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Department of Surgery, University of Vienna, 1090 Vienna, Austria
1Correspondence: Department of Surgery, University of Vienna, Währinger Gürtel 18–20, 1090 Vienna, Austria. E-mail: Michael.Bergmann{at}akh-wien.ac.at
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß receptor
antagonists showed that the effect is mediated by ß1- and
ß2-adrenergic receptors. Thus, endothelial cells might be
a possible source of increased IL-6 production observed in situations
such as stress or septic shock, in which catecholamines are elevated
due to endogenous production or exogenous application.—Gornikiewicz,
A., Sautner, T., Brostjan, C., Schmierer, B., Függer, R., Roth,
E., Mühlbacher, F., Bergmann, M. Catecholamines up-regulate
lipopolysaccharide-induced IL-6 production in human microvascular
endothelial cells.
Key Words: immunomodulation • transcription • adrenoreceptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. However, a significant number of clinical trials that
were designed to inhibit proinflammatory cytokines have failed to
increase the prognosis of septic shock (2)
. These results
might relate to the fact that proinflammatory cytokines such as tumor
necrosis factor (TNF-) and interleukin 1ß (IL-1ß) are only
initially elevated in sepsis and are found later on at low levels
(3)
due to an increase in immunosuppressive hormones and
cytokines. This hyporeactive state of the immune system is designated
as the compensatory anti-inflammatory response syndrome, which is now
thought to be of pathognomonic relevance (4)
. Effective
therapies for septic shock should therefore consider the clinical
course of sepsis, including the multiple interactions of cytokines and
the modulation of the cytokine network by hormones and therapeutic
drugs.
The sepsis-associated immunosuppression can be explained by an
elevation of anti-inflammatory cytokines such as IL-10 (5
, 6)
and transforming growth factor (7)
, by an
increase in soluble TNF- and IL-1 receptors (8)
, and by
an elevated level of immunosuppressive hormones such as catecholamines
and glucocorticoids. IL-6, a cytokine with pro- and anti-inflammatory
activities (9)
, is not only highly elevated at the onset
but remains up-regulated throughout the disease. In fact, serum levels
of IL-6 correlate with the prognosis of sepsis (10)
and
are currently being used as an inclusion criterion for clinical trials
in septic shock. The anti-inflammatory activities of IL-6 might be even
more relevant for the pathomechanism of sepsis than proinflammatory
actions such as the induction of fever and B cell proliferation. The
main features of IL-6-induced immunosuppression are the down-regulation
of lipopolysaccharide (LPS) -stimulated TNF- and IL-1ß expression
(11)
and the induction of soluble TNF- receptor p55 and
IL-1 receptor antagonist (12)
in hematopoietic cells.
Catecholamines are elevated in severe sepsis and septic shock due to
endogenous production as well as exogenous application. In septic shock
they are an indispensable part of the therapy to restore adequate blood
pressure. Catecholamines were also shown to modulate cytokine
expression. For example, epinephrine decreases TNF- serum levels and
enhances those of IL-10 in human endotoxemia (13)
. With
respect to IL-6, the subcutaneous application of epinephrine was shown
to increase the cytokine serum level in the rat (14)
.
Furthermore, in an isolated rat liver perfusion model, epinephrine
increased IL-6 expression (15)
. Kupffer cells were
suggested to be the source of IL-6 production in this model. Szabo et
al. (16)
confirmed a ß-adrenergic enhancement of IL-6
stimulation by the administration of isoproterenol in a mouse
endotoxemia model. We have recently demonstrated that therapeutically
applied catecholamines increase IL-6 mRNA levels in liver, abdominal
lymph nodes, and spleen in a porcine endotoxemia model (T. Sautner, A.
Gornikiewicz, and M. Bergmann, unpublished results). Thus,
sepsis-related IL-6 production could be up-regulated by epinephrine to
a significant extent. However, when whole blood of healthy volunteers
was stimulated ex vivo with LPS, catecholamines increased
protein levels of IL-10 but down-regulated mRNA and protein levels of
TNF-, IL-1ß, and IL-6 (17
18
19)
. Thus, hematopoietic
cells do not seem to mediate epinephrine-induced IL-6 production.
We hypothesize that tissue-derived cells might be the source of the
catecholamine-induced IL-6 levels. A likely candidate is the
endothelial cell, since endothelial cells are involved in cytokine
regulation in sepsis. They are in close contact with substances
circulating in the bloodstream and have the potential to produce IL-6
(20)
. We have investigated the catecholamine-induced
modulation of LPS-stimulated IL-6 expression and the underlying
molecular mechanism in a human microvascular endothelial cell line
(HMEC-1) (21)
. We have confirmed our findings in a primary
endothelial cell culture obtained from human skin capillaries.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
HMEC-1, an immortalized human dermal microvascular endothelial
cell line (21)
, was kindly provided by Dr. Edwin W. Ades,
Francisco J. Candal (Centers for Disease Control and Prevention,
Atlanta, Ga.) and Dr. Thomas Lawley (Emory University, Atlanta, Ga.)
and was cultured in Dulbecco’s modified Eagle’s medium (Bio
Whittaker, Verviers, Belgium) supplemented with 10% fetal calf serum
(FCS), 2 mM glutamine, 1 µg/ml hydrocortisone (Sigma), 10 ng/ml human
epidermal growth factor (Sigma), 50 U/ml penicillin, and 50 µg/ml
streptomycin (Gibco BRL, Life Technologies, Paisley, U.K.). To isolate
primary human skin microvascular endothelial cells (HSMECs), skin
tissue was digested with trypsin for 30 min, mechanically broken up,
and passed through a cell strainer. Microvascular endothelial cells
were isolated with anti-CD31 antibodies conjugated to magnetic beads,
placed into culture, and subsequently subjected to a second round of
selection with antibody-conjugated beads. HSMECs were cultured with
medium 199 containing 150 mg/l L-alanyl-L-glutamine (Gibco BRL, Life
Technologies) supplemented with 20% FCS, 0.03 mg/ml endothelial cell
growth supplement (Upstate biotechnology, Lake Placid, N.Y.), 50 U/ml
penicillin and 50 µg/ml streptomycin (Gibco BRL, Life Technologies)
in tissue culture dishes precoated with 1% gelatin. Cells were
maintained at 37°C and 5% CO2.
Enzyme-linked immunosorbent assay (ELISA)
To study IL-6 release, endothelial cells were grown to
confluence and stimulated for indicated time intervals. Supernatants
were collected, centrifuged at 250 g to remove debris, and
frozen until further analysis. IL-6 concentrations were determined by
ELISA (Amersham, Little Chalfont, U.K.) according to the
manufacturer’s protocol.
RNA extraction and Northern blot analysis
For RNA analysis, cells were grown to confluence, stimulated,
and total RNA was extracted with Trizol reagent (Gibco BRL, Life
Technologies) according to the manufacturer’s protocol. Total RNA (40
µg) was denatured with 5.5 M glyoxal at 50°C and the RNA was
separated on a 1.5% agarose gel by electrophoresis. RNA was blotted
overnight onto a Hybond N nylon membrane (Amersham) by capillary action
in a buffer containing 20 x SSC. The RNA was fixed on the
membrane by UV cross-linking and hybridized to radiolabeled cDNA probes
of human IL-6 and ß-actin. IL-6 cDNA was obtained by reverse
transcription polymerase chain reaction (RT-PCR). The primers for
IL-6 bind to mRNA positions 13–35 and 944–968, respectively, giving a
product length of 955 bp. They read as follows:
5'CGAAAGAGAAGCTCTATCTCCCC-3' and 5'CAAAGGATTCAAACTGCATAGCC-3'. For
ß-actin we used the primer pair at mRNA positions 294–325 and
1131–1100 (Clontech, Palo Alto, Calif.). RT-PCR products were
gel-purified from a low melting agarose gel. Purified cDNA (50 ng) was
labeled with a random priming kit (Amersham) and
32P-dCTP. Labeled cDNA was then purified from
unincorporated nucleotides by nick columns (Pharmacia Biotech, Uppsala,
Sweden) and measured in a beta counter. 5 x
105 counts/ml were used for hybridization. RNA
was hybridized at 65°C overnight in a buffer containing 5 x
TEN (75 mM Tris/chloride, 5 mM EDTA and 0.75 M NaCl),
5 x Denhardt’s solution and 0.2% sodium dodecyl sulfate (SDS).
Membranes were washed twice with 2 x TEN and 0.2% SDS at 65°C
for 15 min, followed by one washing step with 0.2 x TEN and 0.2%
SDS at 65°C for 30 min. For rehybridization, membranes were stripped
by rinsing them four times with 0.1 x SSC and 0.1% SDS at
95°C. Bands obtained by autoradiography were quantitated with a
densitometer (PDI, Huntington Station, N.Y.). All Northern blot
experiments were carried out twice and gave comparable results. For
statistical analysis, mRNA quantitation of HMEC-1 stimulated with
LPS ± epinephrine for 2 h was done three times.
mRNA stability assay
Cells were stimulated with LPS (1 µg/ml) in the presence or
absence of epinephrine (100 ng/ml). After 2 h, actinomycin D was
added at a final concentration of 10 µg/ml. At various time points
after the addition of actinomycin D, total RNA was isolated and 40 µg
of each sample was subjected to Northern blot analysis as described
above. Autoradiographic signals were quantitated by laser densitometry
and normalized to ß-actin signals.
Statistical analysis
ELISA data are presented as the mean and the standard error of
the mean (SE). For the time course ELISA experiment,
P values were determined by repeated measures analysis of
variance (ANOVA). For concentration dependency experiments, blockage
experiments, and stimulation experiments on HSMEC, P values
were determined by ANOVA. For Northern blot analysis, data obtained
from laser densitometry are presented as the mean and the
SE; the P value for Northern blot data
was calculated by Students’ t test. P values of
<0.05 were considered as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The level of this synergistic up-regulation was 8.3-fold
(P<0.0005) when compared to stimulation with LPS alone and
6.6-fold (P<0.0005) when compared to incubation with either
catecholamine alone, respectively. Keeping the LPS concentration at a
constant level while varying the epinephrine-concentration and vice
versa revealed a dose dependency of the synergistic effect of
catecholamines and LPS (Fig. 2
). A statistically significant synergism was reached for 1 ng/ml of
epinephrine and for 10 ng/ml of LPS, respectively. To investigate
whether the catecholamine-induced IL-6 production was also observed in
primary endothelial cells, stimulation experiments were performed with
primary HSMECs (Fig. 3
). Incubation of cells with epinephrine and LPS resulted in IL-6 levels
that were 13.5-fold (P<0.0005) higher than with LPS alone
and 5.5-fold (P<0.0005) higher than with epinephrine alone,
respectively. In the primary endothelial cells, norepinephrine
increased IL-6 to lower amounts than epinephrine did. The cytokine
production was augmented 7.9-fold (P<0.0005) by
costimulation as compared to monostimulation with LPS and 6.9-fold
(P<0.0005) as compared to monostimulation with
norepinephrine, respectively.
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Increase in IL-6 mRNA by catecholamines and LPS
We further investigated the effect of LPS and catecholamines on
the expression of IL-6 mRNA. HMECs were stimulated with LPS in the
presence and absence of epinephrine and norepinephrine or treated with
catecholamines alone for 1, 2, 3, or 4 h (Fig. 4
). Catecholamines alone did not induce detectable levels of IL-6 mRNA
whereas LPS treatment did. When endothelial cells were stimulated with
LPS in the presence of either catecholamine, epinephrine augmented the
IL-6 mRNA to a level comparable to norepinephrine. The peak of
up-regulation was at 2 h. The mean level of IL-6 mRNA
up-regulation by epinephrine at this time point was 6.4 ± 1.3
(P<0.005) when compared to mRNA induction by LPS alone.
ß-actin mRNA was used as the internal reference.
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To determine whether IL-6 up-regulation by catecholamines in
LPS-stimulated HMECs is mediated by newly synthesized proteins, the
effect of cycloheximide on IL-6 mRNA accumulation was studied
(Fig. 5
). In contrast to an untreated control, addition of cycloheximide alone
caused a slight accumulation of IL-6 mRNA. The up-regulation of
LPS-induced IL-6 mRNA by epinephrine was not affected by preincubation
with cycloheximide, suggesting that de novo protein
synthesis is not required for the synergistic effect of LPS and
catecholamines.
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Effect of epinephrine on IL-6 mRNA stability
To determine whether catecholamines act at the level of mRNA
stability, HMECs were stimulated with LPS in both the absence and
presence of epinephrine for 2 h before transcription was stopped
by adding actinomycin D. Over a period of 6 h, total RNA was
extracted and IL-6 mRNA was evaluated by Northern blot analysis
(Fig. 6
). IL-6 mRNA was reduced in a biphasic manner. An initial rapid phase of
degradation was followed by a slow decline pointing to loss of a
short-lived nuclease. The rate of degradation was even minimally
increased in the presence of epinephrine, which suggests that
up-regulation of IL-6 by epinephrine is most likely due to
transcriptional induction.
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Adrenergic receptors mediating catecholamine-induced IL-6
expression
To elucidate which class of adrenergic receptors is responsible
for the up-regulation of IL-6 by epinephrine or norepinephrine,
respectively, experiments using receptor antagonists were performed
(Fig. 7
). HMECs were incubated with LPS (1 µg/ml) alone, LPS and epinephrine
(100 ng/ml), or LPS and norepinephrine (100 ng/ml), respectively, in
the absence or presence of a receptor antagonist. The antagonists
applied were metoprolol (a ß1 receptor
antagonist), butoxamine (a ß2 receptor
antagonist), propranolol (a ß1 and
ß2 receptor antagonist), urapidil (an
1 receptor antagonist), or phenoxybenzamin (an
1 and 2 receptor
antagonist). Receptor antagonists were used at concentrations ranging
from 10-5 to 10-9 M and
added to cell cultures for 30 min prior to treatment with LPS and
catecholamines. Alpha receptor antagonists had no apparent effect on
the up-regulation of IL-6 by either catecholamine (data not shown). In
contrast, metoprolol and butoxamine partly inhibited the effect of
epinephrine at 10-5 M (Fig. 7A, B
).
However, this inhibition did not reach statistical significance.
Propranolol completely abolished the effect of epinephrine (Fig. 7C
), suggesting that the up-regulation of IL-6 by
epinephrine is dependent on ß1 and
ß2 receptor stimulation. Metoprolol completely
inhibited the up-regulation of IL-6 by norepinephrine (Fig. 7D
), suggesting that the stimulation of the
ß1 receptor is sufficient for the IL-6
stimulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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What is especially striking is the fact that LPS and
catecholamines show synergistic rather than additive effects on IL-6
production. Septic shock is also characterized by an excessive
activation of IL-6, which reaches serum concentrations of over 2000
pg/ml. Until now, leukocytes were mainly discussed to be the source of
IL-6 production in sepsis. However, a number of laboratories including
our own have shown that leukocytes are severely hyporeactive in
clinical sepsis and produce only low levels of this cytokine as a
reaction to an adequate stimulus (19
, 22)
. This was
observed at mRNA as well as protein levels. Since catecholamines are
elevated in septic shock, we now propose that the observed increase of
IL-6 levels in the disease is induced in endothelial cells by
catecholamines in the presence of LPS or an LPS-equivalent stimulus.
This argument is supported by the fact that the enhancement of IL-6
production by catecholamines in endothelial cells was observed at a
concentration as low as 1 ng/ml of epinephrine, which equals
approximately the serum concentration of endogenously produced
epinephrine in severe sepsis. Continuous bypass therapy can further
increase this level by a factor 10 to 20. Thus, endogenous production,
but also continuous bypass therapy, could account for the sustained
levels of IL-6 in septic shock.
Up-regulation of IL-6 serum concentrations to 15–40 pg/ml are observed
in stress such as severe exercise (23
, 24)
or during an
operation (25)
. Again, the lower adrenergic stimulation of
endothelial cells in the absence of LPS in vitro corresponds
to these stress situations. It appears that our data obtained in
vitro in endothelial cells closely mimic human physiological and
pathological situations.
We have found that the catecholamine-induced IL-6 expression is a
ß1- and ß2-adrenergic
effect. Since norepinephrine does not stimulate
ß2-adrenoreceptors, its potency of IL-6
induction could be completely neutralized by the selective
ß1 antagonist metoprolol. However, the
down-regulation of LPS-stimulated IL-6 expression in leukocytes could
also be blocked by ß1 receptor antagonists
(18)
. This suggests that the choice of the receptor cannot
explain the differential regulation observed in leukocytes vs.
endothelial cells.
Our data show that the enhancement of LPS-stimulated IL-6 production by
catecholamines is most likely mediated on a transcriptional level and
does not require new protein synthesis. Thus, an activation of a
preformed transcription factor or a cofactor seems likely. The promoter
of IL-6 contains several known binding sites for transcription factors
such as NF-IL6, NF-
B, and AP1 (26
, 27)
. LPS-induced
IL-6 transcription was shown to be primarily mediated by activation of
NF-B. One possible signal pathway of catecholamine-mediated
transcriptional enhancement is via an up-regulation of intracellular
cAMP. A catecholamine-induced increase in cAMP is well documented in a
variety of cell types (17)
. Stimulation of cAMP correlates
with binding of inducible factors to the AP1, NF-IL6, and NF-B
recognition elements in the IL-6 promoter (28)
.
Furthermore, the IL-6 promoter also contains a binding site for the
cAMP-response element binding protein (CREB) (28)
. This
protein becomes activated as a transcription factor on phosphorylation
(29)
. It was shown that ß-adrenergic blockage decreased
a hemorrhage-induced activation of CREB in pulmonary intraparenchymal
mononuclear cells (30)
. The catecholamine-induced
production of IL-6 in HMECs could also be blocked by ß antagonists.
Thus, the ß-adrenergic induction of IL-6 in endothelial cells might
involve an up-regulation of intracellular cAMP and/or the activation of
CREB. However, the elucidation of the signal cascade is further
complicated by the fact that LPS-induced IL-6 mRNA is reduced by
catecholamines in leukocytes. Additional studies are certainly required
to determine the exact mechanism of this pathway.
At an immunological level, IL-6 supports the anti-inflammatory
character of catecholamines. In previous work we have shown that the
immunosuppressive role of catecholamines is attenuated in the blood of
septic patients as compared to blood of healthy volunteers
(19)
. However, the induction of IL-6 in endothelial cells
might restore the immunosuppressive role of catecholamines in late
clinical septic shock. Furthermore, IL-6 increases the
catecholamine-induced activation of acute-phase proteins in the liver
and enhances glycogenolysis. Due to its long half-life, IL-6 prolongs
the metabolic and immunological effects of epinephrine and
norepinephrine.
With regard to microcirculation, the production of IL-6 in endothelial
cells might lead to a pathological disorder of the microvasculature
since IL-6 is induced in high levels throughout the body. For example,
IL-6 itself leads to gap formation (31)
and it induces
vascular endothelial growth factor (VEGF) in endothelial cells
(32)
. The expression of VEGF is associated with an
increased vascular permeability. Since endothelial cells provide such a
large surface in the body, the excessive activation of this ‘endocrine
organ’ might be an important step in the development of a generalized
microvascular leakage and a systemic disease. Furthermore, a systemic
impairment of the microcirculation system might lead to a generalized
acidosis, which would perpetuate the course of sepsis.
The induction of IL-6 by catecholamines in vascular endothelial cells describes a novel interaction between systemic vasoactive hormones and tissue activation of the cytokine network. The interactions within this hormone–cytokine axis seem to be important in the understanding of physiological and pathological stress situations. A low induction of IL-6 as it occurs in severe exercise can be interpreted as a physiological support of adrenergic activities. In contrast, an excessive systemic activation of IL-6 in endothelial cells appears to be of pathological relevance in septic shock. In this disease, IL-6 is likely to potentiate the immunological catastrophe and might aggravate the dysfunction of microcirculation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-6 and endotoxin in the early hepatic reperfusion period after human liver transplantation. Transplantation 58,179-182[Medline]
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