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Am. J. Respir. Cell Mol. Biol., Volume 25, Number 5, November 2001 636-643

Modulation of Chemokine Production in Lung Microvascular Endothelial Cells by Dopamine Is Mediated via an Oxidative Mechanism

Grietje Ch. Beck, Ralph Oberacker, Silke Kapper, Detlef von Zabern, Jutta Schulte, Klaus van Ackern, Fokko J. van der Woude, and Benito A. Yard

Institute for Anaesthesiology, and the V. Medical Clinic, University of Mannheim, Mannheim, Germany

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Serum concentrations of catecholamines are high in patients with sepsis or acute respiratory distress syndrome (ARDS). Because chemokines mediate the recruitment of neutrophils into inflammatory sites, we addressed the question of whether dopamine (DA) is able to influence chemokine production in endothelial cells under basal and proinflammatory conditions. To this end, lung microvascular endothelial cells (LMVEC) were stimulated or not for 24 h with the bacterial toxins lipopolysaccharide (LPS) (1 µg/ml) or lipoteichonic acid (LTA) (10 µg/ml) in the presence or absence of various concentrations of DA (1-100 µg/ml). Whereas under basal and stimulatory conditions, the addition of DA to endothelial cells dose-dependently increased IL-8 production, the production of ENA-78 and Gro-alpha was significantly inhibited (P < 0.01). This effect could still be demonstrated when the cells were stimulated for up to 3 h with LPS before DA administration. Similar findings were detected for the mRNA expression of these chemokines. The influence of DA on chemokine production was not receptor mediated and could be prevented by antioxidants or radical scavengers. Moreover, addition of H2O2 to endothelial cells gave results similar to those observed with DA stimulation, suggesting a pivotal role for reactive oxygen species in DA-mediated modulation of chemokine production in endothelial cells. Our data thus demonstrate that DA administration results in the induction of oxidative stress, with profound effects on endothelial chemokine production.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Acute respiratory distress syndrome (ARDS) is characterized by an increased vascular permeability for plasma proteins and is observed after severe trauma or sepsis induced by gram-negative or gram-positive bacterial toxins, e.g., lipopolysaccharide (LPS) and lipoteichonic acid (LTA). The prevalence of ARDS in patients suffering from multiple organ dysfunction or sepsis is high (1). Despite increased understanding of the pathophysiology of ARDS and increased therapeutic progress, the high mortality of 36-52% in these patients still remains a clinical problem (2, 3).

Clinical and experimental studies have demonstrated that neutrophil recruitment is one of the fundamental steps in the initiation of ARDS (4). Neutrophils adhere to the vessel wall and subsequently extravasate into the inflamed tissue along a chemotactic gradient. It is believed that chemotaxis is maintained through the production of chemokines, synthesized by activated epithelial and endothelial cells. Moreover, perpetuation of this response may depend on the production of inflammatory cytokines that are secreted by the extravasating mononuclear cells. Previously we have demonstrated that the microvascular endothelium is more susceptible to LPS stimulation than macrovascular endothelial cells in regard to chemokine production (5). Most chemokines do not display cell specifity; there are, however, some chemokines, all belonging to the CXC family, that selectively attract neutrophils. Three such chemokines, i.e., interleukin (IL)-8, growth-related gene alpha  (Gro-alpha ), and epithelial neutrophil activating protein-78 (ENA-78), were found to be elevated in serum (6) and bronchial lavage-fluid (7) in patients with sepsis, emphasizing the role of chemokines and neutrophil inflammation in ARDS.

In patients with sepsis, endogenous catecholamines are released as consequence of stress or inflammation (8, 9). Due to hemodynamic instability in these patients, treatment with catecholamines is frequently performed to restore adequate circulatory conditions. Consequently, serum catecholamine levels may increase more than 10-fold (10, 11) compared with healthy individuals. In addition to the hemodynamic properties of catecholamines, catecholamine-mediated modulation of the cytokine network has been demonstrated in leukocytes. Although modulation of the cytokine network via beta -adrenergic receptors is well documented (12, 13), the role of oxidative stress induced by catecholamines has not been extensively studied in this respect. Oxidative stress may be generated through the monoamine oxidase (MAO) pathway (14), or, alternatively, through autooxidation of the catecholamine, resulting in the formation of semi-quinones (15) and reactive oxygen species (ROS) (16). Given the serum catecholamine concentrations reached in septic patients, it is likely that high concentrations of ROS may be generated in vivo, and hence may oxidatively stress the endothelium.

Inasmuch as ROS are known to influence chemokine production in a variety of cells, the present study was conducted to address the question of the extent to which Dopamine influences the production of three endothelial-derived chemokines, i.e., IL-8, Gro-alpha , and ENA-78, that specifically attracts neutrophils to sites of inflammation. Moreover, the involvement of ROS herein was investigated.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reagents

In this study the following reagents were used: essential growth medium for microvascular cells (EGM-MV), fetal bovine serum (FBS), human epidermal growth factor (10 ng/ml), human fibroblast growth factor (4 ng/ml), vascular endothelial growth factor (2 ng/ml), ascorbic acid (75 µg/ml), hydrocortisone (0.2 mg/ml), heparin (1 mg/ml), insulin (5 ng/ml) and Trypsin/EDTA (T/E) were all from Cell Systems, Remagen, Germany. Trizol-Reagent, DNase, and dNTPs were from Gibco BRL, Eggenstein, Germany.

LTA, LPS, propanolol, sulpiride, SCH 23390, pargyline, and L-ascorbic acid were from Sigma, Deisenhofen, Germany. Dopamine (DA) and N-acetylcysteine (NAC) were obtained from Ratiopharm, Ulm, Germany. Superoxide dismutase (SOD), 1st Strand cDNA Synthesis Kit and Fast Start DNA Master SYBR Green I were from Roche, Mannheim, Germany. H2O2 was obtained from Merck, Darmstadt, Germany. Anti-hemoxygenase-1 mAb was from Biomol, Hamburg, Germany. Phentolamine was purchased from Fluka, Taufkirchen, Germany. IL-8, Gro-alpha , and ENA-78 Immunoassays were obtained from R&D Systems GmbH, Wiesbaden, Germany. All Primers were aquired from Perkin Elmer applied Biosystems, Weiterstadt, Germany. SuperScript TM II Preamplification System was purchased from Life Technologies, Karlsruhe, Germany. GBR 12909 was from Tocris, Bristol, UK.

Cell Culture

LMVEC were purchased from Cell Systems at passage 3-4. The cells were seeded in a density of 5,000 cells/cm2 in T25 flasks (Falcon) and cultured at 37°C, 95% relative humidity and 5% CO2 in EGM-MV according to the manufacturer's recommendation. After confluence, they were passaged by Trypsin/EDTA and subcultured (17) until the experiments were perfomed. All experiments were performed with LMVEC at passage 5.

Characterization of endothelial cells was performed on the basis of a positive uptake of acetylated LDL, Factor VIII-related antigen expression and PECAM (CD31), and a negative staining for alpha -smooth muscle actin.

Chemokine Production

LMVEC (1 × 105 cells/ml) were seeded in 24-well plates and grown till confluence. The cells were either stimulated or not for various time periods with LPS (1 µg/ml) or LTA (10 µg/ml) in the presence or absence of DA (1-100 µg/ml). In some experiments, the cells were stimulated for 1, 3, or 6 h with LPS before the addition of DA. To study the involvement of receptors and ROS in the mechanism by which DA exerts its effects, endothelial cells were preincubated for 1 h with the following receptor antagonists: the alpha -receptor antagonist phentolamine, the beta -receptor antagonist propanolol, the D1-receptor antagonist SCH 23390, and the D2/3 receptor antagonist sulpiride, in concentrations ranging from 10-3 to 10-7 M or the antioxidants SOD (10-100 µg/ml), NAC (10-500 µg/ml) and L-ascorbic acid (10-1,000 µM). Furthermore, the influence of H2O2 on chemokine production was tested in a dose-dependent fashion (100-900 µM). At the end of each experiment supernatants were collected and assessed for IL-8, ENA-78, and Gro-alpha production by means of ELISA. All ELISAs were performed according to manufacturer's instructions. Sensitivity of the ELISA were < 0.8 pg/ml for IL-8, < 15 pg/ml for ENA-78, and < 5 pg/ml for Gro-alpha . Each experimental condition was performed in triplicate and each experiment was confirmed at least three times.

Real-Time Polymerase Chain Reaction

LMVEC were grown to confluence in T25 flasks and either stimulated or not with LPS in the presence or absence of dopamine in various concentrations. Total RNA was isolated using Trizol-Reagent according to manufacturer's instructions. DNase digestion was performed before reverse transcription to exclude amplification of genomic DNA.

500 ng of total RNA was reversed transcribed into cDNA according to the manufacturer's instructions, using the 1st Strand cDNA Synthesis Kit. cDNA was diluted in 20 µl DEPC-treated water and stored at -30°C until quantitative real-time polymerase chain reaction (PCR) was performed. The oligonucleotides used for PCR are listed in Table 1.

Specific DNA standards were generated by PCR amplification of cDNA, purification of the amplified products, and quantification by spectrophotometry. Real-time PCR of cDNA specimens and DNA standards were conducted in a total volume of 25 µl, containing 2 µl FastStart DNA Master SYBR GreenI, 10 pMol of gene-specific forward and reverse primers, 2 mM MgCl2 for ENA-78, and 4 mM MgCl2 for IL-8 and Gro-alpha and 11.5 µl of water. The amplification profile for each sample was as follows: 2 min at 50°C, 5 min at 95°C, followed by 45 cycles of amplification for IL-8 and ENA-78, and 55 cycles for Gro-alpha , each cycle consisting of denaturation at 95°C for 15 s and annealing/extension at 55/72°C for 1 min. Standard curves were generated for all chemokines. PCR efficiency was assessed from the slopes of the standard curves and was found to be between 90 and 100%. Linearity of the assay could be demonstrated by serial dilution of all standards and cDNA. All samples were normalized for an equal expression of GAPDH. Each experiment was repeated three times with similar results.

Statistical Analysis

Statistical analysis was performed using Stata Statistical software (Mann-Whitney test). A value of P < 0.05 was considered significant.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Influence of DA on the Production of Chemokines in Endothelial Cells Under Basal Conditions

Previously we have demonstrated that microvascular endothelial cells produce IL-8, ENA-78, and Gro-alpha under basal and inflammatory conditions. To test if DA could influence the production of LMVEC, the latter were stimulated with different concentrations of DA for various time intervals. Whereas the production of IL-8 in LMVEC was increased by DA in a dose-dependent fashion, the production of ENA-78 and Gro-alpha was decreased under these conditions (P < 0.01) (Figure 1A). In concordance with our results obtained by ELISA, it was found that DA upregulated IL-8 mRNA (P < 0.01), while reducing the expression of ENA-78 and Gro-alpha mRNA (P < 0.01) (Figure 1B).


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  		Figure 1.   IL-8, Gro-alpha , and ENA-78 production (A) and mRNA expression (B) by LMVEC after dopamine stimulation. LMVEC were unstimulated or stimulated for 24 h with dopamine in concentrations ranging from 10-100 µg/ml. (A) The supernatants were collected and assessed for chemokine production by ELISA. (B) Total RNA was isolated and real-time PCR for IL-8, Gro-alpha , and ENA-78 mRNA expression was performed as described in MATERIALS AND METHODS. Results are expressed as pg/ml (A) or fg of cDNA template (B). *P < 0.05 compared with unstimulated control group.

Maximal upregulation and inhibition occurred with a concentration of 100 µg/ml of DA (P < 0.01), a concentration at which no cell toxicity was observed as determined by trypan blue exclusion. Kinetic studies demonstrated that the upregulation in IL-8 production in DA-stimulated LMVEC preceded the inhibition in ENA-78 and Gro-alpha production in these cells. Although a significant upregulation in IL-8 production was already found between 6 and 14 h of DA stimulation (P < 0.05), DA did not significantly influence the production of ENA-78 and Gro-alpha at this time point (Figure 2).


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  		Figure 2.   Kinetics of IL-8, Gro-alpha , and ENA-78 production in unstimulated and DA-stimulated LMVEC. Cells were grown to confluence in 24-well plates and stimulated (dotted line) or not (straight line) with dopamine (100 µg/ml) for up to 72 h. Significant differences between stimulated and unstimulated cells were reached for IL-8 between 6 and 14 h of stimulation, and for both Gro-alpha and ENA-78 between 14 and 24 h of stimulation. Results are expressed as mean production ± SD of triplicate cultures. *P < 0.05 compared with unstimulated control group.

Influence of DA on Chemokine Production in LPS- or LTA-Stimulated Endothelial Cells

In patients with sepsis, endothelial cells may be activated by LPS or LTA from gram-negative and -positive bacteria, respectively. We therefore investigated whether DA could also influence the production of chemokines under these conditions. Similar to the inhibitory effect of DA on ENA-78 and Gro-alpha production in unstimulated endothelial cells, the upregulation of these chemokines by LPS or LTA was dose-dependently inhibited by DA in LMVEC (P < 0.01). In addition, DA significantly increased LPS-mediated upregulation in IL-8 production (P < 0.01) (Figure 3). Similar findings could be observed for IL-8, ENA-78, and Gro-alpha mRNA expression in LPS- and LTA-stimulated LMVEC (data not shown).


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  		Figure 3.   Influence of DA on chemokine production in LPS- and LTA-stimulated LMVEC. LMVEC were either cultured in medium alone or stimulated with LPS (1 µg/ml) (open bar) or LTA (10 µg/ml) (  filled bar) in the presence or absence of dopamine (10-100 µg/ml) for 24 h. Thereafter the supernatants were collected and assessed for chemokine production by ELISA. Results are expressed as mean production ± SD of triplicate cultures. *P < 0.05 compared with endothelial cells that were stimulated with LPS or LTA.

DA-mediated downregulation in ENA-78 or Gro-alpha production was still observed when the cells were prestimulated with LPS for up to 3 h before addition of DA. This was also found for the increase in IL-8 production (P < 0.05). Preconditioning of endothelial cells for 12 h with DA before LPS stimulation was as effective as the simultaneous addition of DA and LPS with respect to the inhibition of ENA-78 and Gro-alpha and the upregulation of IL-8 production (data not shown). Although the influence of DA preconditioning on chemokine production in LPS-stimulated endothelial cells waned in time, a significant inhibition in ENA-78 and Gro-alpha (P < 0.01) production was still observed when DA preconditioned cells were cultured further in the absence of DA for 8 h before LPS stimulation. Similar findings were observed for the upregulation in IL-8 production (Figure 4).


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  		Figure 4.   Effect of DA preconditioning on chemokine production in time. LMVEC were stimulated with DA (100 µg/ml) (filled bars) or not (open bars) for 12 h. Thereafter the cells were washed and cultured further for various periods (0, 8, and 24 h washout period) in the absence of DA, before LPS (1 µg/ml) stimulation. Thereafter supernatants were collected and assessed for IL-8, ENA-78, and Gro-alpha production by ELISA. Results are expressed as mean production ± SD of triplicate cultures. *P < 0.05 compared with unstimulated control group.

Polarity in Chemokine Secretion

To investigate if DA influenced the release of chemokines either at the apical or basolateral side, LMVEC were cultured in transwells and stimulated or not at the apical side with DA in the presence or absence of LPS. Both LPS alone and DA alone increased the secretion of IL-8 preferentially at the apical side. The combination of DA and LPS was more potent (P < 0.05) than either LPS or DA alone in this respect. In contrast, neither LPS, DA, nor the combination thereof influenced the direction of Gro-alpha secretion (Figure 5).


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  		Figure 5.   Influence of DA on the polarity of IL-8 and Gro-alpha secretion. LMVEC were seeded in transwell plates and cultured till confluence. Hereafter the cells were stimulated or not with DA (100 µg/ml) for 24 h and the culture supernatants in both upper and lower chamber were collected. IL-8 (filled bars) and Gro-alpha (open bars) production was assessed by ELISA. Apical/basolateral ratios were determined by dividing the production in the upper chamber by the production in the lower chamber. Results are expressed as apical/basolateral ratio ± SD of triplicate cultures. *P < 0.01 compared with group without DA stimulation.

Mechanism by which DA Mediates Alterations in Chemokine Production

To study the mechanism by which DA influenced chemokine production, various receptor antagonists and antioxidants were used. Neither adrenergic receptor antagonists (phentolamin, propanolol), nor antagonists for dopaminergic receptors (SCH23390, sulpiride), used in concentrations known to block these receptors, were able to reverse the influence of DA on chemokine production (data not shown). In contrast, NAC, ascorbic acid, and SOD completely prevented the influence of DA on IL-8, ENA-78, and Gro-alpha production, demonstrating that DA exerts its action on chemokine production through the generation of ROS (Figure 6A-6C). Moreover, the addition of H2O2 to LMVEC mimicked the effect of DA stimulation completely (Figure 7). Because convention of DA by MAO results in the generation of H2O2, we tested in two ways to see whether this pathway was involved in the influence of DA on chemokine production. First, by using the DA uptake inhibitor GBR12909, we were not able to inhibit the effect of DA. Second, by using a MAO inhibitor, i.e., pargyline, DA was still able to exert its effect on chemokine production (data not shown).


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  		Figure 6.   Inhibition of DA-mediated IL-8 production in LMVEC by NAC (A), SOD (B), and ascorbic acid (C). LMVEC were either unstimulated (open bar) or stimulated for 24 h with DA (100 µg/ml) alone (filled bar) or in the presence of various concentrations of (A) NAC (100-500 µg/ml), (B) SOD (10-100 µg/ml), or (C) ascorbic acid (100-1,000µM) (hatched bars). The supernatants were collected and assessed for IL-8 production by ELISA. Results are expressed as mean production ± SD of triplicate cultures. *P < 0.05 compared with dopamine-stimulated control group.


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  		Figure 7.   Influence of H2O2 on IL-8 production in LMVEC. LMVEC were either unstimulated (open bar) or stimulated for 24 h with DA (100 µg/ml) alone (filled bar) or in the presence of various concentrations of H2O2 (100-900 µM) (hatched bars). The supernatants were collected and assessed for IL-8 production by ELISA. Results are expressed as mean production ± SD of triplicate cultures. *P < 0.05 compared with dopamine-stimulated control group.

Previously we could demonstrate that DA induces the expression of heme oxygenase 1 (HO-1) in endothelial cells via an oxidative mechanism. Because it is known that HO-1 can inhibit LPS responses in monocytes, we investigated whether the expression of HO-1 was obligatory for DA's action on chemokine production. In LMVEC that were simultaneously stimulated with DA and the HO-1 inhibitor, zinc protoporphorine (ZnPP), no differences in chemokine production were found when compared with LMVEC that were stimulated with DA alone (data not shown), suggesting that DA influenced chemokine production independently of HO-1 expression. Moreover, when endothelial cells were prestimulated with DA in the presence of cycloheximide, upregulation in IL-8 and downregulation in ENA-78 and Gro-alpha production was still observed when the cells were cultured further in the absence of cycloheximide or DA. Thus, these data demonstrate that the influence of DA on chemokine production was not mediated through the induction of a modulatory protein (data not shown).

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Neutrophil recruitment and migration through the endothelial lining is dominated by the local production of chemokines and the expression of adhesion molecules (18, 19). In patients with sepsis, the inflammatory response is mainly initiated at the microvascular system in end organs such as lung and kidney (20). The preference for the microvascular system may reside in the high susceptibility of microvascular endothelial cells to respond to LPS or LTA (21). Indeed, LPS-stimulated LMVEC strongly upregulate the production of IL-8, ENA-78, and Gro-alpha , three chemokines which are found to be elevated in serum and bronchial lavage fluid of septic patients (5, 6). The early phase in sepsis is furthermore characterized by a so-called stress response, in which endogenous catecholamines are released in the circulation (8, 22). Because hemodynamic instability occurs in most patients with sepsis, therapeutic intervention with high catecholamine concentrations is frequently performed; hence, high serum concentrations of catecholamine can be observed in these patients (11).

Apart from their hemodynamic properties, catecholamines are known to influence the immune system via modulation of the cytokine network (23, 24). Although there is ample evidence that this occurs through the activation of beta -adrenergic receptors (25, 26), the role of oxidative stress in this process has not yet been delineated. It has been appreciated that catecholamines are well suited to provide oxidative stress in various ways (27, 28).The ability of catecholamines to produce damaging ROS, e.g., hydroxyl radicals (29, 30), is partly accomplished by action of the enzyme MAO (31, 32, 33). MAO catalyzes the oxidative deamination of DA resulting in the formation of H2O2. In the presence of Fe2+, H2O2 is then further converted to hydroxyl radical (HO·) through the Fenton reaction (34). Due to the unstable nature of the catechol ring, reactive quinone molecules and superoxide (O2-) may be rapidly formed via auto-oxidation, thereby providing a second pathway through which ROS can be generated (35, 36). Auto-oxidation of the catechol ring can be prevented by antioxidants such as NAC. ROS may have both beneficial and deleterious effects on cells, depending on the concentration and system in which they have been studied (37, 38). As a consequence of their aggressive nature, high concentrations of ROS inevitably result in cyto- and genotoxicity. Low concentrations of ROS, however, may improve cellular redox status by increasing the amount of endogenous antioxidants such as superoxide dismutase, HO-1, and ferritin (39). It has been suggested that this adaptive response, particularly in endothelial cells, may underlie, at least in part, the phenomenon of ischemic preconditioning (40). Zahler and colleagues (41) were able to demonstrate that preconditioning of endothelial cells by transient oxidative stress reduced the release of proinflammatory cytokines upon TNF-alpha stimulation. In keeping with this and the high serum concentrations of catecholamines found in patients with sepsis, this study was conducted to investigate the influence of DA on the production of three chemokines that are likely to play an important role in the onset or perpetuation of ARDS.

In the present study, we provide for the first time evidence that DA differentially regulates the production of endothelial-derived chemokines. Whereas the production of IL-8 is increased by DA, the production of ENA-78 and Gro-alpha is downregulated under this condition. Similar to the upregulation in IL-8 production, Gornikiewicz and coworkers (42) have shown that catecholamines and LPS synergistically upregulated IL-6 production. In striking contrast to their study, however, we demonstrated that both up- and downregulation in chemokine production were exclusively mediated via oxidative stress, since antioxidants, but not receptor antagonists, completely prevented the action of DA. The generation of superoxide radicals was largely responsible for DA's action, as was demonstrated by the neutralizing effect of SOD. Although addition of H2O2 to LMVEC gave similar results with respect to chemokine production, our data do not support a role for MAO in this process. Neither the MAO inhibitor pargylin nor the DA uptake inhibitor GBR 12909 were able to inhibit the DA-induced effects, suggesting that reactive products, i.e., O2- and semi-quinones, generated via autoxidation of the catechol ring, are likely responsible for the altered chemokine production we observed. Although ROS were clearly produced by DA, no cell toxicity was detected when the cells were cultured for up to 72 h in the presence of 100 µg/ml of DA, determined by trypan blue exclusion or release of lactate dehydrogenase (data not shown). High concentrations of DA (> 200 µg/ml), however, were cytotoxic in both assays, and have not been studied further. In relation to catecholamine concentrations reached in patients with sepsis, the concentrations of DA used in this study to obtain a maximal effect was relatively high. It should be mentioned, however, that in contrast to our in vitro study in which DA is given only once, in patients with sepsis catecholamines are intravenously infused over longer periods of time. Given the unstable nature of the catechol ring and the changed pharmacokinetics of catecholamines in patients with sepsis (43), it is likely that the absolute amount of reactive products in these patients is much higher than in our cell culture system. Inasmuch as DA transcriptionally modulate chemokine production in unstimulated and LPS-stimulated endothelial cells, and nuclear factor (NF)-kappa B consensus sequences are found in the promoter region of all three chemokines (44, 45), activation of NF-kappa B could be an important proximal event in the modulation of chemokine production by DA. Indeed, DA activates NF-kappa B in a dose- and time-dependent fashion (data not shown). It should be mentioned, however, that NF-kappa B is a positive regulator of chemokine production and thus may explain the upregulation in IL-8, but not the downregulation in Gro-alpha and ENA-78 production unless an inhibitory protein under control of NF-kappa B is induced. It has been demonstrated that the HO-1 gene is under control of NF-kappa B activation (46) and that a high expression of HO-1 is associated with inhibition of cytokine production in LPS-stimulated macrophages (47). Although HO-1 is induced by DA in endothelial cells (48), HO-1 expression was not obligatory for the action of DA because both a HO-1 inhibitor, i.e., ZnPP, and cycloheximide, were not able to revert the influence of DA on chemokine production in endothelial cells.

DA-upregulated IL-8 production was found to be predominantly at the apical side. In contrast, DA did not change the preference of Gro-alpha secretion, although the absolute amount of Gro-alpha was clearly reduced. Inasmuch as chemotaxis of neutrophils occurs along a chemotactic gradient, our observations, with respect to the absolute amount in chemokine production and the preference in chemokine secretion, would suggest that DA may have antiinflammatory properties on neutrophils.

In conclusion, we demonstrate that DA is able to modulate the expression of chemokines in lung microvascular endothelial cells through the generation of ROS. These effects may be important in the treatment of sepsis-associated inflammatory responses such as ARDS, because modulation of chemokine production may have a large impact on the amount of neutrophils that infiltrate the lungs.

                              
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TABLE 1
Primer sequences and base pairs (bp) of the amplification

    Footnotes

Address correspondence to: Dr. Grietje Beck, M.D., Institute for Anaesthesiology and Critical Care Medicine, University of Mannheim, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. E-mail: grietje.beck{at}anaes.ma.uni-heidelberg.de

Abbreviations: acute respiratory distress syndrome, ARDS; dopamine, DA; epithelial neutrophil activating protein-78, ENA-78; growth-related gene alpha , Gro-alpha ; heme oxygenase 1, HO-1; interleukin-8, IL-8; lung microvascular endothelial cells, LMVEC; lipopolysaccharide, LPS; lipoteichonic acid, LTA; monoamine oxidase, MOA; N-acetylcysteine, NAC; polymerase chain reaction, PCR; reactive oxygen species, ROS.
(Received in original form February 27, 2001 and in revised form July 20, 2001)

Acknowledgments: This study was supported by grants from the Forschungsfonds University of Mannheim.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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