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Cardiovascular Research 2002 54(1):175-182; doi:10.1016/S0008-6363(02)00238-9
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure

Arne Yndestada,b,*, Jan Kristian Damåsa,b,c, Hans Geir Eikena,d, Torbjørn Holma,c, Terje Hauge, Svein Simonsenc, Stig S. Frølanda,b, Lars Gullestadf and Pål Aukrusta,b

aResearch Institute for Internal Medicine, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway
bSection of Clinical Immunology and Infectious Diseases, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway
cDepartment of Cardiology, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway
dMSD Cardiovascular Research Centre, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway
eCentre for Occupational and Environmental Medicine, Rikshospitalet University Hospital, University of Oslo, N-0027 Oslo, Norway
fDepartment of Medicine, Bærum Hospital, Sandvika, Norway

* Corresponding author. Tel.: +47-23-073-629; fax: +47-23-073-630 arne.yndestad{at}klinmed.uio.no

Received 23 October 2001; accepted 27 December 2001


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Inflammation may play a pathogenic role in chronic heart failure (CHF). The objective of the study was to characterise the imbalance in the cytokine network in CHF. Methods: cDNA expression arrays were used to analyse the gene expression of cytokines and related mediators in peripheral blood mononuclear cells (PBMC) from CHF patients (n=8) and healthy controls (n=8). Real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR) was used to determine the gene expression of individual genes in additional 12 patients and eight controls. Results: From 375 genes, 34 were upregulated and two downregulated in CHF patients in the cDNA expression array experiments. Regulated genes included chemokines/-receptors, members of the transforming growth factor β superfamily, orphan receptors and in particular several members of the tumor necrosis factor (TNF) superfamily. Thus, 4-1BB ligand (L), APRIL, CD27L, CD40L, FasL, LIGHT, TRAIL-receptor 4 were upregulated, while TRAIL-receptor 3 was downregulated. Real-time quantitative RT-PCR confirmed significantly upregulated gene expression of APRIL, LIGHT, FasL and CD27L in CHF patients and showed in addition significantly enhanced gene expression of TNF{alpha} and TRAIL. Conclusion: The present study demonstrates differential gene expression in PBMC of several members of the cytokine network in CHF. In particular, the enhanced expression of several ligands in the TNF superfamily may reflect a potential pathogenic role of these cytokines in CHF.

KEYWORDS Heart failure; Immunology; Cytokines; Leukocytes; Gene expression


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Persistent inflammation may play a pathogenic role in the progression of chronic heart failure (CHF). Thus, several clinical studies have reported elevated circulating levels of inflammatory cytokines, such as tumor necrosis factor (TNF){alpha}, interleukin (IL)-6 and IL-1 in CHF in direct relation to the clinical severity of the disease [1–3]. Additionally, inflammatory cytokines have been shown to induce events characterising the remodelling process during CHF, including cardiomyocyte hypertrophy, contractile dysfunction and fibrosis in both in vitro and in vivo models [4–6]. For example, cardiac-specific overexpression of TNF{alpha}, as well as infusion of TNF{alpha}, even in concentrations comparable to those found in the circulation during CHF, have been reported to cause a dilated cardiomyopathy-like phenotype mimicking several aspects of clinical heart failure, indicating that both circulating and locally produced cytokines may induce myocardial dysfunction [7,8].

While several reports have demonstrated enhanced plasma levels of inflammatory cytokines in CHF patients [3], few studies have examined these mediators in circulating leukocytes [9–11]. Circulating inflammatory cells may not only contribute to the systemic immune activation in these patients, but may also, by infiltrating the failing myocardium, indirectly promote myocardial dysfunction [12]. Moreover, while much attention have been drawn to TNF{alpha} and IL-6 in CHF, our knowledge of other members of the cytokine network is limited. For example, whereas there is strong evidence for TNF{alpha} as a pathogenic factor in CHF [13], other members of the TNF superfamily may potentially be even more important, but these mediators have hardly been studied in this disorder.

Our objective was to further characterise the imbalance in the cytokine network in CHF. In the present study we used a cDNA array based approach to analyse the gene expression of a large number of cytokines and other inflammatory mediators in peripheral blood mononuclear cells (PBMC) from CHF patients and healthy blood donors.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Patients
Twenty patients with stable CHF for >6 months in NYHA functional class II–III were studied (Table 1). We did not include end-stage CHF patients (i.e. NYHA class IV) in our study to, if possible, avoid that our findings merely reflect cachexia or severe hemodynamic dearrangement. None of the patients had any evidence of myocardial infarction or unstable angina during the past 6 months and none had significant concomitant disease such as infection, or connective tissue disease. Control subjects were 16 healthy age and sex-matched blood donors (Table 1). The investigation conforms with the principles outlined in the Declaration of Helsinki.


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  		Table 1 Clinical characteristics of CHF patients

 
2.2 Isolation of PBMC
PBMC were obtained from heparinised blood by Isopaque-Ficoll (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) gradient centrifugation within 45 min as previously described [14]. Cell pellets were stored in liquid nitrogen until used.

2.3 cDNA expression array hybridisation
Poly-A+-RNA was isolated for cDNA expression array experiments using the Dynabeads mRNA DIRECT Kit (Dynal, Oslo, Norway) as recommended by the manufacturer. Briefly, ~5x106 frozen PBMC from each individual were lysed in Lysis/binding buffer, poly-A+-RNA was hybridised to Dynabeads Oligo(dT)25 and subsequently washed before elution in nuclease-free water. Poly-A+-RNA was quantified using spectrophotometry (OD260/280 nm) and stored at –80 °C until used. Two pools of ~500 ng poly-A+ RNA were prepared from eight controls and eight CHF patients to represent the mean gene expression in the two populations. 33P-labeled cDNA was subsequently prepared using the cDNA Labeling and Hybridisation Kit (R&D Systems, Minneapolis, MN). Unincorporated nucleotides were removed using ProbeQuant G-50 columns (Amersham Pharmacia Biotech, Piscataway, NJ). Hybridisation to Human Cytokine Expression Arrays (R&D Systems) and washing were performed using conditions described by the manufacturer. The array was exposed to a phosphor screen for 1 and 4 days and scanned using Cyclone System (Packard, Meriden, CT). Individual hybridisation signals were identified and quantitated densitometrically using Phoretix Array (Nonlinear dynamics, Newcastle, UK) and normalised to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA signal. The normalised hybridisation signals for the CHF group were subsequently divided to the corresponding signals for the control group. A gene was considered to be upregulated when the ratio CHF to control exceeded 2.0 and considered to be downregulated when the ratio was below 0.5 as recommended by the manufacturer. The cDNA expression array included several ‘housekeeping genes’ and we found only minor changes in the expression of these genes when using GAPDH as control gene in the array analysis. For example, the ratio CHF/blood donor for β-actin, cyclophilin A, {alpha}-tubulin and transferrin receptor, representing other common control genes, was 1.26, 1.21, 0.91 and 1.19, respectively. Based on these data, our use of GAPDH as control gene should be valid.

2.4 Real-time quantitative RT-PCR
Total RNA for real-time quantitative RT-PCR was isolated from frozen PBMC using RNeasy Minikit (Qiagen, Hilden, Germany), subjected to DNase I treatment (RQI DNase; Promega, Madison, WI) and stored at –80 °C. None of the samples showed any signs of DNA contamination using PCR with intron-specific primers for GAPDH. Sequence specific PCR primers and TaqMan probes were designed using the Primer Express software version 1.5 (Applied Biosystems, Foster City, CA), see Table 2 for details. Quantification of mRNA was performed using the ABI Prism 7700 (Applied Biosystems). Reverse transcription (RT) was performed using TaqMan Reverse Transcription reagents (Applied Biosystems). Each RT-reaction contained 100 ng total RNA, 5 µl 10x RT Buffer, 2.5 µl 50 µM Random Hexamers, 1.25 µl MultiScribe Reverse Transcriptase (50 U/µl), 1 µl RNase Inhibitor (20U/µl), 11 µl 25 mM MgCl2, 10 µl dNTP Mixture and nuclease-free water made up to 50 µl. The reaction was incubated at 25 °C for 10 min, followed by 48 °C for 30 min and finally 5 min at 95 °C. Each real-time PCR reaction contained 2.5 µl of cDNA, 12.5 µl TaqMan Universal Master Mix (Applied Biosystems), 300 nM sense and anti-sense primer, 200 nM TaqMan probe, 1.25 µl 20x Human GAPDH Pre-developed Assay Reagents (Applied Biosystems) and H2O for a 25 µl total volume. Each sample was run in triplicate. Cycling parameters were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Standard curves were run on the same plate and the relative standard curve method was used to calculate the relative gene expression as previously described [15]. GAPDH was included as an endogenous normalisation control to adjust for unequal amounts of RNA.


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  		Table 2 Characteristics of the real-time PCR assays used in the study

 
2.5 Statistical analysis
Statistical comparisons were performed using the Mann–Whitney rank sum test. P values were two-sided and considered significant when <0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Identification of differentially expressed genes in PBMC
By using cDNA expression arrays, we analysed the gene expression of 375 cytokines, cytokine receptors and related mediators comparing PBMC from eight CHF patients and eight healthy blood donors. Radioactively labelled cDNA was prepared from pooled RNA from the two populations and hybridised to two array membranes. One hundred and forty-one genes were detected in PBMC from blood donors compared to 208 genes in CHF patients. As described in Table 3, densitometric analysis revealed several differences between these two groups of individuals with upregulation (ratio CHF/blood donor>2.0) of 34 genes and downregulation (ratio CHF/blood donor<0.5) of two genes in the CHF group.


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  		Table 3 Differentially expressed genes comparing peripheral blood mononuclear cells (PBMC) from CHF patients (n=8) and healthy blood donors (n=8)

 
Two gene families were particularly prominent when viewing the individual regulated genes according to their respective cytokine families. Firstly, several members of the TNF superfamily exhibited differential gene expression in CHF patients and healthy controls. Thus, the expression of the TNF superfamily ligands (L) 4-1BBL, APRIL (a proliferation inducing ligand), CD27L, CD40L, FasL and LIGHT (homologous to lymphotoxins, inducible expression, competes with HSV glycoprotein D for HVEM [herpes virus entry mediator], a receptor expressed on T– lymphocytes), in addition to the TNF-related apoptosis inducing ligand (TRAIL) receptor (R) 4, were all upregulated in the CHF group. In contrast, the decoy receptor TRAIL R3 was markedly downregulated in these patients. Secondly, also several chemokines and chemokine receptors were upregulated in CHF patients. Thus, both macrophage inflammatory protein (MIP)-1{alpha} and -1β as well as their corresponding receptor, CCR5, were markedly upregulated in CHF patients compared with healthy controls. Finally, PBMC from CHF patients also showed enhanced expression of some genes in the transforming growth factor (TGF) β family, interferon (IFN)-{alpha} and -β and IFN-{gamma} inducing factor (i.e. IL-18) receptor genes as well as genes for several orphan receptors (i.e., receptors with unknown ligands)(Table 3). In fact, the gene expression of the orphan receptor Bob, possibly related to the chemokine family [16], showed the most marked upregulation (7.4-fold increase) comparing PBMC from CHF patients and healthy controls (Table 3).

3.2 Gene expression of TNF superfamily ligands
A major finding in the cDNA expression array experiment was the upregulation of several genes in the TNF superfamily in PBMC from CHF patients. To further elucidate this finding we used real-time quantitative RT-PCR to determine the message levels of genes of ligands in the TNF superfamily in PBMC from another 12 CHF patients and eight healthy blood donors. In this experiment we examined both the gene expression of the six genes that were upregulated in the array experiment (i.e. 4-1BBL, APRIL, CD27L, CD40L, FasL and LIGHT) as well as the gene expression of other detectable ligands in the TNF superfamily, i.e. CD30L, lymphotoxin-β, TNF{alpha}, TRAIL and TWEAK (TNF-related ligand with weak ability to induce cell death) with CHF/control ratios of 1.7, 1.0, 1.4, 1.6 and 1.7, respectively, in the cDNA array experiment. This analysis was performed both to investigate the precision in the cDNA expression array experiment and to obtain a more complete and accurate picture of the regulation of this family of cytokines in CHF.

The real-time quantitative RT-PCR experiment confirmed the differential gene expression in four out of the six ‘positive’ TNF superfamily ligands from the array experiment. Thus, the gene expression of APRIL (1.6-fold increase, P<0.01), CD27L (2.3-fold increase, P<0.05), FasL (2.5-fold increase, P<0.001) and LIGHT (2.2-fold increase, P<0.001) were markedly upregulated in PBMC from CHF patients, but we failed to confirm the differential gene expression of 4-1BBL and CD40L (Fig. 1). In addition we demonstrated increased mRNA levels of TNF{alpha} (8.8-fold increase, P<0.001) and TRAIL (2.0-fold increase, P<0.01) in the CHF group, while no significant changes in gene expression were found for CD30L, lymphotoxin-β and TWEAK (Fig. 1). There were no differences in the gene expression of the TNF superfamily ligands comparing CHF patients with coronary artery disease and dilated cardiomyopathy (data not shown). Moreover, although there were differences in the medication within the CHF group, we were not able to relate the degree of gene expression of TNF superfamily ligands to the use of any medication as presented in Table 1.


Figure 1
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  		Fig. 1 Gene expression of ligands in the TNF superfamily relative to GAPDH, quantified by real-time quantitative RT-PCR of 4-1BB ligand (L) (A), APRIL (B), CD27L (C), CD30L (D), CD40L (E), FasL (F), LIGHT (G), lymphotoxin (LT)-β (H), tumor necrosis factor (TNF){alpha} (I), TRAIL (J) and TWEAK (K) in peripheral blood mononuclear cells from healthy blood donors (open circles, n=8) and patients with CHF (filled circles, n=12). Horizontal lines indicate median values.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
4.1 Findings
While increased levels of certain inflammatory cytokines (e.g. TNF{alpha}, IL-1 and IL-6) have been demonstrated in CHF [3], few studies have examined the expression of these mediators at the cellular levels. Even more importantly, the imbalance in the cytokine network involving a wide range of mediators is poorly characterised in this disorder. In our screening experiments, demonstrating for the first time a complex set of changes in the cytokine network in PBMC during CHF by using cDNA expression arrays, we have pinpointed several potentially interesting genes and gene families in the cytokine network that should be further investigated for their possible pathogenic role in this disorder. In particular, we found a marked upregulation of several ligands in the TNF superfamily in PBMC from CHF patients as demonstrated by both cDNA expression arrays and real-time quantitative RT-PCR methods. While the pathophysiological role of TNF{alpha} in CHF has been extensively examined and several studies have reported raised TNF{alpha} levels in this disorder [13], the literature is virtually devoid of data concerning other members of the TNF superfamily in human heart failure. Thus, except for some reports of altered Fas/FasL levels in CHF [17,18], the present study is, to our knowledge, the first demonstration of altered expression of several members of the TNF superfamily in CHF patients.

4.2 Methodological considerations
The accuracy and reproducibility of cDNA arrays have been widely discussed [19]. Our findings in the present study underscore the need for confirmation of results from cDNA array experiments by additional and independent methods. Thus, while we confirmed the upregulation of APRIL, CD27L, FasL and LIGHT by real-time quantitative RT-PCR, the upregulation of 4-1BBL and CD40L were not verified. Notably, for both 4-1BBL and CD40L the hybridisation signals were low. Obviously, the quantification of low abundance transcripts will be vulnerable to variations in background levels and sensitivity, in contrast to the more abundant transcripts.

Because our goal in the cDNA array experiments was to identify genes that were markedly differentially expressed in PBMC from CHF patients and controls, pooled mRNA from the two groups of individuals was used. This procedure has both advantages and obvious disadvantages. On the one hand, analysis of pooled mRNA will reflect the mean in the two populations, reducing the signal noise introduced by individual patient variation, being an accurate way to screen for markedly differentially expressed genes in a study population. On the other hand, the use of pooled mRNA can result in loss of interesting variation in gene expression and extreme variations may also affect the mean leading to false positives, particularly for low abundance genes.

4.3 Potential pathological implications
Although the biological role of the TNF superfamily ligands is still unclear, some of their known effects may be of interest in the context of CHF. Notably, receptors for APRIL, FasL, LIGHT, TNF{alpha} and TRAIL have been reported to be expressed in the heart [20–27], and it is therefore not inconceivable that ligand–receptor interactions involving these upregulated TNF superfamily ligands, either soluble or bound to infiltrating leukocytes may represent potential pathogenic pathways in the progression of myocardial failure. For example, abnormal apoptosis is recognised as a potential pathogenic factor in the progression of myocardial failure [28], and known death-inducing ligands such as TNF{alpha}, FasL, TRAIL and LIGHT [29], that all were upregulated in CHF patients in the present study, may well be involved in this process. However, recent reports suggest that myocardial overexpression of some of these ligands (i.e. TNF{alpha} and FasL) promotes inflammation, hypertrophy and ventricular dilatation with fibrosis, rather than apoptosis [30,31]. Additionally, TRAIL was very recently found to induce collagen synthesis in lung fibroblasts [32]. Hence, while the apoptotic effects of TNF superfamily ligands should be further examined, these mediators may also induce other events involved in the remodelling process during CHF. Finally, we also found upregulation of the potent B cell activators APRIL and CD27L in CHF, and although the implications of this finding are unknown, their potential pathogenic role in the development of autoantibodies against cardiac structures [33], should be investigated. Thus, if enhanced expression of several ligands in the TNF superfamily, as demonstrated in the present study, also exists in PBMC infiltrating the failing myocardium, these cytokines may contribute to the development of myocardial failure. This may in turn lead to further activation of leukocytes within the myocardial circulation, representing a vicious circle in the pathogenesis of CHF.

4.4 Conclusion
Several studies have focused on the possible pathogenic role of TNF{alpha} in human heart failure and targeted therapy against this molecule is ongoing. However, anti-TNF therapy may have limitations. Thus, a recent animal study showed that while soluble TNF-R decreased plasma cytokine levels, there was no reduction in IL-6 within the myocardium [34]. Moreover, the two outcome studies of etanercept, a recombinant chimeric soluble TNF-R type 2, RENAISSANCE and RECOVER, were recently stopped due to lack of evidence of beneficial effects [35]. Future research in this area will therefore have to more precisely identify the most important ‘actors’ in the immunopathogenesis of CHF. Despite several limitations such as lack of cytokine data at the protein level and functional studies, the present study identifies several genes in the cytokine network that should be further investigated as potential pathogenic mediators in CHF. In particular, while not necessarily the initial event or the primary cause of CHF, the enhanced expression of ligands in the TNF superfamily may reflect a potential pathogenic role of these cytokines in the progression of this disorder.

Time for primary review 22 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
We thank Bodil Lunden for excellent technical assistance. This study was supported by the Norwegian Council on Cardiovascular Disease and MSD-Cardiovascular Research Centre, Rikshospitalet University Hospital.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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