chf is not only the failure of the heart to generate sufficient cardiac output , but is a multisystemic disorder with immune activation leading to increased concentrations of several cytokines . in chf several studies showed increased concentrations of proinflammatory cytokines such as tnf , interleukin ( il)-1 , il-6 , il-18 , and cardiotrophin-1 ( ct-1 ) [ 25 ] . one of the most examined proinflammatory molecules in chf is tnf. tnf is a trimeric 17-kda polypeptide mainly produced by monocytes and macrophages . short - term tnf expression is thought to be an adaptive mechanism ; whereas prolonged expression causes left ventricular dysfunction and cardiomyopathy leading to chf propagation . however , tnf influences not only the heart itself but causes endothelial dysfunction and peripheral muscle wasting . 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 . all these cytokines bind to a specific receptor chain ( il-6r , il-11r , or lifr for ct-1 , lif , osm ) . following cytokine binding the cytokine / receptor complex associates with glycoprotein 130 ( gp130 ) causing tyrosine phosphorylation of gp130 and the signal is transduced via the janus kinase ( jak)/signal transducer and activation of transcription 3 ( stat3 ) pathway [ 810 ] . 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 [ 8 , 11 ] . increased ct-1 concentrations were detected in patients with acute myocardial infarction and chronic heart failure ( chf ) . furthermore , ct-1 plasma concentrations correlate with the severity of left ventricular dysfunction [ 1114 ] . however , ct-1 has not only effects on myocytes but also on vasculature by decreasing systemic vascular resistance in an animal model , by induction of acute phase proteins in rat hepatocytes , and by attenuation of endotoxin - induced acute lung injury . there are several studies showing that in chf pbmcs produce tnf [ 18 , 19 ] . but so far the mechanisms responsible for tnf production in these cells under these circumstances are not determined .   in this study we investigated whether ct-1 induces tnf expression in human pbmc of healthy volunteers . recombinant human ct-1 was purchased from r&d systems ( wiesbaden , germany ) and dissolved according to the manufacturer 's instruction . actinomycin d , brefeldin a , and parthenolide were purchased from sigma chemicals ( deisenhofen , germany ) . the blocking antibody against ct-1 was purchased from r&d systems ( wiesbaden , germany ) . 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 37c  in a humified 5% co2 atmosphere . cells were cultivated for 24  hour with rpmi 1640 supplemented with 10% fetal calf serum , 1% penicillin , streptomycin . afterwards , cells were subconfluent and medium was replaced by fresh medium . after 24  hours , over 90% of pbmc were alive tested by trypan blue exclusion . primary cultures from human vein endothelial cells were purchased from promocell ( heidelberg , germany ) . cell culture was done according to the manufacturer 's manual in endothelial growth medium with 2% fetal calf serum ( egm , promocell , heidelberg , germany ) . cells were grown to confluence in collagen i coated tissue culture plastic ( becton dickinson , franklin lakes , usa ) . 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 cells for defined time intervals and concentrations . as a control , approval for this study was given by the ethics committee of the friedrich schiller university of jena , and subjects gave their written informed consent according to the university guidelines . total rna from cultivated pbmc was extracted according to the rneasy protocol ( qiagen , hilden , germany ) . one  g of total rna was reversely transcribed into cdna in a volume of 20  l with avian myeloma leukaemia virus ( amv ) reverse transcriptase and oligo dt primers ( promega , madison , usa ) according to the manufacturers manual . real - time pcr measurement of tnf 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 , the specific primer for human tnf was purchased from r&d systems . the amplification program for tnf consisted of 1 cycle of 94c with a 4-minute hold followed by 40  cycles of 95c with a 45-second hold , 59c annealing temperature with a 45-second hold , and 72c with a 45-second hold . the specific primer pair for gapdh was : sense primer 5 ggg aag gtg aag gtc gg 3 , antisense primer 5 tgg act cca cga cgt act cag 3. the amplification program for gapdh consisted of 1 cycle of 95c with a 30-second hold followed by 30 cycles of 95c with a 5-second hold , 59c annealing temperature with a 10-second hold , and 72c with a 20-second hold . each reaction ( 20  l ) contained 2  l cdna , 2.5  mm mgcl2 , 1 pmol of each primer , and 2  l of fast starter mix ( containing buffer , dntps , sybr green dye and taq polymerase ) . a negative control without cdna was run with every pcr to assess the specificity of the reaction . pcr efficiency was determined by analysing a dilution series of 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 tnf transcripts . tnf concentrations in the culture supernatants were determined by elisa ( quantiglo , r&d systems , wiesbaden , germany ) according to the manufacturer 's instructions . nuclear extracts were achieved by the epiquik nuclear extraction kit i ( epigentek , ny , usa ) according to the manufacturer 's manual . afterwards , protein concentrations of nuclear extracts were determined according to the bradford methode . for determination of nfb 2  g of nuclear proteins were used and further analyzed by gel electrophoretic mobility shift assay ( emsa ) according to the suppliers manual . emsa kits and probes were purchased from panomics , redwood city , usa .   for intracellular staining peripheral blood was collected in lithium - heparin tubes . 100  l of blood was added to rpmi-1640 medium including brefeldin a ( final concentration : 1  g / ml ) ( sigma , taufkirchen , germany ) , and incubated for 6  hours time at 37c . were stained with monoclonal antibodies against the surface antigens cd3 ( coulter - immunotech , krefeld , germany ) , cd4 ( caltag , hamburg , germany ) cd8 or cd14 ( bd - pharmingen , heidelberg , germany ) ( 15 minute , rt ) , followed , after a washing step , by fixation with 100  l 2% paraformaldehyde for 10 min at rt . after a wash the cells were incubated in 100  l permeabilisation solution ( 0,1% saponin and 0,1%   nan3 in pbs ) together with 1  l directly conjugated anti - tnf antibody ( bd - pharmingen , heidelberg , germany ) for 15  minute at rt . followed by a wash with permeabilisation solution the cells were resuspended in pbs/2% fcs and fluorescence intensity was analyzed by flow cytometry ( facscalibur , becton - dickinson , heidelberg , germany ) . for analysis regions were defined by forward scatter and side scatter as well as cd3/cd4- or cd3/cd8-lymphocyte populations and cd14 monocyte population . data were analyzed with cellquest software . because the amount of the cytokines produced was different in each experiment , the effects on tnf production were normalized to unstimulated cells , which were set as one . data were analysed by nonparametric methods to avoid assumptions about the distribution of the measured variables . in the first sets of experiments we analysed whether ct-1 is able to induce tnf in pbmc . as shown in figure 1(a ) , ct-1 induced in a concentration - dependent manner tnf in the supernatant determined by a commercial available elisa . maximal tnf concentration was found after 3 to 6  hours and declined afterwards reaching nearly control values after 24  hours , indicating that ct-1 causes only a transient tnf release in pbmc . in the next experiments we determined intracellular tnf protein in monocytes , cd4 and cd8 lymphocytes after stimulation with various concentrations of ct-1 in the presence of brefeldin a using immunofluorescent flow cytometry . intracellular tnf determination in cd4 and cd8 lymphocytes did not show an effect of ct-1 on tnf expression ( data not shown ) . in monocytes we found a concentration - dependent increase of intracellular tnf after ct-1 application ( figure 1(b ) ) . these results showed that ct-1 induced tnf in pbcm independent of culture conditions and independent of determination methodes . on tnf mrna level we found maximal mrna after 1  hour . blocking ct-1 by an antibody against ct-1 inhibited ct-1 induced tnf mrna ( data not shown ) indicating that tnf induction is specifically caused by ct-1 . with the next experiments we addressed the question whether tnf protein expression is dependent on mrna synthesis and intracellular protein transport . in figure 3 it is shown that both inhibition of mrna synthesis by actinomycin d and intracellular protein transport by brefeldin a were able to abolish ct-1 induced tnf protein induction in the supernatant . as shown in figure 4 ct-1 caused a concentration - dependent nfb translocation to the nucleus determined by emsa reaching maximal translocation with 100  ng / ml ct-1 . in the next sets of experiments we used emsa to verify that nfb activation was responsible for ct-1 induced tnf expression in pbmc . human umbilical vein endothelial cells ( huvecs ) stimulated with tnf were used as a control . unstimulated cells did not show significant nfb protein in the nucleus ; whereas ct-1 caused translocation of nfb into the nucleus . parthenolide , an inhibitor of nfb activation , was able to inhibit nfb translocation to the nucleus ( figure 5 ) . nfb translocation is essential for tnf expression as shown in figure 6(a ) and 6(b ) . because parthenolide was able to inhibit tnf expression both on protein and mrna level we conclude that ct-1 not only was responsible for nfb translocation to the nucleus but this translocation was responsible for tnf expression . using flow cytometry we found in monocytes an increase of intracellular tnf after ct-1 application which could be inhibited by parthenolide ( figure 6(c ) ) . these results show that ct-1 induced tnf in pbcm independent of culture conditions and independent of determination methodes and nfb seems to be essential for the effect of ct-1 on tnf induction in pbmc . the first result of our study is that ct-1 is able to induce tnf mrna and protein in pbmc of healthy volunteers . tnf is increased in serum of patients with chf and correlates with the severity of heart failure , cachexia , and clinical outcome . tnf may be involved in progression of chf because high levels of tnf can induce left ventricular dysfunction , ventricular remodelling , cardiomyopathy , and pulmonary edema [ 22 , 23 ] . cultured human pbmc can synthesize and secrete tnf. in heart failure , both the heart itself and activated monocytes are able to secrete tnf [ 18 , 24 ] . furthermore , the capacity of pbmc of chf patients to secrete tnf is increased compared to control . our data are in good agreement with these former studies and in opposite to shimokawa et al .   who found decreased cytokine generation capacity of monocytes in severe heart failure after stimulation with lipopolysaccharide . besides the hypothesis that in chf the failing heart itself is the main source of tnf it is speculated by other groups that activated monocytes are responsible for increased tnf serum concentrations . monocytes may be activated by lps from the gut because the barrier function of the gut by cardial edema is disturbed and bacteria can easily translocate from the gut lumen to the blood stream .   as a third possibility our data suggest at least in theory a new mechanism for tnf production of pbmc in heart failure . ct-1 produced by the failing ventricle   is able to induce tnf in pbmc without lps . the here presented mechanism might also explain why tnf may be still elevated in chf even after edema were treated successfully with diuretics and the integrity of gut mucosa was restored . furthermore , our data support the study of petretta et al .   who found that tnf is not produced by the failing heart or the gut in patients with mild to severe heart failure .   the second result of our study is the fact that ct-1 activates the nfb system in a concentration - dependent manner in pbmc of healthy volunteers our in vitro data are in line with studies that found an activation of the nfb system in peripheral blood cells in chf . jankowska et al . reported an activation of the nfb system in peripheral blood leukocytes in chf patients measured by immuncytochemistry . found an augmented activation of nfb activation in blood mononuclear cells using electromobility shift assay in patients with chf compared to healthy controls . the exact pathway responsible for nfb activation in chf is still unknown and remains to be determined . but within 3  hours , there is no cytotoxic effect as shown by o'neill et al . in . we also used high ct-1 concentrations compared to concentrations reported in patients with chf by ng et al . . on the other hand a paper published in 2008   reported serum ct-1 concentration in healthy controls and patients with metabolic syndrome of about 100  ng / ml . so far serum concentration of ct-1 in healthy controls and patients is a matter of discussion . but independent of reported ct-1 serum concentration the concentration of ct-1 should be much higher in the myocardium which is the source of ct-1 in chf . exact intramyocardial ct-1 concentrations are not determined so far , only mrna and immunohistochemical studies showed increased ct-1 in hearts of patients with chf . in our experiments both tnf mrna expression and tnf protein production of pbmc showed a large standard variation . first one explanation for the large standard deviation may be a different genetic susceptibility of pbmc from different persons to stimuli . second , we used the low basal mrna concentration as the basis of normalization explaining the large standard variation . third , the fact that the increase of tnf mrna expression after ct-1 application is much higher compared to the increase of protein in the supernatant may be explained methodically . because in chf many proinflammatory cytokines are elevated and pbmc are activated , it is not easy to study the effect of a single cytokine in pbmc of patients with chf . for this reason we used pbmc from healthy volunteers in culture and stimulated them with recombinant ct-1 . in conclusion , interestingly , in our study lps is not needed for elevated tnf expression in pbmc . elevated tnf concentrations may be important in the pathogenesis and perpetuation of heart failure by modulating systemic metabolism , causing apoptosis and having a negative inotropic effect . in the light of our results modulating ct-1
chronic heart failure ( chf ) is associated with elevated concentrations of tumor necrosis factor ( tnf )  and cardiotrophin-1 ( ct-1 ) and altered peripheral blood mononuclear cell ( pbmc ) function . 
 therefore , we tested whether ct-1 induces tnf in pbmc of healthy volunteers . 
 ct-1 induced in pbmc tnf protein in the supernatant and tnf mrna in a concentration- and time - dependent manner determined by elisa and real - time pcr , respectively . 
 maximal tnf protein was achieved with 100  ng / ml ct-1 after 36 hours and maximal tnf mrna induction after 1 hour . 
 elisa data were confirmed using immunofluorescent flow cytometry . 
 inhibitor studies with actinomycin d and brefeldin a showed that both protein synthesis and intracellular transport are essential for ct-1 induced tnf expression . 
 ct-1 caused a dose dependent nuclear factor ( nf ) b translocation . 
 parthenolide inhibited both nfb translocation and tnf protein expression indicating that nfb seems to be necessary . 
 we revealed a new mechanism for elevated serum tnf concentrations and pbmc activation in chf besides the hypothesis of pbmc activation by bacterial translocation from the gut .