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1 Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Faculty of Medicine, Department of Pharmacology, 1081 BT Amsterdam, The Netherlands; 2 Division of Endocrinology, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire EN6 3QG, United Kingdom; and 3 Palo Alto Institute of Molecular Medicine, Mountain View, California 94043.
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Peripheral administration of lipopolysaccharide (LPS) may activate the hypothalamus-pituitary-adrenal (HPA) axis by way of both neural and humoral mechanisms. We have investigated whether biologically active endotoxin appears in the general circulation after intraperitoneal administration of LPS (5 or 100 µg/kg) to rats and whether this is a prerequisite for activation of this HPA axis. Within 15 min, endotoxin appeared in the general circulation, whereas elevations of plasma adrenocorticotropic hormone (ACTH), corticosterone, and interleukin (IL)-6 concentrations were not detected until 90 min after LPS injection. At this time, a marked interindividual variation was observed in plasma concentrations of endotoxin, ACTH, corticosterone, and IL-6. Elevated levels of plasma endotoxin were associated with elevated levels of ACTH, corticosterone, and IL-6. Intravenous administration of the LPS antagonist cationic antimicrobial protein 18 (5 mg/kg), which did not affect cytokine production in the peritoneal cavity, markedly reduced plasma ACTH, corticosterone, and IL-6 levels after 5 µg/kg LPS. Our results suggest that circulating endotoxin is required for the activation of the HPA axis. They also favor a role for circulating IL-6 in this response.
interleukin-1
; adrenocorticotropic hormone; cationic
antimicrobial protein
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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IN ANIMALS AND HUMANS, peripheral administration of
gram-negative bacteria-derived lipopolysaccharides (LPS) induces an
array of brain-mediated responses, including fever
(14), sickness behavior (13), hyperalgesia (17), and activation of the
hypothalamus-pituitary-adrenal (HPA) axis (3, 25). Proinflammatory
cytokines, such as tumor necrosis factor-
(TNF-), interleukin
(IL)-1, and IL-6, which are produced by cells of the immune system
and other cells in response to LPS (7, 8), are considered to mediate
these nonspecific symptoms of illness (26, 31, 32).
However, the signaling pathways by which LPS or proinflammatory
cytokines affect the brain to induce these symptoms are still poorly
understood. After intraperitoneal administration of LPS, primary vagal
afferents have been described as crucial in the transduction of immune
signals to the brain. Thus subdiaphragmatic vagotomy blocked the
induction of sickness behavior (5) and the induction of hyperalgesia
(39). In addition, vagotomy reduced the LPS-induced expression of Fos
in hypothalamic corticotropin-releasing hormone neurons, as well as the
concomitant plasma adrenocorticotropic hormone (ACTH) response (10).
Subdiaphragmatic vagotomy also inhibited the elevation of plasma ACTH
concentrations after intraperitoneal administration of the
proinflammatory cytokine IL-1 (12), as well as IL-1-induced
hyperalgesia (40), fever (38), and sickness behavior (4). These results
have led to the hypothesis that intraperitoneal administration of LPS
stimulates the production of IL-1 in the peritoneal compartment,
which, subsequently, stimulates primary sensory fibers of the vagal
nerve.
After intravenous administration of LPS, humoral signaling pathways
have been proposed to play a crucial role in the induction of
nonspecific symptoms of illness. LPS or LPS-induced proinflammatory cytokines may act as humoral signals at the brain either by active transport across the blood-brain barrier, as demonstrated for IL-1
and IL-6 (1, 2), or by passing the barrier at the circumventricular
organs (28). In addition, circulating endotoxin or IL-1 may affect
brain functions indirectly by acting at cerebral endothelial cells.
Indeed, after intravenous administration of LPS, immunoreactive
prostaglandin E2
(PGE2), a major mediator of
nonspecific symptoms of illness (18, 36), has been detected in the
microvasculature of the brain (33). Furthermore, mRNA encoding for type
I IL-1 receptors has been found to be associated with brain vasculature
(9). Accordingly, with respect to
PGE2 production, functional type I
IL-1 receptors have been demonstrated on rat brain endothelial cells in
vitro (34).
Taken together, the above observations support the view that the route of LPS administration determines the predominant signaling pathways involved in the induction of brain-mediated symptoms of illness. However, subdiaphragmatic vagotomy, which effectively blocked the plasma ACTH response to a low dose of intraperitoneally administered LPS, was only partially effective after a high dose of LPS administered by the same route (10). Therefore, it was speculated that low doses of intraperitoneally administered LPS signal the brain primarily by vagal afferents, whereas at high doses LPS might also activate humoral signaling pathways. Because it has been reported that endotoxin concentrations in rat arterial plasma increased gradually in time after the induction of peritonitis, despite a high endotoxin clearance capacity of the rat liver (22, 23), we postulate that endotoxin, even at low doses, may reach the general circulation after intraperitoneal administration to activate vagal afferents.
In the present study, we tested whether intraperitoneally administered
LPS can reach the general circulation and, if so, whether circulating
endotoxin plays a role in the activation of the HPA axis and the
appearance of the proinflammatory cytokines IL-1 and IL-6 in the
blood. We determined plasma endotoxin concentrations, as well as plasma
ACTH, corticosterone (Cort), and IL-6 concentrations, at various time
intervals after intraperitoneal administration of LPS. We also studied
the potential role of systemic bioactive endotoxin in the activation of
the HPA axis and the induction of IL-1 and IL-6 production by
inhibiting the actions of circulating endotoxin. For this, we used a
32-amino acid fragment of the LPS antagonist rabbit cationic
antimicrobial protein 18 (CAP18).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Animals
Male Wistar rats (Harlan CPB, Zeist, The Netherlands) were housed two per cage under controlled temperature (20-22°C) and light-dark conditions (lights on 7 AM, lights off 7 PM) for at least 1 wk before the experiments. Food and water were available ad libitum. At the time of the experiments the animals had body weights of 200-240 g. To reduce nonspecific activation of the HPA axis, rats were habituated to experimental procedures twice daily for 3 successive days before the experiments. The experimental protocol had been approved by the Institutional Committee for Animal Health and Care.Materials
Lipopolysaccharide [LPS; Escherichia coli 055:B5, Westphal, 1 international unit (IU) equals 100 pg; Difco, Detroit, MI] was reconstituted in sterile pyrogen-free saline and stored at
20°C. Appropriate
dilutions were prepared immediately before administration. LPS was
injected intraperitoneally in a volume of 500 µl per rat.
A 32-amino acid fragment of the 18 kDa LPS antagonist rabbit cationic
antimicrobial protein [CAP18-(106
137)], produced by Larrick et al. (16) and designated CAP18 throughout this paper, was
dissolved in sterile pyrogen-free saline and injected intravenously (500 µl) into the lateral tail vein. This fragment has been shown to
inhibit LPS-induced release of cytokines and nitric oxide from macrophages in vitro, as well as to protect mice from LPS lethality (16).
Blood Collection
Animals were killed by decapitation and trunk blood was collected in ice-cold glass tubes that had been made endotoxin-free by heat treatment (200°C for 5 h). This procedure had also been applied to the glass funnels used for collecting the blood. The tubes contained dalteparin sodium (Fragmin, 9 IU/ml of blood, Pharmacia, Uppsala, Sweden) as anticoagulant (0.6 IU endotoxin/1,000 IU heparin). Blood samples were centrifuged (2,000 g, 10 min, 4°C), and aliquots of plasma were stored at20°C until assayed.
Peritoneal Lavage
Within 5 min after rats had been killed, 2 ml of cell culture medium (RPMI 1640, GIBCO BRL Life Technologies, Breda, The Netherlands) was injected intraperitoneally, after which the abdomen was gently massaged. Peritoneal lavage fluid was then collected in ice-cold tubes and centrifuged immediately to separate cells (2,000 g, 10 min, 4°C). Peritoneal lavage fluid was stored at20°C until analyzed.
Assays
All disposables used during the analysis of plasma samples in which endotoxin concentrations were measured were pyrogen free.Endotoxin assay. Endotoxin concentrations in rat plasma were measured using a kinetic chromogenic Limulus amebocyte lysate (LAL) assay (Pyrochrome, Associates of Cape Cod, Woods Hole, MA), according to the supplier's instructions. The First International Standard for Endotoxin (84/650, WHO) was used as a reference, and endotoxin concentrations are expressed as international units per milliliters. Before testing, samples were diluted 1:1,000 or 1:10,000 with endotoxin-free water to reduce interference of plasma components in the LAL assay. Accordingly, detection limits were 10 or 100 IU/ml plasma, respectively.
Hormone assays. Plasma ACTH
concentrations were determined by radioimmunoassay (RIA) as previously
described (21). Synthetic rat ACTH-(139) (Peninsula Laboratories,
Belmont, CA) was used as a standard, and human ACTH-(139) (Peninsula)
was used for labeling. Specific rabbit antiserum, directed to the
mid-portion of ACTH (15), was kindly provided by Dr. G. B. Makara
(Institute for Experimental Medicine, Budapest, Hungary). The
sensitivity of the RIA was 10 pg/ml plasma. Intra- and interassay
variations were 5 and 8%, respectively.
Plasma Cort concentrations were determined, using a commercially available RIA (ImmuChem Double Antibody CORT kit, ICN Biomedicals). The sensitivity of the RIA was 1.0 ng/ml plasma. Intra- and interassay variations were 7 and 8%, respectively.
Cytokine assays. IL-6 bioactivity in plasma and peritoneal lavage fluid was measured using the IL-6-sensitive B9 cell line as described elsewhere (11). Using human recombinant IL-6 (lot 1449-10-01, CLB, Amsterdam, The Netherlands) as a standard, half-maximal stimulation of B9 cell proliferation was defined as one unit of IL-6 bioactivity. The sensitivity of the assay was 12.5 U/ml plasma. Intra- and interassay variations were 5% and 6%, respectively.
Rat IL-1 concentrations in plasma and peritoneal lavage fluid were
measured using an enzyme-linked immunoassay based on a protocol
previously described (27). For this assay, 96-well microtiter plates
(Nunc-Immuno Plate MaxiSorp, Roskilde, Denmark) were coated with
immunoaffinity-purified sheep anti-rat IL-1 antibodies (NIBSC, 2 µg/ml) in 100 µl coating buffer (0.14 M NaCl, 2.7 mM KCl, 1.5 mM
KH2PO4,
8.1 mM
Na2HPO4;
pH 7.2) for 16 h at 4°C. The plates were then washed three times
with 200 µl dilution buffer (0.5 M NaCl, 2.5 mM
NaH2PO4,
7.4 mM
Na2HPO4,
0.1 % vol/vol Tween 20; pH 7.2). Samples and standards (100 µl) were
added and incubated in duplicate for 16 h at 4°C. Standards were
diluted in normal rat plasma or RPMI 1640, depending on the sample to be measured. Appropriate standard curves were included on each plate.
Subsequently, the plates were washed three times with dilution buffer
and incubated for 1 h at room temperature with biotinylated immunoaffinity-purified sheep anti-rat IL-1 antibodies (NIBSC) in
100 µl dilution buffer, containing 1% normal goat serum (DAKO A/S,
Glostrup, Denmark). After the plates had been washed three times with
dilution buffer, streptavidin, conjugated to
poly-horseradish-peroxidase (Poly-HRP-Strepta-E+, 20 ng/ml, CLB, Amsterdam, The Netherlands) was added in 100 µl dilution
buffer and wells were incubated for 30 min at room temperature. Plates
were washed three times (dilution buffer) and wells were incubated with
100 µl of 1,2-(ortho)phenylenediamine dihydrochloride solution
(P-1526 Sigma, Zwijndrecht, The Netherlands) at a concentration of 0.4 mg/ml detection buffer (34.7 mM citric acid, 66.7 mM
Na2HPO4,
containing 0.4 µl 30%
H2O2/ml
buffer; pH 5.0). The reaction was stopped with 150 µl 1.0 M
H2SO4,
and optical densities were determined at 495 nm using a microplate
reader (ICN Biomedicals, Zoetermeer, The Netherlands). The sensitivity of the assay was 1.5 pg/ml plasma. The intra-assay variation was 6%.
Statistical Analysis
Data were analyzed by two-way analysis of variance (2-way ANOVA), followed by a t-test for independent samples when values were normally distributed. If necessary, data were log transformed before analysis. IL-1 data could not be transformed
to meet criteria of ANOVA and were, therefore, tested nonparametrically
by the Mann-Whitney U test and
represented as median values. Significance was defined at the level of
P 
0.05. The statistical evaluation was carried out using the SPSS statistical program (release 6.1) for
Windows.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Plasma Endotoxin, ACTH, Cort, and IL-6 Concentrations After Intraperitoneal Administration of LPS
To determine alterations in plasma endotoxin, ACTH, Cort, and IL-6 concentrations after intraperitoneal administration of LPS, 30 male Wistar rats were divided into five groups (n = 6). Animals from four groups were injected with LPS (100 µg/kg body wt ip) and decapitated 5, 15, 30, or 90 min thereafter. Control animals (0 min) did not receive LPS. As shown in Fig. 1, endotoxin appeared in the general circulation after 15 min and reached an average plasma concentration of 1,480 IU/ml after 90 min. This equals ~150 ng/ml plasma, demonstrating that the amount of endotoxin in the general circulation represented ~10% of the injected dose of LPS. In all groups, one or more animals had plasma endotoxin concentrations below the detection limit of the LAL assay (100 IU/ml plasma). Plasma endotoxin concentrations from control animals (0 min) were all below this detection limit.
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In rats killed 5, 15, and 30 min after LPS administration, plasma concentrations of ACTH, Cort, and IL-6 were not different from control values (Fig. 2). However, markedly elevated plasma ACTH, Cort, and IL-6 concentrations were observed 90 min after intraperitoneal LPS injection. Thus the appearance of endotoxin in the general circulation preceded the elevation of plasma ACTH, Cort, and IL-6 concentrations. At 90 min, a large interindividual variation in plasma endotoxin and in plasma ACTH, Cort, and IL-6 concentrations was observed. As shown in Fig. 3, three of six rats (triangles) had plasma ACTH (Fig. 3A), Cort (Fig. 3B), and IL-6 (Fig. 3C) concentrations similar to those observed in control animals. These "nonresponding" animals all had plasma endotoxin concentrations below the detection limit of the LAL assay. In contrast, "responding" animals with elevated plasma ACTH, Cort, and IL-6 levels all had detectable endotoxin concentrations in the general circulation. These results suggest a correlation between plasma endotoxin concentrations and plasma ACTH, Cort, or IL-6 concentrations. However, because endotoxin concentrations in "nonresponding" animals were undetectable as a result of the detection limit of the LAL assay, correlation coefficients could not be computed. Therefore, we will discuss the data on the basis of an "association" between the appearance of detectable amounts of endotoxin in the circulation and elevated plasma ACTH, Cort, and IL-6 concentrations.
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Effect of CAP18 on LPS-Induced Activation of the HPA Axis and Cytokine Production
To investigate whether the presence of biologically active endotoxin in the general circulation is a prerequisite for the appearance of proinflammatory cytokines in the blood and/or for the activation of the HPA axis, we studied the effect of blockade of the actions of circulating endotoxin by intravenous administration of the LPS antagonist CAP18. For this, 48 rats were divided into six groups (n = 8). Animals from three groups were pretreated with CAP18 (5 mg/kg body wt iv), whereas the other animals received sterile pyrogen-free saline (iv). Four minutes later, animals were injected with 5 or 100 µg/kg body wt ip LPS or with sterile pyrogen-free saline. Ninety minutes later, rats were killed by decapitation, and trunk blood, as well as peritoneal lavage fluid, was collected.Plasma endotoxin concentrations. In
CAP18-pretreated rats, plasma endotoxin concentrations could not be
determined because of interference of CAP18 in the LAL assay. In
vehicle-pretreated rats, intraperitoneal administration of 5 and 100 µg/kg LPS resulted in mean endotoxin concentrations of 120 ± 31 and 5,427 ± 1,614 IU/ml plasma, respectively. Again, at 100 µg/kg
LPS, an association between the appearance of endotoxin in the general
circulation and elevated plasma ACTH, Cort, IL-6, and IL-1
concentrations was observed (Fig. 3, circles). A similar association
was observed at the lower dose of LPS. Seven rats with plasma endotoxin
concentrations above those observed in control animals had increased
plasma ACTH, Cort, and IL-6 concentrations, whereas the one rat with a
control level of plasma endotoxin also had control levels of these
plasma variables (data not shown). Moreover, at both concentrations of LPS, a complete association existed between the appearance of IL-6 in
plasma and the appearance of elevated plasma ACTH and Cort
concentrations.
Plasma ACTH and Cort concentrations. Intraperitoneal LPS administration (5 or 100 µg/kg) significantly increased plasma ACTH and Cort concentrations (2-way ANOVA, P < 0.001; Fig. 4, A and B). CAP18 pretreatment markedly reduced the ACTH response to 5 µg/kg LPS (P < 0.01), but did not attenuate the ACTH response to 100 µg/kg. Similarly, CAP18 reduced plasma Cort concentrations (P = 0.05) in response to the lower dose of LPS, but not in response to the higher dose.
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Plasma IL-1 and IL-6
concentrations. Ninety minutes after intraperitoneal
administration, the low dose of LPS (5 µg/kg) did not affect plasma
IL-1 concentrations (Fig. 4C).
After injection of the higher dose of LPS (100 µg/kg), median plasma
IL-1 concentrations increased from <1.5 (saline-treated animals)
to 52 pg/ml (LPS-treated animals). As illustrated, CAP18 markedly
reduced plasma IL-1 concentrations induced by this dose of LPS
(Mann-Whitney U test, P = 0.05, corrected for ties).
As shown in Fig. 4D, LPS significantly elevated plasma IL-6 concentrations (2-way ANOVA, P < 0.001). In vehicle-pretreated animals, mean plasma IL-6 concentrations increased from <12.5 to 1,720 and 8,466 U/ml after intraperitoneal administration of 5 and 100 µg/kg LPS, respectively (P = 0.05). CAP18 significantly reduced plasma IL-6 concentrations induced by 5 µg/kg LPS (P < 0.05), but not by 100 µg/kg LPS.
IL-1 and IL-6 concentrations in peritoneal
lavage fluid. To investigate whether intravenous
administration of CAP18 affected LPS-induced cytokine concentrations in
the peritoneal cavity, levels of IL-1 and IL-6 were measured in
cell-free peritoneal lavage fluid. Intraperitoneal administration of 5 and 100 µg/kg LPS to vehicle-pretreated animals induced high levels
of both cytokines in the lavage fluid, which were already maximal at
the lower dose of LPS (Fig. 5).
Concentrations of IL-1 ranged from 19 ± 8 in saline-treated rats
to 681 ± 99 and 857 ± 125 pg/ml in 5 and 100 µg/kg
LPS-treated rats, respectively, whereas in the same groups of animals
IL-6 concentrations ranged from 12.5 to 10,825 ± 3,029 and 17,668 ± 3,156 U/ml. These concentrations were not significantly affected
by CAP18, indicating that intravenous administration of this LPS
antagonist did not affect LPS-induced cytokine production within the
peritoneal cavity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have shown that intraperitoneally administered LPS reaches the general circulation and that the appearance of endotoxin in the blood is a prerequisite for the appearance of the proinflammatory cytokine IL-6 in the general circulation, as well as for the activation of the HPA axis.
Previously, we reported that plasma ACTH, Cort, and IL-6 concentrations
peaked ~2 h after intraperitoneal administration of LPS. In the
present study we show that the appearance of endotoxin in the plasma
preceded the IL-6 and HPA responses. In addition, 90 min after
intraperitoneal injection of both a relatively low (5 µg/kg) and a
relatively high dose of LPS (100 µg/kg), our results demonstrate a
marked interindividual variation in plasma endotoxin concentrations.
This variation could not be explained by a biotechnical failure
(unpublished studies involving intraperitoneal injection of blue
dextran). Moreover, without exception, all responding and nonresponding
animals had elevated levels of IL-1 and IL-6 in peritoneal lavage
fluid, which suggests that LPS was appropriately injected. These
observations led us to postulate that in most animals bioactive
endotoxin reaches the general circulation after intraperitoneal
injection of LPS, whereas in some animals it does not. The nature of
these individual differences remains speculative, although similar
observations have previously been published (23). Irrespective of its
exact nature, our results show that the appearance of detectable
amounts of biologically active endotoxin in the blood coincided with
elevated plasma ACTH, Cort, and IL-6 concentrations. Because direct
administration of LPS into the general circulation is known to activate
the HPA axis and to induce elevated plasma levels of IL-1 and IL-6
(6, 19), we considered the possibility that circulating endotoxin in
our experiments may in fact mediate these responses.
Indeed, administration of the LPS antagonist CAP18 into the general
circulation significantly reduced plasma ACTH, Cort, and IL-6
concentrations induced by the low dose of LPS (5 µg/kg). Similarly,
in mice, systemic anti-LPS antibodies markedly reduced plasma TNF-,
IL-1, and IL-6 levels after intraperitoneal injection of live
E.
coli bacteria (41). In contrast to its
effects on the low dose of LPS, CAP18 did not affect plasma ACTH, Cort,
or IL-6 responses to 100 µg/kg ip LPS, possibly because the dose of
CAP18 (5 mg/kg) was insufficient to inactivate all circulating endotoxins. It should be noted that 5 µg/kg is already a maximally effective dose of intraperitoneally administered LPS for activation of
the HPA axis (29) and that mean plasma endotoxin levels after the high
dose of intraperitoneally administered LPS were ~50-fold higher than
those observed after intraperitoneal administration of the low dose. A
limited availability of CAP18 prevented us from testing higher CAP18
doses. Surprisingly, compared with the other variables, the induction
of detectable concentrations of IL-1 in the general circulation was
less sensitive to intraperitoneally administered LPS, because 5 µg/kg
LPS was insufficient to increase plasma IL-1 concentrations.
Accordingly, plasma IL-1 concentrations were reduced by CAP18 at the
higher dose of LPS, suggesting that inactivation of only a part of the
circulating endotoxin was sufficient to diminish the increase in plasma
IL-1 concentrations.
Results from several studies suggest that IL-1 may act as an
intermediate in the LPS-induced activation of the HPA axis. For
example, intraperitoneal administration of the IL-1 receptor antagonist
(IL-1ra) inhibited the ACTH and Cort responses to intraperitoneally administered LPS (29). These results are in line with other data,
suggesting that IL-1 in the peritoneal cavity may directly stimulate
primary sensory fibers of the vagal nerve to activate the HPA axis (12)
as well as to induce other nonspecific symptoms of illness (4, 38, 40).
However, our results, showing that nonresponding rats still had high
levels of both IL-1 and IL-6 in the peritoneal lavage fluid,
challenge the proposed role for peritoneally derived IL-1 (or IL-6)
in the activation of the HPA axis after intraperitoneal LPS
administration. Our results also do not support a role for circulating
IL-1 in the activation of the HPA axis after intraperitoneal LPS
administration. First, after administration of a maximally effective
dose of LPS (5 µg/kg) for HPA axis activation (29), plasma IL-1
concentrations were <1.5 pg/ml (equivalent to ~0.1 pM) and are
unlikely to represent biologically active concentrations. Second, at
the higher dose of LPS, CAP18 reduced plasma IL-1 concentrations,
whereas plasma ACTH and Cort levels remained unaffected. Third, also at
shorter time intervals (30 and 60 min) after intraperitoneal LPS
administration (5 µg/kg), IL-1 was not detected in plasma (own
unpublished observations), which excludes the possibility that we
missed the plasma IL-1 response at 90 min. In line with our results,
other investigators also questioned a role for plasma IL-1 in the
LPS (ip)-induced activation of the HPA axis (7).
Another proinflammatory cytokine that may be involved in the activation
of the HPA axis in response to LPS is IL-6. Intravenous administration
of IL-6 markedly stimulated the ACTH and Cort release in rats (20, 35),
but its potency (on the basis of weight) was shown to be two- to
fivefold lower than that of IL-1 (20; own unpublished results).
However, in the present experiments, plasma IL-6 concentrations were
~2-8 ng/ml (1 U of IL-6 equals ~1 pg of IL-6), which was
almost 200 times higher than plasma concentrations of IL-1.
Therefore, IL-6 is most likely to play a signaling role in the
LPS-induced activation of the HPA axis. Moreover, the LPS dose-response
characteristics of plasma IL-6 concentrations (and not those of plasma
IL-1) were similar to those of plasma ACTH and Cort concentrations
in that 5 µg/kg LPS induced maximal responses for all three
parameters. Furthermore, the reduction in plasma ACTH and Cort
concentrations by CAP18 at the lower dose of LPS was accompanied by a
reduction in plasma IL-6 concentrations, whereas at the higher dose of
LPS, like plasma ACTH and CORT concentrations, plasma IL-6
concentrations remained unaffected by CAP18. These observations,
together with the association between the presence of IL-6 in the
general circulation and the induction of plasma ACTH and Cort, indicate
a possible intermediate role for plasma IL-6 in the activation of the
HPA axis.
Both doses of LPS induced high levels of IL-1 and IL-6 in the
peritoneal lavage fluid, with plateau levels reached after 5 µg/kg
LPS. Therefore, it is unlikely that IL-1 is readily transported from
the peritoneal cavity to the general circulation, because IL-1 could
not be detected in the plasma after the low dose of LPS. In addition,
CAP18 did not affect cytokine levels in the peritoneal cavity, whereas
it suppressed plasma concentrations of both IL-1 and IL-6. These
data further support the idea that cytokines produced in the peritoneal
cavity remain locally in the time frame of our experiment and that
cytokines in the blood are primarily derived from endothelial cells or
monocytes outside the peritoneal cavity (24, 37).
Taken together, the present results suggest that intraperitoneal
administration of a low dose of LPS activates the HPA axis in a manner
dependent on the presence of endotoxin in the blood. Moreover,
circulating IL-6, rather than circulating IL-1, seems to play an
intermediate role in the activation of this axis. Because results from
earlier studies have suggested that low-dose LPS-induced ACTH secretion
is mediated by the vagal nerve, we postulate that vagal afferents are
activated by circulating, rather than by peritoneally derived,
endotoxins or cytokines, which is supported by recent observations
showing that endotoxin in the circulation, or systemically induced
pro-inflammatory mediators, may induce nonspecific symptoms of illness
by a neural route (30).
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ACKNOWLEDGEMENTS |
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We thank S. Selkirk, R. Binnekade, and J. Brevé for excellent technical assistance.
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FOOTNOTES |
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This study was supported by the BIOMED I program "Cytokines in the brain" (PL-391450) of the commission of the European Communities.
Address for reprint requests: F. J. H. Tilders, Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Faculty of Medicine, Dept. of Pharmacology, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.
Received 11 June 1997; accepted in final form 19 August 1997.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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(n = 6), data derived from experiment
described in Figs. 
(n = 8), data derived from experiment described in Fig. 
