Cardiotrophin-1 induces tumor necrosis factor alpha synthesis in human peripheral blood mononuclear cells

Abstract

Chronic heart failure (CHF) is associated with elevated concentrations of tumor necrosis factor (TNF) alpha and cardiotrophin-1 (CT-1) and altered peripheral blood mononuclear cell (PBMC) function. Therefore, we tested whether CT-1 induces TNFalpha in PBMC of healthy volunteers. CT-1 induced in PBMC TNFalpha protein in the supernatant and TNFalpha mRNA in a concentration- and time-dependent manner determined by ELISA and real-time PCR, respectively. Maximal TNFalpha protein was achieved with 100 ng/mL CT-1 after 3-6 hours and maximal TNFalpha 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 TNFalpha expression. CT-1 caused a dose dependent nuclear factor (NF) kappaB translocation. Parthenolide inhibited both NFkappaB translocation and TNFalpha protein expression indicating that NFkappaB seems to be necessary. We revealed a new mechanism for elevated serum TNFalpha concentrations and PBMC activation in CHF besides the hypothesis of PBMC activation by bacterial translocation from the gut.

Figure 1

(a) Concentration- and time-dependent expression of TNFα protein in the supernatant after incubation with CT-1. Human PBMCs were incubated with various concentrations of CT-1 and for different periodes. After the indicated time TNFα protein concentration was determined by a commercial available ELISA. n = 5, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells. (b) Analysis of intracellular TNFα production using immunofluorescent flow cytometry. Human blood was incubated with different concentrations of CT-1 for 6 hours in the presence of brefeldin A. Afterwards erythrocytes were lysed and cells were stained with a monoclonal antibody against CD14 FITC-conjugated and against TNFα PE-conjugated. Monocytes were gated and results are expressed normalized to unstimlulated monocytes. n = 11, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells.

Figure 2

Concentration- and time-dependent induction of TNFα mRNA after incubation with CT-1. Human PBMCs were incubated with various CT-1 concentrations and for various periodes. After the indicated time mRNA was determined by real-time PCR. All TNFα mRNA expression data were normalized to GAPDH. n = 8, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells.

Figure 3

After 3 hours TNFα protein was determined by ELISA in the supernatant. CT-1 0 ng/mL was set as 1. Act: actinomycin D (5 μg/mL), inhibits mRNA transcription, Bre: brefeldin (10 μg/ml), inhibits intracellular protein transport. n = 6, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells.

Figure 4

CT-1 causes a concentration dependent increase of NFκB activity measured by EMSA. Bands corresponding to NFκB activity were quantified by densitometry and expressed in arbitrary units and normalized to unstimulated PBMC. n = 7, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells.

Figure 5

Detection of NFκB activity in human PBMC. Representative EMSA of CT-1 induced NFκB activity in PBMC, which could be inhibited by parthenolide an inhibitor of NFκB activation. Human umbilical vein endothelial cells (HUVECs) stimulated with TNFα were used as control.

Figure 6

Effect of parthenolide on CT-1 induced TNFα expression. (a) Human PBMCs were incubated with CT-1 (50 ng/ml) for 1 hour in the presence of parthenolide. Afterwards cells were lysed and TNFα mRNA expression was determined by real-time-PCR. All TNFα mRNA expression data were normalized to GAPDH. n = 6, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells. (b) Human PBMCs were incubated with CT-1 (50 ng/ml) for 3 hours in the presence of parthenolide. Afterwards TNFα protein concentration in the supernatant was determined by ELISA. n = 5, data are expressed as mean  ±  SEM and normalized to unstimulated cells. *P < .05 compared to unstimulated cells. **not significant compared to unstimulated cells. (c) Analysis of intracellular TNFα production using immunofluorescent flow cytometry. Human blood was incubated with 50 ng/ml CT-1 for 6 hours in the presence of brefeldin A and parthenolide. Afterwards erythrocytes were lysed and cells were stained with a monoclonal antibody against CD14 FITC-conjugated and against TNFα PE-conjugated. Monocytes were gated and results are expressed normalised to unstimlulated monocytes. n=6, data are expressed as mean  ±  SEM. *P < .05 compared to unstimulated cells, **P < .05 compared to cells stimulated with 50 ng/ml CT-1.