therapy in patients with septic shock may reduce the need for vasopressor medication and attenuate complement and contact activation (31, 32). Moreover, effects of C1-inh have been observed in several animal models of sepsis: C1-inh supplementation abrogated endoxin-induced disseminated intravascular coagulation and hypotension in rabbits (33, 34) and pulmonary dysfunction in endotoxemic dogs (35). In this study, we evaluated the effect of i.v. administration of C1-inh on hemodynamic, coagulant, inflammatory, and cell injury responses in an established model of severe septic shock in nonhuman primates. Our results indicate that in this experimental model, exogenous administration of C1-inh may exert benificial effects, in part through modulation of cytokine release, and support the notion that activation of complement and/or contact system proteases is associated with organ injury and impending lethality.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pasteurized human C1-inh was provided by Behringwerke AG (Marburg, Germany). On SDS-PAGE, this preparation consisted of >95% native C1-inh.
Experimental and infusion procedures
Experiments were performed on 10 juvenile baboons (Papio anubis/Cynocephalus), each with a hematocrit exceeding 36% and free from tuberculosis. The animal-handling procedures and Escherichia coli (type B) preparation were performed using the methodology described in previous publications (36, 37). Briefly, baboons were fasted overnight before each experiment and given water ad libitum. Each animal was sedated with ketamine hydrochloride (14 mg/kg, intramuscularly) on the morning of the study and anesthetized with sodium pentobarbital (2 mg/kg) via a percutaneous catheter positioned in the cephalic vein. The femoral artery and both femoral veins were cannulated aseptically and used for measuring aortic pressure, obtaining blood samples, infusing live organisms, C1-inh, and for fluid and anesthetic administration as reported elsewhere (36, 37). Gentamicin was given (9 mg/kg) as a 75-min infusion immediately after the E. coli infusion had been stopped, and then (4.5 mg/kg) as a 30-min infusion at 6 and 9 h after the start of the E. coli infusion. Additional gentamicin (4.5 mg/kg) was given as an intramuscular injection at 10 h after the start of the infusion and twice daily for the subsequent 3 days.
Each animal was i.v. challenged with a lethal dose of E. coli (4 x 1010 CFU/kg of body weight), given as a 2-h infusion. The time point at which the infusion was started is further indicated as T + 0, a time point of n hours thereafter referred to as T + n h. Time points before the start of the challenge are indicated as T - n h.
Two experimental E. coli groups were studied: one group
consisted of five baboons that received an initial dose of 500 U/kg
C1-inh (1 U is the amount of C1-inh present in 1 ml of normal human
plasma, which is equal to 2.50 µM/L or 270 mg/L; 30
administered i.v. over a 30-min period before the start of the lethal
E. coli challenge, followed by a continuous infusion of 200
U/kg for 9 h (treatment group). C1-inh doses were based on studies
in human volunteers (38) in which human native C1-inh was cleared from
the circulation with a fractional catabolic rate of 2.5% of the plasma
pool per hour and has an apparent t1/2 of 
28
h; thus, presumably this scheme of administration would maintain
concentrations of C1-inh at 10 times the level observed in normal
human plasma during the first 9 h of the experiment. The second
group consisted of five animals that received saline according to a
similar scheme of administration before and after lethal E.
coli infusion (control group).
All animals were maintained under anesthesia and monitored for 10 h. They were observed continuously for an additional 36 h and daily for a maximum of 7 days. Blood samples were collected at given time points for hematology, clinical chemistry, and C1-inh determinations. Additional samples were collected on EDTA/soy bean trypsin inhibitor (final concentrations, 10 mM and 0.1 mg/ml, respectively) before (T + 0), and at 0.5, 1, 2, 3, 4, 6, 8, and 10 h after E. coli challenge for plasma determination of cytokines, complement activation products, contact system proteins, neutrophil degranulation products, and coagulation/(anti)fibrinolytic parameters. Baboons surviving for 7 days were considered permanent survivors and were subsequently killed with sodium pentobarbital for necropsy on the eighth day.
Assays
All assays used in this study were developed with mono- or polyclonal Abs raised against human proteins. When possible, standards were prepared with baboon plasma or serum to correct for differences in affinities of the Abs for baboon vs human proteins. When human standards had to be used, we established that dilution curves of these standards were parallel to those obtained with baboon plasma samples. Notably, the lower affinity of the Abs for baboon proteins may have led to an underestimation of the baboon proteins.
Antigenic C1-inh. Plasma levels of exogenously administered C1-inh were measured by nephelometer (Behringwerke Nephelometer Analyzer, Behringwerke AG) and expressed as mg/L. Functional C1-inh levels were measured by RIA as described previously (30).
Complement activation products. C3b/c and C4b/c in baboon plasma were quantified by RIA as reported elsewhere (39, 40, 41). C3b/c was expressed as a percentage of the amount present in normal baboon serum aged (NBA), i.e., normal baboon serum (NBS) incubated for 7 days at 37°C in the presence of 0.02% (w/v) NaN3. Results for C4b/c were expressed as a percentage of the amount generated in NBS by incubation with heat-aggregated IgG (NBS-AHG). Levels of the terminal complex of complement (C5b-9) were measured by ELISA according to the instructions of the manufacturer (Behringwerke AG, Marburg, Germany), and were expressed as µg/L with reference to a serially diluted standard of human C5b-9.
Cytokines.
Plasma concentrations of TNF-, IL-6, IL-8, and IL-10 were measured
by ELISA as previously described (42, 43, 44).
Coagulation and (anti)fibrinolytic parameters.
Levels of tissue-type plasminogen activator (t-PA), plasminogen
activator inhibitor type 1 (PAI-1), and thrombin-antithrombin III (TAT)
complexes were determined by ELISA as described previously (41, 45, 46). Values were expressed as ng/ml.
Plasmin-2-antiplasmin (PAP) complexes were measured
by RIA as described (46). PAP complex levels were expressed as
percentage of the level present in normal baboon plasma in which a
maximal amount of complexes was generated by incubation with an equal
volume of urokinase (50 µg/ml) in the presence of 0.2 mol/L
methylamine (final concentration) to inactivate
2-macroglobulin, further referred to as NBP-MA-UK.
Measurement of prekallikrein and factor XII in plasma. Plasma prekallikrein and factor XII were determined by a sandwich-type ELISA. Flat-bottom microtiter (96-well) plates (Dynatech, Plochingen, Germany) were coated overnight at room temperature with 100 µl of 2.5 µg/ml anti-human prekallikrein mAb K15, or mAb OT-2 against human factor XII, in carbonate buffer, pH 9.5, and blocked for 30 min with 150 µl PBS containing 2% (v/v) cow’s milk. All subsequent incubations were in 100-µl volumes at room temperature, and plates were washed after each incubation with PBS/0.02% (w/v) Tween-20. The plates were then incubated for 2 h with baboon plasma samples diluted in 100 µl high performance ELISA (HPE) buffer (CLB, Amsterdam, The Netherlands). Bound prekallikrein and factor XII were detected by subsequent 1-h incubation with HPE buffer containing 1 µg/ml of biotinylated mAbs 13G11 (kindly provided by Dr. R. W. Colman, Temple University, Philadelphia, PA) and F3, respectively, followed by a 1:10,000 dilution of streptavidin-polymerized horseradish peroxidase (polyHRP; CLB) in PBS/2% (v/v) cow’s milk for 30 min. The plates were developed with a solution of 100 µg/ml of 3,5,3',5'-tetramethylbenzidin (Merck, Darmstadt, Germany) with 0.003% (v/v) H2O2 in 0.11 mol/L sodium acetate, pH 5.5. The reaction was stopped by the addition of an equal volume of 2 mol/L H2SO4 to the wells. Serial dilutions of normal pooled baboon plasma was used as a standard. Values were expressed as percentage of the amount present before E. coli infusion (T + 0).
Neutrophil degranulation products.
Elastase-1-protease inhibitor complexes were
determined with a RIA that has been described in detail elsewhere (47).
Results were expressed as nanograms of elastase per milliliter by
reference to a standard curve that consisted of normal baboon plasma to
which human neutrophil elastase (Elastin Products Co., Pacific, MO) was
added at a final concentration of 2 µg/ml. In this standard, >95%
of the elastase is complexed to 1-antitrypsin.
Statistical analysis
Results are expressed as mean ± SEM. Statistical analysis was performed using a commercial statistical package (StatView; Abacus Concepts, Inc., Berkeley, CA). Comparisons between groups during the course of the observation period were performed using repeated measures analysis of variance (ANOVA). Data were analyzed by two-tailed ANOVA to determine the significance of differences in means between groups at given times. Within one group, differences from baseline levels were determined with ANOVA using Fischer’s least significant difference (Fischer LSD). Statistical significance was designated at the 95% confidence level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C1-inh was infused into baboons to achieve a concentration of
10 times the level observed in normal human plasma (i.e., 270 mg/L)
just before the start of the E. coli infusion (T + 0).
Figure 1
shows the course of antigenic
levels of C1-inh in the treatment group. Baseline levels of endogenous
C1-inh 30 min before E. coli challenge (T-0.5 h) were <200
mg/L, which probably resulted from poor cross-reactivity of the
employed anti-human C1-inh Abs with Papio C1-inh. C1-inh
levels at T + 0 were 2243 ± 86 mg/L (range: 2016–2644), and
remained elevated for at least 10 h. During this entire
observation period, >95% of circulating human C1-inh in both groups
consisted of native, uncleaved C1-inh, as determined by RIA, based on
the binding capacity of functional C1-inh to C1s (Ref. 30; not shown).
In the control group, levels of antigenic C1-inh remained below the
limit of detection (not shown).
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Table I shows the conditions, the
number of organisms infused and found circulating in the blood at
T + 2 h, and survival times of animals in control and
treatment groups. The mean E. coli dosage of C1-inh-treated
and control groups was similar, i.e., 9.45 x 1010 and
8.70 x 1010 CFU/kg, respectively
(p > 0.05). Moreover, no significant
difference was noted in the number of organisms circulating at T +
2 h (4.64 x 107 and 4.77 x 107
CFU/ml in treated and control animals, respectively), indicating that
C1-inh administration did not interfere with bacterial clearance.
Administration of C1-inh rescued one of five baboons in the treatment
group. Moreover, one animal receiving C1-inh survived for 62.5 h,
an unusual occurrence in this model of sepsis. None of the excipient
control animals survived beyond 27 h, and the mean survival time
in this group was 19.4 h. However, possibly due to the limited
number of animals included in this study, comparison of the survival
curves using the likelihood ratio test failed to indicate a significant
difference in survival time of treated vs control groups
(p > 0.05).
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. Infusion of E. coli
produced a severe hypotensive shock in all baboons, which was
unaffected by supplementation with C1-inh. In both control and
treatment groups, a significant decline in mean systemic arterial
pressure was noted at 2 h, remaining below baseline values during
the 10-h observation period. Mean heart rate and temperature were
similarly elevated in all animals. E. coli-infusion with or
without C1-inh induced a prompt fall in leukocyte count to 40% of
control at T + 1 h, a steady decline in platelet count to
30% of control at T + 10 h, a small rise in hematocrit from
T + 6 h and on, and a progressive decrease in fibrinogen
levels over the entire observation period. Changes in mean systemic
arterial pressure, heart rate, temperature, white blood cell, platelet
counts, and hematocrit were not different between the nonsurviving
C1-inh-treated animals and the animal surviving treatment (not shown).
However, the rate of fibrinogen consumption appeared to be modestly
decreased in the surviving C1-inh-supplemented animal (Table II).
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shows that
levels of blood urea nitrogen, creatinine, uric acid, lactate
dehydrogenase (LDH), serum glutamic oxaloacetic transaminase (SGOT),
and serum glutamic pyruvate transaminase (SGPT) were increased in all
animals at 10 h. The magnitude of the increases, however, tended
to be lower in the C1-inh treatment group, especially in the treated
baboon that was rescued from lethal challenge, and in the animal
surviving for >60 h after C1-inh supplementation. At 10 h
postchallenge, these animals exhibited only moderate changes in uric
acid, LDH, SGOT, and SGPT, while a pronounced increase in the levels of
these parameters was observed in the treated and control animals that
died within 14 to 27 h.
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Effect of high dose C1-inh supplementation on inflammatory response patterns in lethal E. coli sepsis
Complement activation.
Plasma levels of C4b/c, C3b/c, and C5b-9 were measured to evaluate the
effect of C1-inh supplementation on E. coli-induced complement
activation (Fig. 2,
A-C). In the control group, circulating levels of
C4b/c and C3b/c continued to rise during the entire observation period,
reaching maximal levels of 13.3 ± 3.1% and 10.9 ± 2.8%,
respectively, of fully activated NBS at T + 10 h (Fig. 2, A and B). Administration of high doses of
C1-inh almost completely abrogated the appearance of C4b/c
(p < 0.0001) in all animals thus treated,
indicating efficient inhibition of Papio C1 by human C1-inh.
Moreover, C1-inh markedly attenuated the appearance of C3b/c at all
time points (p < 0.01), suggesting that at
least part of the C3 activation had occurred via the classical pathway.
Plasma levels of the terminal complex of complement, i.e., C5b-9, also
rapidly increased upon E. coli challenge (Fig. 2C). In control animals, peak levels of 3691 ±
221 µg/L were noted at T + 2 h, remaining elevated until
the end of the observation period. C1-inh treatment only modestly
affected concentrations of C5b-9 (p = 0.05),
and peak levels of 3061 ± 289 µg/L were measured at 2 h
after the E. coli infusion was started. A uniform response
was observed for all activation products in the treatment group, and
this response appeared unrelated to survival and/or organ damage.
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A–C). In both control and treatment
groups, a protracted increase of t-PA concentrations was noted from
T + 1 h and on, to reach a maximum level of 77.8 ± 16.1
and 117.6 ± 15.2 ng/ml at T + 10 h in control and
C1-inh treatment groups, respectively (Fig. 3A).
Concentrations of circulating PAP complexes, which reflect the
generation of plasmin, the key enzyme of the fibrinolytic system,
transiently increased upon E. coli infusion, peak levels
being 10.3 ± 3.5% and 15.7 ± 4.2% of maximally activated
plasma at T + 2 h in controls and treated animals,
respectively (Fig. 3B). This difference in time
course of t-PA and PAP complexes was not statistically significant
between both groups (p > 0.05). In contrast,
C1-inh administration markedly attenuated the appearance of PAI into
the circulation, especially at and beyond T + 6 h (Fig. 3C; p < 0.0001). In both groups, peak
levels of PAI were noted at 8 h postchallenge, and were 5180
± 877 and 2379 ± 446 ng/ml in controls and C1-inh-supplemented
animals, respectively.
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). Consistent with a pronounced decline
in fibrinogen and platelets observed upon lethal E. coli
administration (Table II
), indicating the onset of diffuse
intravascular coagulation (DIC), circulating TAT complexes sharply
increased from T + 1 h and on, and maximal levels of
2236 ± 314 and 1668 ± 452 ng/ml were measured at 6 h
postchallenge in control and treatment groups, respectively. Although
the course of TAT complexes was not statistically different between
both groups, the magnitude of increase was markedly lower in the
surviving C1-inh-treated animal that also showed a retarded fibrinogen
consumption (animal No. 9; Tables II and IV).
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A). Consistent with
our previous studies (48, 49), peak TNF levels of 16.3 ± 1.9
ng/ml were observed in control animals at T + 2 h, i.e., at
the end of the E. coli infusion. Administration of lethal
E. coli supplemented with C1-inh elicited a similar, but
significantly lower TNF response, with peak plasma concentrations of
11.0 ± 1.7 ng/ml (p < 0.05). Notably,
the lowest TNF peak was observed in the treated animal surviving
E. coli challenge (No. 9; 4.1 ng/ml at T + 2
h).
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B). In excipient control baboons, IL-10 levels
sharply increased to reach a maximum of 3564 ± 772 pg/ml 3 h
after the start of the experiment and were still elevated at T +
10 h. Upon C1-inh treatment, peak levels of IL-10 were noted at
T + 3 h (2, 266 ± 767 pg/ml), to become near baseline
values at the end of the observation period (p
< 0.05). In addition, administration of C1-inh significantly
attenuated the sustained release of IL-6 and IL-8 into the circulation,
and maximal levels of 279.6 ± 74.9 and 167.4 ± 19.1 ng/ml
were noted at 8 and 4 h, respectively, i.e., a two- to fivefold
reduction compared with peak control levels (Fig. 4, C and
D; p < 0.001).
Contact system proteases.
Mean plasma levels of factor XII and prekallikrein declined by 20 to
25% after 1 h in the untreated control group, which was
statistically different from baseline values (Fig. 5, A and B; T
+ 1 to T + 10 h vs baseline: p < 0.05 by
Fischer LSD). In contrast, in the animals receiving C1-inh treatment,
factor XII levels remained relatively stable until the end of the
observation period and were only significantly reduced at T +
10 h (Fig. 5A). Similarly, plasma prekallikrein
did not decline until after 6 h, although this decrease was not
statistically different from initial levels (Fig. 5B). Comparison of the untreated and treated groups
indicated a significant difference in the 10-h course of factor XII and
prekallikrein (p < 0.05).
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1-antitrypsin complexes were assayed in
the plasma of both groups to study the effects of C1-inh administration
on E. coli-induced neutrophil degranulation. In control
animals, levels of these complexes steeply increased shortly after
start of the bacterial challenge, reaching plateau levels from T +
3 h and on, with maximal concentrations of 1491 ± 82 ng/ml
at T + 10 h. C1-inh treatment neither affected kinetics nor
levels of elastase complexes, and the highest concentrations were noted
at 6 h (1448 ± 109 ng/ml; not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Metabolic studies with radiolabeled C1-inh in human volunteers have
yielded a fractional catabolic rate of 2.5% of the plasma pool of
C1-inh per hour (38). Based on this catabolic rate, we calculated that
a continuous infusion of 200 U/kg over 9 h would be necessary to
sustain the increase in circulating C1-inh induced by a loading dose of
500 U of C1-inh/kg of body weight. Constant levels after the loading
dose were indeed observed (Fig. 1), indicating that the fractional
catabolic rate in humans can be used to calculate dosing of C1-inh to
baboons.
A rise in activation products of various components of the complement system was observed immediately after starting the E. coli infusion. C1-inh supplementation abrogated the increase of plasma C4b, indicating efficient inhibition of classical pathway activation. By contrast, the appearance of C3b and C5b-9 was incompletely blocked by C1-inh, which indicates that circulating organisms may have directly activated the alternative pathway and/or that only a small percentage of classical pathway zymogens need to be activated to cleave their substrates in a catalytic manner, resulting in less efficient inhibition of activation downstream the cascade. In addition, the more pronounced effect of C1-inh administration on the generation of C3b as compared with C5b-9 generation may also support a bypass mechanism of activation of C5, which is mediated by reactive oxygen species with the formation of a novel terminal complex containing oxidized C5 rather than C5b (52, 53). Notably, reduced activation of the complement cascade had no effect on the clearance of the infused E. coli bacteria, since circulating numbers of these organisms at 2 h postchallenge were similar in treatment and control groups. Thus, although complete inhibition of complement at the level of C3 may impair bacterial clearance (54), inhibition at the level of C1 apparently does not.
C5a is generally regarded to be the most powerful anaphylatoxin: i.v. administration of purified C5a to animals can induce hypotension (55), and pretreatment with anti-C5a Abs in a primate model of sepsis results in a recovery in mean arterial pressure (56). Moreover, it potently stimulates neutrophils to generate toxic oxygen radicals, to degranulate, and to aggregate (13). Our data show that downstream complement activation was only modestly blocked by C1-inh supplementation; effects of C1-inh are therefore not likely related to inhibition of C5a generation.
Infusion of E. coli was associated with a protracted drop in
antigenic levels of factor XII and prekallikrein within 1 h of
starting the experiment, which was impeded by supplementation with
C1-inh (Fig. 5). Reduced levels of factor XII and prekallikrein in the
control group likely reflected activation of the contact system (51).
Despite the apparent inhibition of contact system activation in the
treatment group, C1-inh administration was unable to prevent the severe
hypotension observed upon E. coli infusion. This finding
does not agree with a study by Pixley et al. showing that pretreatment
with a mAb that inhibits activation of factor XII abrogated the
secondary decline in arterial pressure observed in this model (51).
Moreover, in further contrast to the data presented here, we
demonstrated that blockade of the contact system by use of this
anti-factor XII mAb modestly reduced the release of neutrophil
elastase and t-PA and inhibited the generation of PAP complexes,
suggestive of the involvement of the contact system in neutrophil
activation and fibrinolysis (41). The findings described here need to
be reconciled with those data as well as with reported hemodynamic
effects of C1-inh in septic patients (31, 32) and endotoxemic rabbits
(34). In the present study, some degree of factor XII and prekallikrein
activation is likely to have occurred at the C1-inh concentrations used
(as suggested by slightly reduced factor XII and prekallikrein levels
at the end of the observation period; Fig. 5). In addition, although
bacteria or their products are likely candidates to have initiated
activation of the contact system shortly after start of the challenge,
negatively charged surfaces such as cell membranes and extracullar
matrix exposed as a consequence of sepsis-induced tissue damage may
have contributed to contact system activation during later stages of
the septic process. We have previously reported that in vitro, various
glycosaminoglycans may alter the relative contribution of serpins to
inactivation of contact proteases, and a twofold protection of
inhibition of -factor XIIa and ß-factor XIIa by C1-inh could
be demonstrated in the presence of dextran sulfate (57). Moreover,
activator-bound factor XIIa may not be accessible by C1-inh, whereas it
can still be inhibited by Abs (58). The inactivation rate of C1-inh may
therefore depend on the identity, localization, and phase (fluid or
solid) of the in vivo activator, and parameters such as severity and
distribution of organ damage and/or the model of sepsis employed may
influence the relative efficacy of C1-inh supplementation. Thus, some
activation of the contact system and the generation of bradykinin may
have escaped inhibition by C1-inh, but not by anti-factor XII mAb,
in this baboon model. We suggest that this may explain observed
differences between the effects of C1-inh and anti-factor XII
mAb (51).
Administration of high-dose C1-inh reduced circulating levels of TNF,
IL-6, IL-8, and IL-10 elicited by lethal infusion with E.
coli. Differences in cytokine release were not due to variations
in bacterial challenge, since the number of organisms in the infusion
fluid was similar for both treatment and control groups. Rather, our
data suggest the possibility of links between activated complement
and/or contact proteases and the cytokine response during severe
sepsis. In vitro, the anaphylatoxins as well as bradykinin and factor
XIIa can stimulate the synthesis and release of early response
cytokines such as TNF, IL-1 and IL-6 (16, 17, 18, 19, 20, 21, 22). Recently, engagement of
monocyte receptors for the fixed C3 fragments iC3b and C3b has been
shown to induce IL-1 synthesis or synthesis and secretion, respectively
(59, 60). Our results do not allow conclusions regarding the mechanism
of reduced cytokine release upon 1-inh administration. However,
considering the mild effects of this treatment on the activation of C3
and C5 (see Fig. 2), we favor the explanation that the attenuated
cytokine release resulted from reduced generation of contact activation
products.
Many symptoms of septic shock can be reproduced by direct infusion of
TNF into animals, and anti-TNF Abs reduces LPS-induced mortality
and abrogates many of the attendant manifestations of sepsis (61, 62).
Among other effects, TNF has been shown to increase endothelial
procoagulant activity (63) and induce the secretion of pro- and
antiinflammatory cytokines such as IL-10, IL-6, and IL-8 (64, 65, 66). We
show here that lowest TNF levels were measured in a treated surviving
animal displaying retarded fibrinogen consumption and reduced thrombin
formation. Survival benefit in this animal may therefore be interpreted
to result indirectly from an impediment of systemic TNF release. On the
other hand, we have previously reported that the progressive
elaboration of IL-6 and IL-8 during the later stages in this model is
associated with sepsis-induced tissue damage and death, which may occur
despite the absence of circulating immunoreactive TNF (43, 49).
Accordingly, the ability of C1-inh to modulate ongoing production of
IL-6 and IL-8 may go beyond a reduction of TNF release, and protective
effects on E. coli-induced organ injury during C1-inh
treatment may be linked to a direct interruption of these protagonists
in the proinflammatory merge of cascades. Modulatory effects of C1-inh
treatment on the cytokine response may also explain observed effect on
PAI-1 release (Fig. 2C). Synthesis and release of
PAI-1 from endothelial cells and hepatocytes is induced by IL-1, IL-6,
and TNF (67, 68, 69). PAI-1 belongs to the serpin family and acts as a
pseudosubstrate for t-PA and urokinase-type plasminogen activator,
forming inactive t-PA/PAI or urokinase-type plasminogen activator/PAI
complexes, respectively (70). However, since t-PA levels were
unaffected by C1-inh supplementation, and PAP concentrations peaked
before inhibitory effects on PAI became apparent, i.e., not until
4 h postchallenge, the attenuating effects of C1-inh on
(anti)fibrinolysis are likely of secondary importance in this E.
coli model.
Hematologic and biochemical response profiles revealed that the lethal
effects of E. coli were related to the occurrence of
disseminated intravascular coagulation and organ damage. None of the
control animals lived beyond 27 h, while in the treatment group,
one animal survived the challenge and another lived beyond 48 h.
Moreover, pathologic examination revealed less severe damage to various
organs in four of the five animals receiving C1-inh, consistent with
reduced organ dysfunction during the later stages (Table III). Notably,
we administered a twofold lower dose of C1-inh to two additional
animals, one of which survived (data not shown). These findings,
together with the observed effects on the elaboration of cytokines,
support the notion that C1-inh does interfere with reactions that occur
in the microenvironment of the plasma/target cell interface and show
that C1-inh supplementation has a beneficial, although mild effect on
the inflammatory and physiologic sequelae of lethal E. coli
challenge.
In conclusion, we demonstrate here that administration of C1-inh to baboons suffering from lethal sepsis blocked classical complement activation and reduced the decrease in plasma levels of factor XII and prekallikrein. We suggest that, in this model, activated complement and/or contact system proteases may promote E. coli-induced organ injury and lethality, at least in part, by augmentating the cytokine response.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. C. Erik Hack, CLB, Department of Pathophysiology of Plasma Proteins, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
3 Abbreviations used in this paper: C1-inh, C1-inhibitor; NBA, normal baboon serum aged; NBS, normal baboon serum; NBS-AHG, normal baboon serum incubated with heat-aggregated IgG; NBP-MA-UK, normal baboon plasma incubated with methylamine and urokinase; PAP, plasmin-2-antiplasmin complex; TAT, thrombin-antithrombin complex; tPA, tissue-type plasminogen activator; Fischer LSD, Fischer least significant difference.
Received for publication April 21, 1997. Accepted for publication September 23, 1997.
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References |
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) groups.
C3b/c and C4b/c are expressed as percentage of NBA and NBS-AHG,
respectively. Differences between both group by ANOVA repeated
measurements were significant at p < 0.05.
