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Catecholamines potentiate LPS-induced expression of MMP-1 and MMP-9 in human monocytes and in the human monocytic cell line U937: possible implications for peri-operative plaque instability¹

WALTER S. SPEIDL, WOLFGANG G. TOLLER^*, CHRISTOPH KAUN, THOMAS W. WEISS, STEFAN PFAFFENBERGER, STEFAN P. KASTL, ALEXANDER FURNKRANZ, GERALD MAURER, KURT HUBER, HELFRIED METZLER^* and JOHANN WOJTA²

Department of Internal Medicine II, University of Vienna, Vienna, Austria;
^* Department of Anaesthesiology and Intensive Care Medicine, University of Graz, Graz, Austria; and
Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria

²Correspondence: Department of Internal Medicine II, University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: johann.wojta{at}univie.ac.at

SPECIFIC AIMS

Plaque destabilization leading to myocardial infarction is often observed after surgery even if the intervention is not cardiovascular. We examined whether catecholamines and LPS, as possible mediators of peri- or postoperative stress, alone or in combination could affect the expression of MMPs in human monocytes/macrophages.

PRINCIPAL FINDINGS

1. Catecholamines potentiate LPS-induced production of MMP-1 in human monocytes
Catecholamines alone showed only moderate effects on MMP-1 production by peripheral blood-derived monocytes (PBM) and monocyte-derived macrophages (MDM) isolated from three different donors. LPS alone significantly increased the production of MMP-1 whereas a combination of LPS and the respective catecholamines further enhanced these effects up to 2.5-fold in PBM as well as in MDM from these donors (Fig. 1 A, B).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of LPS and a combination of LPS and catecholamines on MMP-1 production in human primary peripheral blood monocytes (PBM, A) and monocyte-derived macrophages (MDM, B): PBM or MDM were incubated for 48 h in the absence or presence of LPS (1 µg/mL) alone or combination of LPS (1 µg/mL) and epinephrine (1 µg/mL) or LPS (1 µg/mL) and norepinephrine (1 µg/mL). Conditioned media of such treated cells were collected and MMP-1 was determined. Values are given in ng/106 cells/48 h and represent mean values ± SD of 3 independent determinations. Experiments were performed three times with PBM and MDM isolated from an individual donor. A representative experiment is shown. ***P < 0.0001 compared with control, P < 0.0001 compared with LPS-treated cells.

2. Catecholamines potentiate LPS-induced production of MMP-1 and MMP-9 in U937 cells
Incubation of U937 cells with epinephrine (1 µg/mL) and norepinephrine (1 µg/mL) for 48 h induced MMP-1 protein production 2.6- and 2.4-fold and MMP-9 protein production 2.8- and 2.3-fold. Induction of MMP-1 and MMP-9 by LPS (1 µg/mL) was 10.4- and 16.9-fold. When U937 were incubated with catecholamine in the presence of LPS, MMP-1 was up-regulated 8.8-fold with epinephrine and 6.7-fold with norepinephrine (P<0.00001); MMP-9 was up-regulated 9.3-fold with epinephrine and 7.4-fold with norepinephrine (P<0.00001) compared with incubation with LPS alone. Incubation of U937 cells with epinephrine (1 µg/mL) or norepinephrine (1 µg/mL) synergistically increased LPS (1 µg/mL) -induced MMP-1 and MMP-9 activity.

3. Catecholamines potentiate LPS-induced upregulation of MMP-1 and MMP-9 mRNA in U937 cells
Epinephrine and norepinephrine caused an up to 4-fold increase in mRNA levels of MMP-1 and MMP-9 in U937 whereas LPS up-regulated MMP-1 specific mRNA 11-fold and MMP-9 specific mRNA 6-fold. The combination of the respective catecholamine and LPS further enhanced the increase of mRNA levels of MMP-1 and MMP-9 up to 80-fold. Cycloheximide completely inhibited the effects of epinephrine or LPS or the combination of both on mRNA levels of MMP-1 and MMP-9, suggesting that protein synthesis is required for induction of MMP-1 and MMP-9 mRNA. After addition of actinomycin D, MMP-1 and MMP-9 specific mRNA of epinephrine-treated U937 seems to decline even more rapidly than that of cells treated with LPS alone, but at 8 h mRNA levels decline to 40% in cells treated with LPS alone or with LPS and epinephrine, suggesting that increase of MMP-1 and MMP-9 mRNA after stimulation with catecholamines is not induced by increased mRNA stability but rather by increased transcription.

4. Epinephrine potentiates LPS-induced c-Fos binding to consensus AP-1 site
Nuclear extracts were prepared from U937 treated with epinephrine or LPS or a combination of both for 8 h and examined for the increase of the specific binding of c-Fos and c-Jun to the consensus AP-1 site of the target DNA. Whereas LPS had no effect on specific binding of c-Jun to the consensus AP-1 site at this late time, epinephrine increased the specific binding of c-Jun by 74%. The combination of LPS and epinephrine showed no further effect compared with epinephrine alone. Cells treated with LPS showed an 40% increase of specific binding of c-Fos to the consensus AP-1 site, epinephrine treatment increased specific binding of c-Fos by 68% and the combination of LPS and epinephrine synergistically up-regulated the specific binding of c-Fos to the consensus AP-1 site 2.5-fold compared with unstimulated control.

5. ß-Antagonists modulate the effect of catecholamines on LPS-induced production of MMP-1 and MMP-9 in U937 cells
The combined ß₁ and ß₂ antagonist propranolol completely inhibited the effects of epinephrine or norepinephrine on LPS-induced MMP-1 and MMP-9 production in U937. The ß₁ antagonist metoprolol abolished the stimulatory effects of norepinephrine on LPS-induced up-regulation of MMP-1 and MMP-9 whereas the ß₂ antagonist butoxamine showed no effect. In MDM the ß antagonists had similar effects on the stimulatory activity of catecholamines on LPS-induced up-regulation of MMP-1. Neither of the antagonists had any effect on the stimulation of LPS-induced production of MMP-1 and MMP-9 by catecholamines.

CONCLUSIONS AND SIGNIFCANCE

In this paper we present, to our knowledge for the first time, evidence that the catecholamines epinephrine and norepinephrine, respectively, potentiate the stimulating effect of LPS on the production of MMP-1 in human peripheral blood monocytes and in human monocyte-derived macrophages from different donors. We used the human monocytic cell line U937 to further characterize this effect. We found that epinephrine and norepinephrine up-regulated MMP-1 and MMP-9 up to 2.8-fold. Bacterial LPS alone significantly up-regulated the production of both MMPs in these cells. When catecholamines were added to the cells together with LPS, a marked potentiation in the stimulation of the production of MMP-1 was observed. In contrast to the results obtained with primary human cells, in U937 a potentiation of effects between the catecholamines and LPS was evident on production of MMP-9. Data on the effects of LPS on MMP-9 expression in the latter cells is contradictory. Based on our data, we think that the relevance of U937 cells to serve as a model for primary human monocytes and macrophages for studying MMP-9 regulation should be interpreted with caution. The synergistic effect increased LPS-induced expression of metalloproteinases in U937 up to ninefold over the values obtained with LPS alone. This effect was significant at catecholamine concentrations 10 ng/mL. Whereas peri-operative plasma levels of catecholamines of 5 ng/mL have been measured and systemic plasma levels of epinephrine have been shown to reach 1 ng/mL in sepsis, one could speculate that the local catecholamine concentration in the vicinity of sympathetic endings after sympathetic neuron discharge in the vessel wall might reach higher values.

The increase in MMP-1 and MMP-9 protein induced by a combination of LPS and catecholamines was paralleled by an increase in specific MMP-1 and MMP-9 activity. To investigate whether potentiation of effects between LPS and catecholamines is due to catecholamine-induced increased mRNA stability, we performed real-time PCR in the presence of actinomycin D. Specific MMP-1 and MMP-9 mRNA in cells treated with epinephrine and LPS declined even more rapidly than that of cells treated with LPS alone. This suggests that the increase of MMP-1 and MMP-9 mRNA after stimulation with catecholamines and LPS is not induced by increased mRNA stability but by increased transcription. The stimulating effect of catecholamines on LPS-induced MMP-1 and MMP-9 expression was completely blocked by cycloheximide, indicating it was dependent on de novo protein synthesis. The promoters of most matrix metalloproteinases contain consensus sites for AP-1. To further investigate any role of transcription factor AP-1, we used an ELISA-based assay to measure DNA binding activity of c-Fos and c-Jun. Whereas LPS had no effect on specific binding of c-Jun to the consensus AP-1 site at this late time, epinephrine up-regulated specific binding of c-Jun. We could also show that treatment with epinephrine increased, and the combination of LPS and epinephrine further up-regulated specific DNA binding of c-Fos. The data suggest that the transcription factor AP-1 might play a role in regulating the increase of MMP-1 and MMP-9 after stimulation with catecholamines and LPS.

To test the involvement of subsets of adrenergic receptors, we studied the effect of specific and ß antagonists on the synergistic action of catecholamines and LPS on MMP-1 and MMP-9 expression. Whereas the combined ß₁ and ß₂ antagonist propranolol completely blocked the effects of epinephrine and norepinephrine on LPS-induced MMP-1 and MMP-9 production, the ß₁ antagonist metoprolol abolished only the effect of norepinephrine and the ß₂ antagonist butoxamine showed no effect; antagonists did not block the stimulation of LPS-induced production of MMP-1 and MMP-9 by catecholamines. This indicated that receptors do not participate in the modulation of potentiation of the effects of catecholamines and LPS resulting in increased MMP-1 and MMP-9 production. On the other hand, the effect seems to be modulated via both ß receptors with epinephrine acting via either ß₁ receptors or ß₂ receptors. Our data show that norepinephrine can act only through the former receptor-type, which is in agreement with the known low affinity of norepinephrine for ß₂ receptors. Since the potentiating effect of epinephrine on LPS-induced MMP-1 and MMP-9 expression is not blocked by the ß₁ antagonist metoprolol, we hypothesize that this effect can be mediated via the interaction of epinephrine with ß₂ receptors alone. From our results demonstrating that the ß₂ antagonist butoxamine showed no effect we conclude that the potentiation of LPS-effects on MMP-1 and MMP-9 production can be mediated via the interaction of catecholamines with ß₁ receptors alone. Based on this data one could speculate that a combined ß₁ and ß₂ antagonist might even give superior results to that seen with a specific ß₁ antagonist in the clinical setting.

We have shown that modulators of peri- or postoperative stress more than additively enhance expression of matrix metalloproteinases in human monocytes. A similar potentiation of effects of LPS and catecholamines has been described to regulate IL-6 expression in human endothelial cells. A recent study has established a link between psychosocial stress and monocyte activation by showing that catecholamines activate peripheral blood mononuclear cells via the NF-B pathway. We conclude that if such potentiation of effects of LPS and catecholamines stimulates the production of matrix metalloproteinases in monocytes and macrophages in vivo this mechanism might contribute to post surgery plaque destabilization (Fig. 2 ). Thus, our findings might help explain the observation that cardiac events are important causes of morbidity and mortality after noncardiac surgery.

View larger version (22K): [in this window] [in a new window]   Figure 2. Schematic diagram.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0454fje

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