Cardiotrophin-1 induces interleukin-6 synthesis in human monocytes

www.elsevier.com/locate/issn/10434666 Cytokine 38 (2007) 137–144

Cardiotrophin-1 induces interleukin-6 synthesis in human monocytes Michael Fritzenwanger *, Katharina Meusel, Martin Foerster, Friedhelm Kuethe, Andreas Krack, Hans-R. Figulla Department of Internal Medicine I, Division of Cardiology, Friedrich-Schiller-University Jena, Erlanger Allee 101, 07740 Jena, Germany Received 14 August 2006; received in revised form 21 May 2007; accepted 25 May 2007

Abstract Background. Patients with congestive heart failure (CHF) show increased serum concentrations of cytokines like interleukin-6 (IL-6) and cardiotrophin-1 (CT-1). Additionally, monocyte function is modulated in CHF. The aim of this study was to examine if CT-1 is able to induce IL-6 in human monocytes and to investigate the underlying pathway. Methods. Separated peripheral blood monocytes of healthy volunteers were cultured with increasing concentrations of CT-1 for different periods. IL-6 mRNA was determined by RT-PCR or real-time PCR and IL-6 protein concentration in the supernatant by ELISA. Phosphorylation of signal transducer and activation of transcription (STAT) 3 was analyzed by western blot or by FACS analysis. To clarify the signalling pathway of CT-1 induced IL-6 expression various inhibitors of possible signal transducing molecules were used. Results. CT-1 induced IL-6 mRNA in monocytes in a time- and concentration-dependent manner. Maximal mRNA induction was detectable after 6 h with 100 ng/ml CT-1. IL-6 protein also increased in a time- and concentration-dependent manner with a maximum after 48 h with 100 ng/ml CT-1. AG490 as well as SB 203580 and parthenolide blocked CT-1 induced IL-6 expression completely. AG 490 was able to inhibit STAT3 phosphorylation in western blot analysis completely. This indicates that JAK2/STAT3, p38 and nuclear factor jB (NFjB) are involved in this pathway. To exclude a possible influence of plastic adherence of monocytes on CT-1 induced IL-6 expression, we determined intracellular STAT3 phosphorylation in whole blood samples by FACS analysis and observed independently of culture conditions a CT-1 concentrationdependent STAT3 phosphorylation. Conclusion. CT-1 induces IL-6 mRNA and protein expression in a time- and concentrationdependent manner. The underlying pathway is Janus kinase (JAK)2/STAT3, p38 and NFjB dependent. These data may explain increased IL-6 serum concentrations and altered monocyte function found in patients with CHF. Modulation of the CT-1 pathway might be a interesting strategy in the treatment of CHF.  2007 Elsevier Ltd. All rights reserved. Keywords: Cardiotrophin-1; Interleukin-6; Monocyte; Heart failure; Signalling

1. Introduction Cardiotrophin-1 (CT-1) is a member of the IL-6 cytokine family that consists of IL-6, IL-11, ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), neuropoietin (NPN) and oncostatin M (OSM) and has recently been supplemented by the addition of two newly characterized cytokines IL-27 and IL-31 [1]. *

Corresponding author. Fax: +49 3641 9324102. E-mail address: [email protected] Fritzenwanger).

(M.

1043-4666/$ - see front matter  2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2007.05.015

All these cytokines bind to a specific receptor chain (IL6R, IL-11R or LIFR for CT-1, LIF, OSM). Afterwards the cytokine/receptor complex associates with glycoprotein 130 (gp130). This leads to tyrosine phosphorylation of gp130 and the signal is transduced via the Janus kinase (JAK)/signal transducer and activation of transcription 3 (STAT3) pathway [2–4]. CT-1 is expressed in a time-dependent manner during embryogenesis of organs, is expressed in the heart during life, induces cardiac myocyte hypertrophy, and is able to prevent myocyte apoptosis via a mitogen dependent kinase pathway [2,5]. Interestingly, increased CT-1 concentrations were detected in patients with acute myocardial infarction and chronic heart failure

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(CHF). Furthermore, CT-1 plasma concentrations correlate with the severity of left ventricular dysfunction [5–8]. However, CT-1 has not only effects on myocytes but also on the vasculature by decreasing systemic vascular resistance in animal models [9], induction of acute phase proteins in rat hepatocytes [10], and by attenuation of endotoxin-induced acute lung injury [11]. IL-6, a 185 amino acid polypeptide, which is produced by a variety of cells and organs in health and disease has pleiotrophic effects. IL-6 is able to stimulate B-cell differentiation [12], activates thymocytes and T-cells for differentiation [13], activates macrophages [14] and natural killer cells [15], and stimulates hepatocytes to produce acute phase proteins [16]. IL-6 also possesses anti-inflammatory properties [17]. Additionally, IL-6 is involved in cardiovascular physiology and pathology [18]. For example, in left ventricular dysfunction independent of its origin elevated IL-6 concentrations are found and are a predictor of subsequent clinical outcomes. Since atherosclerosis is an inflammatory process many studies show the important role of IL-6 in this disease. IL-6 concentrations are elevated in patients with acute myocardial infarction and unstable angina but not stable angina [19]. IL-6 causes myocyte hypertrophy, myocardial dysfunction and a decrease of muscle mass. On the other hand IL-6 inhibits cardiomyocyte apoptosis [20]. Although IL-6 plays a central role in cardiovascular disease it is not clear whether IL-6 is a beneficial or detrimental cytokine so far. In CHF many cytokines and their receptors are up-regulated indicating that CHF is also a disease which influences the immune system. There are some studies showing that monocyte function is modulated in CHF [21,22]. But so far the mechanisms responsible for altered monocyte function are not determined. In this study, we investigated whether CT-1 is able to induce IL-6 expression in human monocytes of healthy volunteers. Furthermore, we used different inhibitors of signal transduction to clarify the underlying pathway. 2. Materials and methods 2.1. Reagents Recombinant human CT-1 was purchased from R&D Systems (Wiesbaden, Germany) and dissolved according to the manufacturer’s instruction. Wortmannin, staurosporin, parthenolide and PD98059 were purchased from Sigma chemicals (Deisenhofen, Germany), AG490 from Calbiochem (Darmstadt, Germany), SB203580 from Upstate (Dundee, UK). The blocking antibody against CT-1 was purchased from R&D Systems (Wiesbaden, Germany). 2.2. Cell culture Human peripheral blood mononuclear cells were obtained from healthy volunteers by Ficoll-paque (Amersham Bioscience, Uppsala, Sweden) centrifugation. The

cells were washed three times with PBS, resuspended in RPMI 1640 supplemented with 10% fetal calf serum, 1% penicillin, streptomycin (all from Biochrom AG, Berlin, Germany) and cultured in plastic dishes at 37 C in a humified 5% CO2 atmosphere. After 2 h non adherent cells such as lymphocytes were removed by changing the culture medium. Cells were cultivated overnight. Stimulation and pharmacological studies were done next morning. All stimulants, inhibitors and media were without significant endotoxin levels according to the manufacturers’ instructions. Pharmacological agents, dissolved in fresh medium, were added to the monocytes for defined time intervals and concentrations. As a control, fresh medium was added to the cells. Approval for this study was given by the Ethics Committee of the Friedrich-Schiller-University, Jena, and subjects gave their written informed consent according to University guidelines. 2.3. Real-time PCR Total RNA from cultivated monocytes was extracted according to the RNeasy protocol (Qiagen, Hilden, Germany). One microgram of total RNA was reversely transcribed into cDNA in a volume of 20 ll with avian myeloma leukaemia virus (AMV) reverse transcriptase and oligo dT primers (Promega, Madison, USA) according to the manufacturer’s manual. Real-time PCR measurement of IL-6 cDNA was performed with the Light Cycler Instrument using the Fast Start DNA Master SYBR Green I kit (Roche Diagnostics, Mannheim, Germany). For verification of the correct amplification product, PCR products were analyzed on a 2% agarose gel stained with ethidium bromide. The specific primer pair for IL-6 resulting in a 542-bp PCR product was: sense primer 5 0 GCC TTC GGT CCA GTT GCC TT 3 0 , antisense primer 5 0 GCA GAA TGA GAT GAG TTG TC 3 0 . The amplification program for IL-6 consisted of 1 cycle of 95 C with a 30-s hold followed by 45 cycles of 95 C with a 5-s hold, 56 C annealing temperature with a 10-s hold and 72 C with a 20-s hold. The specific primer pair for GAPDH was: sense primer 5 0 GGG AAG GTG AAG GTC GG 3 0 , antisense primer 5 0 TGG ACT CCA CGA CGT ACT CAG 3 0 . The amplification program for GAPDH consisted of 1 cycle of 95 C with a 30-s hold followed by 30 cycles of 95 C with a 5-s hold, 59 C annealing temperature with a 10-s hold and 72 C with a 20-s hold. Each reaction (20 ll) contained 2 ll cDNA, 2.5 mM MgCl2, 1 pmol of each primer and 2 ll of Fast Starter Mix (containing buffer, dNTPs, Sybr Green dye and Taq polymerase). Amplification was followed by melting curve analysis to verify the correctness of the amplicon. A negative control without cDNA was run with every PCR to assess the specificity of the reaction. Analysis of data was performed using Light Cycler software version 3.5. PCR efficiency was determined by analyzing a dilution series of

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2.4. Reverse transcriptase-PCR Total RNA from monocytes was extracted as described above for real-time PCR and reversely transcribed into cDNA. Semiquantitative PCR was carried out by nomalizing all cDNAs to GAPDH. IL-6 primer sequences were: sense 5 0 GGG AAG GTG AAG GTC GG 3 0 and antisense 5 0 TGG ACT CCA CGA CGT ACT CAG 3 0 . The RT-PCR program consisted of 1 cycle of 95 C with a 3-min hold followed by 30 cycles of 96 C with a 30-s hold, 55 C annealing temperature with a 45-s hold and 72 C with a 45-s hold. Afterwards 72 C with a 5-min hold. GAPDH primer sequences were from Clontech (Heidelberg, Germany). PCR fragments were separated on 1.5% agarose gel containing ethidium bromide and visualized by UV irradiation. Gels were photographed and analyzed desitometrically (Herolab Easy Win 32 Software). 2.5. IL-6 ELISA Cultured monocytes were treated with various concentrations of CT-1 for various time periodes. IL-6 secretion in the culture supernatants was determined by ELISA (QuantiGlo, R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. 2.6. Western blot analysis Monocytes were lyzed with 600 ll of boiling buffer consisting of one part Roti-Load (Roth, Germany) and two parts aqua dest. Lysates were sonificated two times for 5 s. Proteins were separated by SDS–PAGE with 40 ll lysate per lane and transferred to nitrocellulose membranes (Amersham, Little Chalford, England). Membranes were incubated with primary antibodies and peroxidase conjugated secondary antibodies (Amersham, Little Chalford, England). Visualization was achieved by an enhanced chemiluminescence system (Amersham, Little Chalford, England). 2.7. Flow cytometric analysis of Stat3-phosphorylation For intracellular staining peripheral blood was collected in lithium-heparin tubes. PBMC were isolated as described above and suspended in RPMI (2 · 106 cells/ml). Cells were stimulated with CT-1 (10, 50, 100 ng/ml) at 37 C. After 15 min the cells were fixed with 1 ml 2% paraformaldehyde for 10 min at RT. After washing with 2 ml PBS for 4 min at 1400 rpm cells were permeabilized with 200 ll 90%

ice-cold methanol for 10 min at 20 C. After another washing step the cells were resuspended in 50 ll PBS/ FCS. Ten microliters of a Stat3 (pS727) antibody (clone 49/pStat3, Becton–Dickinson, Heidelberg, Germany) directly conjugated to Alexa Fluor 488 was added for 30 min at RT in the dark. Isotype control was from Caltag/Invitrogen (Karlsruhe, Germany). Cells were washed in 2 ml PBS/0.2% Tween-20 for 10 min, centrifuged for 4 min at 1400 rpm. The supernatant was discarded and the cells resuspended in 300 ll PBS/FCS. Fluorescence intensity was analyzed by flow cytometry (FACSCalibur, Becton–Dickinson, Heidelberg, Germany). For analysis the monocyte region was defined by forward scatter and side scatter. Datas were analyzed with CellQuest Software (Becton–Dickinson, Heidelberg, Germany). 2.8. Statistical analysis Because the amount of the cytokines produced was different in each experiment, the effects on IL-6 production were normalized to unstimulated cells, which were set as one. Data were analyzed by non-parametric methods to avoid assumptions about the distribution of the measured variables. Comparisons between groups were made with the Wilcoxon test. All values are reported as means ± SD. Statistical significance was considered to be indicated by a value of P < 0.05. 3. Results 3.1. Effect of CT-1 on IL-6 mRNA expression by monocytes In the first set of experiments we examined whether CT1 could induce IL-6 mRNA in human monocytes isolated from peripheral blood. Monocytes where incubated with CT-1 for 0.5, 1, 2, 4, 6, 16 and 24 h. Unstimulated cells showed only scarce IL-6 mRNA expression measured by real-time PCR. Basal IL-6 mRNA expression was set as 1. CT-1 (100 ng/ml) caused a time-dependent expression of IL-6 mRNA with a maximum effect at 6 h (8.2 ± 7.4 fold compared to control) (Fig. 1). As a next step rising concentrations of CT-1 (5, 10, 50 and 100 ng/ml) were tested for

IL-6 mRNA expression (arbitrary units)

cDNA (external standard curve). The identity of the PCR product was confirmed by comparing its melting temperature (Tm) with the Tm of amplicons from standards or positive controls. GAPDH was analyzed in parallel to each PCR and the resulting GAPDH values were used as standards for presentation of IL-6 transcripts.

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Fig. 1. Time-dependent expression of IL-6 mRNA by human monocytes after incubation with CT-1 (100 ng/ml). After the indicated time RNA was isolated and IL-6 mRNA expression was determined by RT-PCR. n = 5, data are expressed as means ± SD. *P < 0.05 vs. control.

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their ability to induce IL-6 mRNA expression in monocytes. As shown in Fig. 2 CT-1 (5, 10, 50 and 100 ng/ml) enhanced IL-6 mRNA compared to control with a maximum for 100 ng/ml employing both real-time PCR and RT-PCR. These data indicate that CT-1 induces IL-6 mRNA in a time- and concentration-dependent manner. 3.2. Effect of CT-1 on IL-6 protein secretion by monocytes To examine the effect of CT-1 on IL-6 protein expression we exposed monocytes to rising CT-1 concentrations for 6, 16, 24 and 48 h. IL-6 protein in the supernatant was determined with a commercially available ELISA. IL-6 protein concentration was set one for control conditions (unstimulated monocytes at the indicated time). IL-6 protein increased with time and CT-1 concentration (Fig. 3). Already after 6 h a significant increase of IL-6 after CT-1 application could be determined. Maximum

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Fig. 2. (a) Concentration-dependent production of IL-6 mRNA by human monocytes after incubation with CT-1. Monocytes were incubated with various concentrations of CT-1. After 6 h RNA was isolated and IL-6 mRNA was determined by real-time PCR. n = 6, data are expressed as means ± SD. *P < 0.05 vs. control. (b) Representative RT-PCR agarose gel of IL-6 mRNA expression in monocytes stimulated by CT-1 for 6 h. GAPDH served as loading control.

3.3. Signalling pathway of CT-1 induced IL-6 expression Based on the results of other groups [23,24] we used different inhibitors to clarify the pathway responsible for CT-1 induced IL-6 expression in monocytes: AG490 for JAK2/STAT3 inhibition, SB203580 for p38 MAPK inhibition, PD98059 for MEK, wortmannin for PI3K and staurosporine for proteinkinase C. Monocytes were incubated with CT-1 (50 ng/ml) for 6 h in the presence of these inhibitors. After this period cells were lyzed and IL-6 mRNA expression was determined by real-time PCR. All inhibitors were applied 30 min before CT-1 application. We used inhibitor concentrations which had been shown to be appropriate [25,26]. The inhibitors alone did not influence basal IL-6 expression in monocytes significantly (data not shown). CT-1 induced IL-6 expression could be completely inhibited by AG490 (50 lM). SB203580 (5 lM) was also able to inhibit IL-6 mRNA. PD98059 (30 lM), wortmannin (100 nM, data not shown) and staurosporin (10 ng/ ml, data not shown) had no inhibitory effect. These data indicate that JAK2/STAT3 and p38 are involved in this pathway whereas MEK, PI3K and PKC are not involved directly (Fig. 4a). To confirm that AG490 blocks JAK2/STAT3 we determined STAT3 phosphorylation in monocytes by western blot. CT-1 induced STAT3 phosphorylation in cultured monocytes. CT-1 had no influence on total STAT3 protein concentration. STAT3 phosphorylation could be blocked completely by AG490 indicating that CT-1 induced IL-6 in monocytes via JAK2/STAT3 (Fig. 4b). In another set of experiments we tested the effect of parthenolide (50 lM), an inhibitor of NFjB activation, on CT-1 induced IL-6 protein expression. We found that the upregulation of IL-6 after CT-1 application could be

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IL-6 protein induction was achieved after 24 h with a concentration of 100 ng/ml CT-1 (41.7 ± 20.1 fold compared to unstimulated monocytes). These data show that CT-1 induced IL-6 mRNA expression is followed by an increased IL-6 protein expression in monocytes.

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Fig. 3. Concentration-dependent production of IL-6 protein by monocytes after incubation with increasing concentrations of CT-1 for various periods. Monocytes were incubated for 6, 16 or 24 h unstimulated or with CT-1 50 or 100 ng/ml. Supernatants were tested with a specific ELISA for the presence of IL-6. n = 6, data are expressed as means ± SD. *P < 0.05 vs. control.

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Fig. 4. (a) Monocytes were incubated with SB203580 (5 lM, p38 inhibitor), AG490 (50 lM, JAK2 inhibitor) and PD98059 (30 lM, MEK inhibitor). After 30 min CT-1 (50 ng/ml) was added for additional 6 h (0: control). RNA was isolated and IL-6 mRNA was determined by real-time PCR. n = 6, data are expressed as means ± SD. *P < 0.05 (CT-1 50 ng/ml compared to inhibitor). (b) Representative western blot of STAT3 phosphorylation in monocytes after CT-1 stimulation and the effect of AG490 (50 lM). Quiescent monocytes were stimulated with 100 ng/ml CT-1 for 15 min. Afterwards protein was isolated and STAT3 and pSTAT3 were determined by western blot analysis.

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Fig. 5. Effect of NFjB inhibition on CT-1 induced IL-6 protein expression. Monocytes were incubated with medium or parthenolide (50 lM). After 30 min CT-1 (50 ng/ml) was added for additional 6 h (0: control). IL-6 protein in the supernatant was determined by a commercially available ELISA. n = 5, data are expressed as means ± SD. *P < 0.05 compared to 50 ng/ml CT-1.

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completely blocked by NFjB inhibition emphasizing that IL-6 is regulated on transcriptional level under these culture conditions (Fig. 5). To confirm that IL-6 expression is a specific effect of CT-1 we used a neutralizing antibody against CT-1. We found that incubation of CT-1 with this antibody is able to block CT-1 induced IL-6 mRNA expression completely. This confirms that CT-1 is specifically responsible for IL-6 expression (Fig. 6). To exclude that culture conditions are responsible for the observed CT-1 induced IL-6 expression we did some experiments with whole blood and examined STAT3 phosphorylation. Determining intracellular phosphorylation with FACS analysis shows that also in whole blood mono-

0 -

Fig. 6. Effect of a neutralizing antibody on CT-1 induced IL-6 mRNA expression. Quiescent layers of human monocytes were incubated for 6 h with medium or CT-1 (50 ng/ml) or CT-1 (50 ng/ml) and a monoclonal antibody against human CT-1 (10 lg/ml). After 6 h RNA was isolated and IL-6 mRNA was determined by real-time PCR. Data are expressed as means ± SD. Data are normalized to the unstimulated control, n = 4.

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Fig. 7. Flow cytometry histogram of intracellular STAT3 phosphorylation in monocytes of whole blood. Peripheral blood mononuclear cells were stimulated for 15 min with different CT-1 concentrations. Using FACS analysis intracellular STAT3 phosphorylation was determined with a monoclonal antibody against pSTAT3. Unstimulated monocytes’ fluorescence was set one and results are expressed as fold-stimulation of unstimulated cells, n = 3.

cytes show a CT-1 induced concentration-dependent STAT3 phosphorylation (Fig. 7). Because STAT3 phosphorylation via JAK2 is necessary for IL-6 expression these data show that culture conditions did not affect the CT-1 effect. 4. Discussion In the last decades the pathophysiological concepts of CHF have changed from a hemodynamical view to a multisystem disease. Now it is assumed that nearly all clinical signs of CHF are induced by inflammatory cytokines. TNF-a and NO were shown to induce left ventricular dysfunction, left ventricular remodelling and lung edema in experimental models [20,27]. In addition IL-6 is found increased in CHF patients and indicates poor prognosis [28–31]. Like Robledo [32] who found a time- and concentrationdependent IL-6 expression after CT-1 application in the KB epidermoid cell line we could show that CT-1 is able to induce IL-6 in monocytes in a time- and concentration-dependent manner. So far the precise mechanism and the cells which are responsible for increased cytokine expression in CHF are not determined. Some authors

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found that in addition to the myocardium also endothelial cells are able to produce IL-6 in severe CHF [31]. Activation of leukocytes and migration of these cells from blood vessels to areas of myocardial inflammation seems to be an important factor in the pathophysiology of CHF [33,34]. So far only few reports examined the importance of monocytes in CHF. Aukrust et al. [21] found that not only monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1a (MIP-1a) and regulated on activation normally T-cell expressed and secreted (RANTES) are elevated in CHF. Monocytes of patients with CHF generate more reactive oxygen species compared to control persons. That study also showed that spontaneous monocyte O2  generation is dependent on MCP-1 concentration. This is in good agreement with data of Vonhof et al. [22] who could determine elevated plasma neopterin concentration, a marker of monocyte activation, in CHF compared to control persons and that these leukocytes were prone to LPS stimulation. The importance of leukocytes and monocytes in the development of CHF caused by ischemia has been emphasized by Askari et al. [35]. This group showed in acute myocardial infarction after coronary artery ligation that mice deficient in myeloperoxidase, a protein found in granules of leukocytes and monocytes, showed decreased leukocyte infiltration, reduction in left ventricular diameter and marked preservation of left ventricular function. However, the role of monocytes as a source of cytokines in CHF is still a matter of discussion and the available data are discrepant so far. Shimokawa and coworker reported that after whole blood stimulation, there was significant lower TNFa, IL-1a and IL-1b in patients with severe CHF (NYHA class IV) vs. moderate heart failure and controls [36]. On the other hand Conraads et al. found that CD14 expression and monocyte cytokine production, both unstimulated and after LPS stimulation, were increased in moderate–severe when compared to mild CHF [37]. Our results suggest that in CHF the cytokine CT-1 is not only able to activate endothelial cells [38] but also monocytes. The relatively high values for SD compared to mean values result from the normalization of IL-6 concentrations to the basal IL-6 concentration which showed a great variability from experiment to experiment and donor to donor due to biological diversity. We as Robledo [32] used relatively high CT-1 concentrations to detect IL-6 expression compared to serum concentrations published for healthy subjects (range 6.9– 48.3 fmol/ml) or patients with CHF (74.3–182.8 fmol/ml) [39]. But so far the exact concentration of CT-1 in myocardial vessels is not reported. Because CT-1 is mainly produced in the heart in CHF [40], we speculate that intracoronary CT-1 concentration is much higher than CT-1 concentrations measured in venous serum and that blood monocytes may become activated while they pass the myocardial circulation. On the basis of the here presented data we propose a mechanism by which elevated CT-1 concentration inde-

pendent of its origin may induce IL-6 in monocytes and thus may be responsible for increased vascular permeability [41] and muscle wasting in CHF [42]. Tsutamoto et al. [31] also showed that subcutaneous administration of IL-6 for 7 days caused heart dilatation, reduced endsystolic pressure and decreased myocardial contractility [42]. In contrast there are observations indicating that IL-6 induction by CT-1 may be a beneficial counterregulatory mechanism. In a rat model of reperfusion injury Matsushita et al. [43] could reduce infarct size and myocyte apoptosis if IL-6/soluble IL-6R complex was given a short time before the left coronary artery was occluded. Myocyte apoptosis, which is detected in end stage heart failure [44] may be prevented by CT-1 induced IL-6 expression in infiltrating monocytes. In this study, we could also clarify the pathway responsible for CT-1 induced IL-6 expression in monocytes. The members of the IL-6 superfamily are known as gp130 cytokines because they share gp130 as a common transducer protein within their functional receptor complexes. Several members of the IL-6 family, among them CT-1, use the LIF receptor as a component of their receptor complex [3]. In this study we found that CT-1 induces IL-6 via JAK2 and p38 activation. This is in good agreement with our data in endothelial cells where we could show that CT-1 induces IL-6 in HUVEC via JAK2 and p38 [45]. In contrast to our recent study in endothelial cells JAK2/STAT3 phosphorylation is necessary for IL-6 induction in monocytes [38]. Our data are supported in part by Devaraj et al. who found that high glucose concentrations increase IL-6 expression in monocytic cells (THP-1) via a PKC-a and -b and p38 and NFjB dependent mechanism [46]. Devaraj and we did not find a direct involvement of the MEK pathway. In contrast to Devaraj we could not find that PKC inhibition could block IL-6 expression, which indicates that PKC activation is not necessary for IL-6 induction. Our data in monocytes are also supported by Kuldo et al. who could show that in HUVEC IL-6 expression is at least done by a p38 and NFjB dependent mechanism [47]. NFjB dependence was expected, because in the promoter region of the IL-6 gene a NFjB binding site is found [48]. LIF induced IL-6 transcription was shown to be linked to activation of NFjB [49]. The involvement of p38 might be explained in analogy to the findings of Burysek et al. [26] because in the IL-6 promotor a AP-1 binding site is reported. SB203580 inhibits AP-1 activation and prevents IL-6 induction by CT-1 via this pathway. The exact mechanism how JAK2/STAT3 activation is involved in CT-1 induced IL-6 expression is not clear. A direct activation of the IL-6 gene seems unlikely because there is no STAT3 binding site in the IL-6 promotor. In the light of our results modulation of CT-1 induced IL-6 expression in monocytes might be a new tool in the treatment strategy of CHF.

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