Department of Emergency Medicine, University of Tsukuba School of Medicine, Tennohdai, Tsukuba, 305 Japan
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ABSTRACT |
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Trauma victims may suffer from repeated hemorrhage, but
responses of cytokines to it have not been described. To study this question, we first detected the time course of changes in serum tumor
necrosis factor (TNF) activity and hepatic TNF- mRNA by cytotoxicity
against L929 cells and by reverse transcription (RT) and polymerase
chain reaction (PCR), respectively, after different sizes of hemorrhage
(10-20 ml/kg) with chronically cannulated rats. Then we examined
the changes in TNF-
mRNA when two sequential 10 ml/kg hemorrhages
were performed. TNF activity showed no significant increases after
either size of hemorrhage. At mRNA level, both 15 ml/kg and 20 ml/kg
hemorrhages induced significant increases after hemorrhage, whereas a
10 ml/kg hemorrhage did not. When the 10 ml/kg hemorrhage was repeated
24 h later, however, TNF-
mRNA showed a significant increase. There
were no significant differences in blood pressure and heart rate after
single and repeated 10 ml/kg hemorrhage. This potentiation persisted
for
48 h. These results show that responses of hepatic TNF-
mRNA are augmented when moderate hemorrhage is repeated.
cytokine; hemorrhagic shock; hypothalamus-pituitary-adrenal axis
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INTRODUCTION |
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CYTOKINES are small polypeptides or glycoproteins
secreted by a cell that affects growth and metabolism either of the
same (autocrine) or of another (paracrine) cell. Since 1980, when
interferon- became the first cytokine to be isolated, the number has
grown to >50 distinct molecules (1). Cytokines show pleiotropy and redundancy in their actions and form a complex network by either inducing or suppressing the expression of other cytokines. In responding to external stimuli, the vertebrates set in motion molecular
and cellular interactions designed to facilitate a return to
physiological homeostasis and to aid tissue repair, and the cytokine
network is deeply involved in these complex interactions. Tumor
necrosis factor-
(TNF-
) is one of the proinflammatory cytokines;
it has a great influence on deciding temporal and spatial patterns of
inflammatory responses. Its role in the development of organ failure
after massive hemorrhage as well as sepsis is strongly suggested (3,
12, 26, 29).
In clinical settings, trauma victims often suffer simultaneously from
different kinds of stimuli, including pain, tissue injury, and
hemorrhage. They may also suffer from repeated stimuli, for example,
rebleeding. Therefore, it is important to study the physiological responses to the combination of stimuli and to repeated stimuli, because the responses may differ from those observed after a single stimulus. It is well-known that the hypothalamus-pituitary-adrenal (HPA) axis shows a potentiated response to repeated hemorrhage as well
as to a combination of stimuli (5, 13, 17, 18). Because the cytokine
network is subject to endocrine and neural control and exerts a
reciprocal effect on neuroendocrine systems (19), responses of the
cytokine network may be modified by a combination of stimuli and by
repeated stimuli. It has already been reported that the cytokine
network shows a potentiated response to the combination of trauma and
hemorrhage (3). To our knowledge, however, responses of the cytokine
network to repeated hemorrhage have not yet been described. To study
this question, we first detected the time course of changes in serum
TNF activity and hepatic TNF- mRNA after different sizes of
hemorrhage (10-20 ml/kg) by using chronically cannulated conscious
rats. Then we examined the changes in hepatic TNF-
mRNA when rats
were subjected to two sequential 10 ml/kg hemorrhages spaced 24-72
h apart.
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MATERIALS AND METHODS |
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Animal preparation. Male Wistar rats (Charles River, Tsukuba, Japan), weighing 300-400 g, were anesthetized with pentobarbital sodium (50 mg/kg ip), and a femoral arterial cannula was placed using sterile procedures as previously described (11). After recovery, all rats were housed individually in a temperature-controlled environment with a 12:12-h light-dark cycle and were allowed to move freely. Access to food and water was given ad libitum. On the morning of the 4th day after cannulation, the following three experiments were begun. All procedures were performed by manipulating the cannulas outside the cage so as not to disturb the rats in any way. All the experiments were approved by the Animal Care Committee of the University of Tsukuba.
Experiment 1.
Ninety chronically cannulated rats were subjected to either 10, 15, or
20 ml/kg hemorrhage over 3 min, and the blood shed into a sterile
heparinized (25 U) syringe was reinfused 60 min later. At
time 0 or 0.5, 1, 2, 4, or 24 h after
hemorrhage, rats were killed, and livers were removed and snap-frozen
in liquid nitrogen for extraction of total cellular RNA
(n = 5 livers for each time point).
Immediately before the slaughter, 0.4 ml of blood was withdrawn from
the arterial catheter for measurement of TNF activity by cytotoxicity
against L929 cells (24). An endotoxin-treated rat, killed 2 h after 100 g/kg iv endotoxin, was used as a positive control
(Escherichia coli lipopolysaccharide, serotype 0111: B4; Sigma Chemical, St. Louis, MO). The time course of
changes in TNF- mRNA was detected by reverse transcription (RT) and
polymerase chain reaction (PCR), as previously described (28, 29).
Briefly, the first strand of cDNA was synthesized by reverse
transcriptase (MMLV-RT, GIBCO BRL, Rockville, MD) and pooled. The
resulting cDNA samples were adjusted to PCR buffer conditions and were
run for PCR simultaneously. The primers for TNF-
were
5'-TCAGCCTCTTCTCATTCCTGC and
5'-GTGCAGCATCGTTTGGTGGTT (15). A three-temperature step PCR cycle
program was carried out with an OmniGene Temperature Cycler (0.5 min
annealing at 54°C, 1 min extension at 72°C, and 1 min
denaturation at 94°C). The length of the PCR product was 203 base
pairs and was checked by electrophoresis with 1 kb DNA Ladder (GIBCO
BRL). After PCR, the products were quantitated by HPLC as previously
described (28). Twenty-five to thirty-five PCR cycles were run to
determine the dependency of the number of PCR products on cycle
numbers. Linearity between the amount of amplified cDNA and that of
applied RNA was checked in the preplateau exponential phase. The
relative multiple-increase changes in TNF-
mRNA at each time point
after hemorrhage compared with the level of normal untreated rats were determined after normalization for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The primers for GAPDH were
5'-TGATGACATCAAGAAGGTGGTGAAG and
5'-TCCTTGGAGGCCATGTAGGCCAT (28).
Experiment 2.
First, to detect the changes in mean arterial blood pressure (MABP) and
heart rate (HR), fifteen chronically cannulated rats were divided into
three groups and were subjected to either a 10 ml/kg hemorrhage or sham
hemorrhage on two successive days; these were the HH group (10 ml/kg
hemorrhage on days 1 and
2), the SH group (sham-hemorrhage on
day 1 and a 10 ml/kg hemorrhage on
day 2), and the SS group
(sham-hemorrhage on days 1 and
2). The changes in MABP and HR were
recorded on day 2 by connecting the
arterial cannula to a hemodynamic monitor (Life Scope II, Nihon Kohden,
Tokyo, Japan) through a high-pressure transducer (Gould, Oxnard, CA).
Another 75 cannulated rats were divided into the three (HH, SH, and SS)
groups for detection of the changes in hepatic TNF- mRNA. Rats were
killed and livers were removed on day
2 at time 0 or at 0.5, 1, 2, and 4 h for extraction of RNA (n = 5 for each group). The time course of changes in hepatic TNF-
mRNA
was detected by the method described in experiment 1.
Experiment 3.
Ten chronically cannulated rats underwent two sequential 10 ml/kg
hemorrhages spaced by 48 and 72 h (n = 5 for each group). Rats were killed and livers were removed 1 h after
the second hemorrhage for extraction of RNA. The increases in TNF-
mRNA were detected by the method used in experiment
1 and were compared with the results in
experiment 2.
Statistics. Data were analyzed by two-way ANOVA, corrected for repeated measures over time. Differences between HH, SH, and SS groups at the individual time points were analyzed by the Newman-Keuls test after two-way ANOVA. Differences between control, single, and repeated 10 ml/kg hemorrhage were also analyzed by the Newman-Keuls test after two-way ANOVA. Significant differences are indicated (P < 0.05).
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RESULTS |
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Experiment 1. One blood sample taken 2 h after 20 ml/kg hemorrhage showed 0.2 U/ml TNF activity, but all the other samples showed no detectable TNF activity (<0.1 U/ml). Thus the changes in TNF activity were not significant after either size of hemorrhage. On the contrary, the blood sample taken 2 h after endotoxin treatment showed >64 U/ml.
When reverse-transcribed RNA from an endotoxin-treated rat was subjected to an increasing number of PCR cycles, a period of exponential increase in PCR products was followed by a period of saturation between 25 and 35 cycles. At 29 cycles, good linearity between the amount of reverse-transcribed RNA and that of the resulting PCR product was acquired over a range of 0.0063-0.2 µg. When 0.2 µg of reverse-transcribed RNA from hemorrhaged rats was amplified by RT-PCR at 29 cycles, the peak areas of the PCR products were within the linear range. All further PCR experiments were performed using 0.2 µg of reverse-transcribed RNA at 29 cycles. Reproducibility of PCR was good, and the PCR products for TNF-
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Experiment 2.
MABP and HR remained constant in the SS group throughout the
experiment. However, in the HH and SH groups, 10 ml/kg hemorrhage caused a prompt decrease in both MABP and HR (Fig.
2). By 1 h after hemorrhage, MABP and HR
recovered to the prehemorrhage level and stabilized. The responses of
MABP and HR were similar in the HH and SH groups, and there were no
significant differences between these two groups at any time point. The
changes in TNF- mRNA after hemorrhage were small and not significant
in the SS and SH groups, whereas the changes were significant in the HH
group; the peak level in the HH group was ~70% of that after single
15 ml/kg hemorrhage (Fig. 2,
bottom). The differences between the HH and SH groups were significant at 0.5 and 1 h.
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Experiment 3.
When two sequential 10 ml/kg hemorrhages were spaced 48 h apart, the
increase in TNF- mRNA at 1 h was still significant, whereas when two
sequential hemorrhages were spaced 72 h apart, the increase in TNF-
mRNA was small and not significant compared with the prehemorrhage
level (Fig. 3).
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DISCUSSION |
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In traumatized patients, control of bleeding is sometimes difficult,
and bleeding may occur repeatedly. In such cases, the responses of the
neuroendocrine system, including the HPA axis and the sympathoadrenal
system, are augmented, even if hemorrhage size is small (17, 18). To
our knowledge, the response of the cytokine network to repeated
hemorrhage has not yet been described. In this study, single 10 ml/kg
hemorrhage did not induce a significant increase in hepatic TNF-
mRNA. When the same size of hemorrhage was repeated 24 h later,
however, TNF-
mRNA showed a higher peak, and the increase after
hemorrhage was significant. These facilitative effects of the first
hemorrhage persisted for
48 h. These results show that the cytokine
network also shows a potentiated response when moderate hemorrhage is
repeated.
It is now widely believed that TNF- is a crucial factor in the
development of the systemic inflammatory response syndrome (SIRS) and
organ failure after surgical insult (4, 12). Many studies have
demonstrated elevated circulating levels of TNF-
in patients with
clinically overwhelming infections and in animals subjected to
intravenous injections of live bacteria or endotoxin (6, 27). However,
in clinical SIRS and even in animal models, increases in serum TNF-
concentrations are often modest or absent (2, 7). Our previous study
also showed that neither hemorrhagic shock (MABP = 40 mmHg for 60 min)
nor 20 ml/kg hemorrhage induces significant changes in serum TNF
activity (29). It seems that circulating levels of TNF-
do not
always reflect the magnitude of insult. One of the reasons for these
results is that induction of TNF-
is usually transient. Its mRNA is
unstable because of an adenine/uracil-rich sequence in the
3'-untranslated region, and both protein and message are easily
degraded when its role is finished (1). Another reason is that TNF-
mainly acts at local sites in an autocrine and/or paracrine
manner. Interestingly, a recent study has shown that gene expression of
1-acid glycoprotein in the
liver correlates well with intrahepatic TNF-
abundance but not with
circulating TNF-
levels in cecal ligation and puncture models of
rats (2). Liver is a major site for synthesis of TNF-
as well as of
acute phase proteins and blood coagulation factors (10, 22). Although
TNF-
mRNA levels may not accurately reflect protein secretion, they
correlate with TNF synthesis in general (1, 6). On the basis of these
findings, we detected the changes in hepatic TNF-
mRNA in this
study.
Responses of the mammalian body to external stimuli are exquisite and complicated, and both feedback inhibition and facilitation can occur after repeated hemorrhage. The changes observed after the second hemorrhage are the result of a complex interplay of the facilitative and feedback effects elicited by the first stimulus (17, 18). For example, the dog shows an inhibition of adrenocortical response after two sequential 10 ml/kg hemorrhages spaced 90 min apart, whereas it shows a facilitative response after two 7.5 ml/kg hemorrhages spaced 24 h apart (13, 18). Which phenomenon, inhibition or facilitation, is dominant is influenced by many factors, including magnitude of hemorrhage, rate of hemorrhage, interval of sequential hemorrhages, anesthesia, and dietary factors (18). When a delay of 24 h is selected between two small hemorrhages, this period provides an adequate time for resolution of the feedback effects after the first hemorrhage and reveals the residual facilitative effects in the HPA axis (18). We also adopted an interval of 24-72 h between two sequential 10 ml/kg hemorrhages in this study. Another important factor is an interval between cannulation and hemorrhage. We began the hemorrhage experiments on the morning of the 4th day after anesthesia and cannulation, whereas experiments were performed immediately after anesthesia and cannulation in most of the other studies detecting the changes in cytokines. In such an acute setting, the first stimuli of anesthesia and surgical intervention may exert complex influences on the following treatment and may obscure a net effect of the treatment. Accordingly, chronically cannulated rats like ours seem to be more adequate for analysis than acutely prepared models.
"Two-hit theory" has been proposed in the development of organ
failure after surgical insult (12). According to this theory, priming
of monocytes/macrophages is achieved after the first insult. When the
second attack is delivered in this state, even if it is not so severe,
primed cells produce excessive inflammatory mediators and bring about
organ failure. Although excessive production of TNF- may harm the
body, induction of TNF-
to external stimuli is originally a
teleological response. For example, mild endotoxin administration
results in the release of TNF-
into the local tissue milieu, where
TNF-
initiates and orchestrates many of the beneficial responses
aimed at improving antimicrobial function and reducing tissue damage
(21). The physiological significance of cytokine induction after
hemorrhage is unknown, but one possibility is activation of the blood
coagulation system to avoid further blood loss. Although vascular
endothelial cells are antithrombogenic in the normal state, they lose
their antithrombogenic properties when they are perturbed by cytokines
(10). We have shown that clotting time shortens and plasminogen
activator inhibitor (PAI) activity increases after 20 ml/kg hemorrhage
and that both tissue factor mRNA and PAI-1 mRNA are induced in the
liver (28, 30). The augmented responses of hepatic TNF-
mRNA to
repeated hemorrhage may prepare the body for prompt hemostasis.
The mechanism whereby augmented responses of hepatic TNF- mRNA occur
to repeated hemorrhage is also unknown. In this study, the responses of
MABP and HR were similar in the SH and HH groups, and no significant
differences were noted between these two groups. Although there is a
possibility that we could not detect small hemodynamic differences by
monitoring MABP and HR, blood volume is completely restored within 24 h
after 10 ml/kg hemorrhage even without reinfusion (14), and no change
in blood volume was detected in experiments using a similar hemorrhage
and reinfusion protocol (McCloud and Gann, unpublished observations
cited in Ref. 17). Therefore, it is unlikely that the potentiation in
hepatic TNF-
response was due to differences in the hemodynamic
response. After repeated exposure to endotoxin, suppressed TNF-
production by macrophages is observed (16), and this "endotoxin
tolerance" is reported to be associated with alterations in guanine
nucleotide binding protein activity and in arachidonic acid turnover
(8, 23). Tolerance to myocardial ischemia is also
reported, and this phenomenon seems to be associated with alterations
in receptor subtypes and with phosphorylation of intracellular enzymes
(20). The augmented response of hepatic TNF-
mRNA to repeated
hemorrhage may also result from alterations in intracellular signaling
pathways at several levels. Recently, it has been shown that gene
expression of TNF-
is transcriptionally regulated by nuclear
factor-
B (NF-
B), a transcription factor that is activated by a
variety of conditions, including oxidant stress and endotoxin (9, 25).
In response to these stimuli, I
B, an inhibitory protein, is removed
from the latent NF-
B, and the active transcription factor is then translocated from the cytoplasm to the nucleus, where it binds to a
specific cis-acting regulatory element
in target genes. Interestingly, in vitro studies have shown that
NF-
B is activated by TNF-
(25) and that dexamethasone treatment
diminishes the basal nuclear level of NF-
B (9). Changes in nuclear
factor as a consequence of prior stimulation might play a role in the
augmented response of hepatic TNF-
mRNA to repeated hemorrhage.
Further study is needed to elucidate the precise mechanism.
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FOOTNOTES |
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Address for reprint requests: M. Yamashita, Dept. of Emergency Medicine, Univ. of Tsukuba Hospital, 2-1-1, Amakubo, Tsukuba, 305-8576 Japan.
Received 3 November 1997; accepted in final form 31 March 1998.
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