Catecholamines up-regulate lipopolysaccharide-induced IL-6 production in human microvascular endothelial cells ALEXANDER GORNIKIEWICZ, THOMAS SAUTNER, CHRISTINE BROSTJAN, ¨ GGER, ERICH ROTH, BERNHARD SCHMIERER, REINHOLD FU ¨ FERDINAND MUHLBACHER, AND MICHAEL BERGMANN1 Department of Surgery, University of Vienna, 1090 Vienna, Austria ABSTRACT The catecholamine-mediated modulation of the cytokine network has primarily been demonstrated for leukocytes. Whereas catecholamines decrease the LPS-induced production of IL-6 by leukocytes, serum levels of IL-6 are dramatically increased by the catecholamine epinephrine in animal endotoxemia models. We now demonstrate that epinephrine as well as norepinephrine can induce IL-6 in an endothelial cell line (HMEC-1). Furthermore, these catecholamines could even potentiate the LPS-induced IL-6 protein production. The synergistic effect of catecholamines and LPS could be reproduced in primary human skin microvascular endothelial cells. The catecholamine-induced IL-6 stimulation is based on increased IL-6 mRNA levels. RNA stability assays revealed that this regulation is not a result of enhanced RNA stability and therefore is most likely due to an increased transcription. Treatment with cycloheximide indicated that new protein synthesis is not necessary for this transcriptional up-regulation of IL-6 mRNA. Preincubation with ␣ and  receptor antagonists showed that the effect is mediated by 1- and 2-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., Fu¨gger, R., Roth, E., Mu¨hlbacher, F., Bergmann, M. Catecholamines up-regulate lipopolysaccharide-induced IL-6 production in human microvascular endothelial cells. FASEB J. 14, 1093–1100 (2000)
Key Words: immunomodulation 䡠 transcription 䡠 adrenoreceptors
Septic shock accounts for a mortality of 30 – 40% in intensive care unit patients. The pathomechanism is best described as a systemic inflammatory response syndrome (SIRS) and is characterized by an initial excessive activation of proinflammatory cytokines (1). However, a significant number of clinical trials 0892-6638/00/0014-1093/$02.25 © FASEB
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 1 (IL-1) 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 antiinflammatory 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-1 expression (11) and the induction of soluble TNF-␣ receptor p55 and IL-1 receptor antagonist (12) in hematopoietic cells. 1 Correspondence: Department of Surgery, University of Vienna, Wa¨hringer Gu¨rtel 18 –20, 1090 Vienna, Austria. Email:
[email protected]
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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 -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-1, and IL-6 (17–19). 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.
MATERIALS AND METHODS Reagents LPS (Escherichia coli 0111:B4), actinomycin D, and cycloheximide were purchased from Sigma (St. Louis, Mo.). Adrenergic agents were obtained from the following manufacturers: epinephrine-hydrochloride and norepinephrine-hydrochloride from Hoechst (Frankfurt am Main, Germany); metoprolol-tartrate from Astra (Wedel, Germany); butoxamine from Sigma; propranolol-hydrochloride from Zeneca (Macclesfield, U.K.); urapidil-hydrochloride from Byk Gulden (Konstanz, Germany); and phenoxybenzamin-hydrochloride from Procter and Gamble (Weiterstadt, Germany). Dilutions were 1094
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made in isotonic saline. All adrenergic agents were tested to be endotoxin-free. 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 g/ml hydrocortisone (Sigma), 10 ng/ml human epidermal growth factor (Sigma), 50 U/ml penicillin, and 50 g/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-Lglutamine (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 g/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 g) 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 ⫻ SSC. The RNA was fixed on the membrane by UV cross-linking and hybridized to radiolabeled cDNA probes of human IL-6 and -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 -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 ⫻ 105 counts/ml were used for hybridization. RNA was hybridized at 65°C overnight in a buffer containing 5 ⫻ TEN (75 mM Tris/chloride, 5 mM EDTA and 0.75 M NaCl), 5 ⫻ Denhardt’s solution and 0.2% sodium dodecyl sulfate (SDS). Membranes were washed twice with 2 ⫻ TEN and 0.2% SDS at 65°C for 15 min, followed by one
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washing step with 0.2 ⫻ TEN and 0.2% SDS at 65°C for 30 min. For rehybridization, membranes were stripped by rinsing them four times with 0.1 ⫻ 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 g/ml) in the presence or absence of epinephrine (100 ng/ml). After 2 h, actinomycin D was added at a final concentration of 10 g/ml. At various time points after the addition of actinomycin D, total RNA was isolated and 40 g of each sample was subjected to Northern blot analysis as described above. Autoradiographic signals were quantitated by laser densitometry and normalized to -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.
RESULTS Induction of IL-6 protein expression by catecholamines and LPS Both 100 ng/ml of epinephrine and 100 ng/ml of norepinephrine induced IL-6 protein in the microvascular endothelial cell line HMEC-1 to similar levels. These levels were not statistically different from those obtained by stimulation with 1000 g/ml of LPS. When HMECs were incubated with either catecholamine in the presence of LPS, the production of IL-6 levels was synergistically enhanced in a time-dependent manner for up to 24 h (Fig. 1). 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 ADRENERGIC INDUCTION OF IL-6 IN ENDOTHELIAL CELLS
Figure 1. Time course of IL-6 protein production as determined by ELISA. HMECs were incubated for up to 24 h with 1 g/ml of LPS alone, LPS and 100 ng/ml epinephrine, LPS and 100 ng/ml norepinephrine, or catecholamines alone. Relative values are presented since the absolute level of up-regulation varied depending on the status of the cell line. The level of IL-6 production by epinephrine and LPS after 24 h was designated as 100%, which corresponds to 2267 ⫾ 834 pg/ml. Mean and se of 3 independent experiments are shown. epi, epinephrine; nor, norepinephrine; LPS, lipopolysaccharide.
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. 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 upregulation by epinephrine at this time point was 6.4 ⫾ 1.3 (P⬍0.005) when compared to mRNA induction by LPS alone. -actin mRNA was used as the internal reference. To determine whether IL-6 up-regulation by catecholamines in LPS-stimulated HMECs is mediated 1095
rine 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 upregulation of IL-6 by epinephrine is most likely due to transcriptional induction. Adrenergic receptors mediating catecholamineinduced IL-6 expression
Figure 2. Concentration dependency of IL-6 protein production as determined by ELISA. A) HMECs were stimulated for 24 h with the indicated concentrations of epinephrine in ng/ml and 1000 ng/ml of LPS. Relative values are indicated as percentage of levels obtained by stimulation with the highest epinephrine concentration, which was 3206 ⫾ 693 pg/ml. B) HMECs were stimulated for 24 h with the indicated concentrations of LPS in ng/ml and 100 ng/ml of epinephrine. Relative values are indicated as percentage of levels obtained by stimulation with the highest LPS concentration, which was 3108 ⫾ 678 pg/ml. Means and se of three independent experiments are shown. Concentrations of epinephrine and LPS are indicated at the bottom. *P⬍0.005 as compared to monostimulation, **P⬍0.0005 as compared to monostimulation. epi, epinephrine; LPS, lipopolysaccharide.
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 g/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 1 receptor antagonist), butoxamine (a 2 receptor antagonist), propranolol (a 1 and 2 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. Pro-
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. 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 epineph1096
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Figure 3. IL-6 protein production as determined by ELISA in primary human skin microvascular endothelial cells (HSMECs). HSMECs were stimulated for 24 h with 1 g/ml LPS, LPS and 100 ng/ml epinephrine, LPS and 100 ng/ml norepinephrine, or catecholamines alone. Means and se of three independent experiments are shown. epi, epinephrine; nor, norepinephrine; LPS, lipopolysaccharide.
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which underlines the significance of the finding. Since catecholamines are known to down-regulate the LPS-stimulated IL-6 production in leukocytes, we propose that endothelial cells are the source of catecholamine-induced IL-6 production in endotoxemia models. 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. Figure 4. Time course of IL-6 mRNA expression as detected by Northern blotting. HMECs were stimulated with 1 g/ml LPS in the presence or absence of 100 ng/ml epinephrine or norepinephrine as well as with catecholamines alone for 1, 2, 3, and 4 h. Total RNA was extracted and Northern blot analysis was performed. epi, epinephrine; nor, norepinephrine; LPS, lipopolysaccharide; C, unstimulated control; h, hours.
pranolol completely abolished the effect of epinephrine (Fig. 7C), suggesting that the up-regulation of IL-6 by epinephrine is dependent on 1 and 2 receptor stimulation. Metoprolol completely inhibited the up-regulation of IL-6 by norepinephrine (Fig. 7D), suggesting that the stimulation of the 1 receptor is sufficient for the IL-6 stimulation. DISCUSSION Catecholamines induce IL-6 to high levels in LPSstimulated microvascular endothelial cells. We demonstrate this effect not only in a human microvascular endothelial cell line, but also in a primary endothelial cell culture of human skin capillaries, ADRENERGIC INDUCTION OF IL-6 IN ENDOTHELIAL CELLS
Figure 5. Influence of cycloheximide on the induction of IL-6 mRNA. HMECs were stimulated for 2 h with 1 g/ml LPS, LPS and 100 ng/ml epinephrine in the presence or absence of cycloheximide or with cycloheximide alone. Total RNA was extracted and Northern blot analysis was performed. Cycloheximide was used at a final concentration of 10 g/ml and added to cell cultures for 30 min prior to treatment with LPS and catecholamines. epi, epinephrine; LPS, lipopolysaccharide; CHX cycloheximide; C, unstimulated control. 1097
Figure 6. Effect of epinephrine on the degradation of IL-6 mRNA. HMECs were incubated in the presence of 1 g/ml LPS or 1 g/ml LPS and 100 ng/ml epinephrine for 2 h before actinomycin D was added at a final concentration of 10 g/ml to stop RNA synthesis. At indicated time points after addition of actinomycin D, total RNA was extracted and subjected to Northern blot analysis. A) Northern blots. B) Blot of relative values of IL-6 mRNA. Autoradiographic signals for IL-6 mRNA were quantitated by laser densitometry and normalized to -actin mRNA levels. Values are given as percentage of IL-6 mRNA levels observed before addition of actinomycin D. epi, epinephrine; LPS, lipopolysaccharide; Act D, actinomycin D.
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 1- and 2-adrenergic effect. Since norepinephrine does not stimulate 2-adrenoreceptors, its potency of IL-6 induction could be completely neutralized by the selective 1 antagonist metoprolol. However, the down-regulation of LPSstimulated IL-6 expression in leukocytes could also be blocked by 1 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 LPSstimulated 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). LPS1098
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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 -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  antagonists. Thus, the -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-
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Figure 7. Adrenergic receptors mediating IL-6 up-regulation as determined by ELISA. HMECs were incubated for 6 h with LPS (1 g/ml) alone, LPS and 100 ng/ml epinephrine, or LPS and 100 ng/ml norepinephrine in the presence or absence of the following -adrenergic receptor antagonists: A) metoprolol, B) butoxamine, C) propanolol, D) metoprolol. Antagonists were used at concentrations indicated on the x axis (ranging from 10⫺5 to 10⫺9 M) and added to cell cultures for 30 min prior to treatment with LPS and catecholamines. Three independent experiments were carried out. Mean and se are shown as the percentage of the IL-6 value, which was obtained by stimulation with LPS and epinephrine. This value was 1386 ⫾ 65 pg/ml for panel A, 1220 ⫾ 215 pg/ml for panel B, 1260 ⫾ 230 pg/ml for panel C, and 1211 ⫾ 302 pg/ml for panel D, respectively. Mean and se of three independent experiments are shown. *P⬍0.005 as compared to stimulation with catecholamine and LPS, **P⬍0.0005 as compared to stimulation with the catecholamine and LPS. epi, epinephrine; nor, norepinephrine; LPS, lipopolysaccharide, M, metoprolol; B, butoxamine; P, propanolol.
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. ADRENERGIC INDUCTION OF IL-6 IN ENDOTHELIAL CELLS
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 1099
catastrophe and might aggravate the dysfunction of microcirculation.
17. 18.
We thank Ines Haberl and Elke Waldmann for technical ¨ sterreichhelp. A.G. was supported by grant # 6944 of the O ische Nationalbank.
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The FASEB Journal
Received for publication June 14, 1999. Revised for publication January 4, 2000.
GORNIKIEWICZ ET AL.
