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European Journal of Heart Failure 2005 7(6):984-990; doi:10.1016/j.ejheart.2005.05.013
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© 2005 European Society of Cardiology

Activation of the NF-{kappa}B system in peripheral blood leukocytes from patients with chronic heart failure

Ewa A. Jankowskaa,*, Stephan von Haehlingb, Anna Czarnyc, Ewa Zaczynskac, Agnieszka Kusa, Stefan D. Ankerb,d, Waldemar Banasiaka and Piotr Ponikowskia

a Cardiology Department, Military Hospital ul. Weigla 5, 50-981 Wroclaw, Poland
b Clinical Cardiology, National Heart and Lung Institute Imperial College, London, United Kingdom
c Institute of Immunology and Experimental Therapy, Polish Academy of Sciences Wroclaw, Poland
d Division of Applied Cachexia Research, Department of Cardiology Charite, Berlin, Germany

* Corresponding author. Tel./fax: +48 71 7660 250. E-mail address: Ewa.Jankowska{at}antro.pan.wroc.pl


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Disscusion
 References
 
Aim: To evaluate the activation of transcriptional nuclear factor kappa-B (NF-{kappa}B) in peripheral blood leukocytes (PBL) from patients with chronic heart failure (CHF). In vitro experiments were used to elucidate the role of lipopolysaccharide (LPS) as a stimulus for the NF-{kappa}B system in PBL.

Methods and results: We examined 46 CHF patients (age: 62±1 years, LVEF: 31±1%, NYHA class: 2.7±0.1), 11 coronary artery disease (CAD) patients without CHF, and 13 healthy young subjects. The immunocytochemical localisation of NF-{kappa}B in PBL was assessed using a polyclonal rabbit IgG anti-c-Rel-subunit antibody. NF-{kappa}B activation was expressed as the percentage of PBL nuclei stained positively for c-Rel (NF-{kappa}B(+)cell). PBL from healthy controls were exposed in vitro to the following concentrations of LPS from Escherichia coli (strain O111:B4): 0.1, 10 and 5000 ng/mL. CHF patients demonstrated the highest NF-{kappa}B activation in PBL (NF-{kappa}B(+)cells [%]: 37.1±1.5) as compared to CAD patients (29.1±3.0%) and controls (12.6±1.5%) (all p<0.05). There were three main clinical determinants of NF-{kappa}B activation in PBL from CHF patients: peak oxygen consumption (r=0.53, p=0.025), presence of peripheral oedema (r=0.37, p<0.05) and serum C-reactive protein (r=0.40, p=0.02). In PBL from healthy subjects, LPS at all concentrations increased NF-{kappa}B activity towards the pattern detected in CHF.

Conclusions: The NF-{kappa}B system is highly overactive in PBL from CHF patients. LPS at low concentrations in peripheral blood may be involved in NF-{kappa}B activation in PBL, and is a potential target for future therapeutic applications.

Key Words: Nuclear factor kappa-B • Immune activation • Chronic heart failure

Received October 25, 2004; Revised November 25, 2004; Accepted May 16, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Disscusion
 References
 
There is increasing evidence to support the role of immune mechanisms in the pathogenesis and progression of chronic heart failure (CHF) [1–3]. The overexpression of proinflammatory cytokines, elevated levels of chemokines, adhesion molecules and autoantibodies, and abnormal regulation of nitric oxide production, are all components of immune dysfunction, and contribute to the central and peripheral manifestations of the disease [4–6]. Despite recognition of the relevance of immune activation in CHF, its underlying mechanisms remain largely unknown. Only recently has attention been directed towards a potential role of transcriptional nuclear factor kappa-B (NF-{kappa}B), a central mediator of immune response [7–9].

The NF-{kappa}B family of pleiotropic transcriptional factors controls the transcription of a series of genes involved in innate and adaptive immune response [8–10]. The NF-{kappa}B system can be activated through receptor-mediated pathways with the involvement of many ligands, such as cytokines, mitogens, and lipopolysaccharide (LPS) [7,8,11].

There are only a few studies showing augmented activation of the NF-{kappa}B system with accompanying cytokine activation in the failing myocardium [12,13] and in the skeletal muscle and increased expression of inducible nitric oxide synthase, in subjects with CHF [14]. It is prudent to expect overactivation of the NF-{kappa}B system in immunocompetent blood cells, which may be partially responsible for their abnormal function, a phenomenon well described in CHF [15–18].

The aim of this study was therefore to evaluate the pattern of activation of the NF-{kappa}B system in PBL from CHF patients according to the severity and aetiology of CHF, and to relate it to clinical status, exercise intolerance and inflammatory status. Additionally, in a series of in vitro experiments, the role of LPS as a stimulus for the NF-{kappa}B system in PBL was evaluated. There is some evidence that in CHF patients, elevated LPS levels in peripheral blood can be a strong immune activator, through action on circulating immunocompetent cells [15,19,20], with downstream activation of the NF-{kappa}B system as a signal transducer of immune response to LPS.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Disscusion
 References
 
2.1. Study population
This prospective study recruited patients who were admitted to our Cardiology Department or who attended the outpatient clinic, as follows: i) 46 patients with a documented history of CHF of at least 6 months duration, with impaired left ventricular systolic function (left ventricular ejection fraction (LVEF) ≤ 45%); ii) 11 patients with coronary artery disease (CAD) confirmed by coronary angiography, with preserved LV function (LVEF > 50%), and without symptoms of CHF; and iii) 13 healthy young subjects (mean age 29 years). Patients were included irrespective of their underlying CHF aetiology. Ischaemic aetiology was confirmed by coronary angiography. Non-ischaemic aetiology was established only when ischaemic aetiology was excluded by normal coronary angiogram, relevant valvular pathologies were excluded by echocardiography. None of the patients had experienced an acute coronary event and/or cardiac revascularisation within the previous 6 months, and none of the subjects had any signs of acute/chronic infection, autoimmune disease, allergy or cancer. Subjects taking non-steroidal anti-inflammatory drugs or cortisol were excluded. The clinical characteristics of the study population are presented in Table 1.


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  		Table 1 Clinical characteristics of the study population

 
The study protocol was approved by the local Ethics Committee. All subjects gave written informed consent. The study conforms with the principles outlined in the Declaration of Helsinki.

2.2. Isolation of peripheral blood leukocytes
Venous blood samples were taken in the morning following an overnight fast, and after a supine rest of at least 15 min. PBL were isolated from heparinised whole blood using the gradient centrifugation method (Gradisol, Aqua Medica, Poznan, Poland, 1.115 g/mL, 20 min at 2000 rpm).

2.3. Immunocytochemical staining for NF-{kappa}B
A well established immunocytochemical method was used for the assessment of activation of the NF-{kappa}B system in PBL [21–23]. PBL were placed on poly-L-lysine-coated microscope slides using cytocentrifugation (Cytospin 4, Termo Shandon, Cheshire, United Kingdom) (5 min at 500 rpm). PBL were fixed at room temperature using 4% paraformaldehyde solution and air dried. After washing in distilled water, endogenous peroxidase activity was blocked by incubating slides in 3% hydrogen peroxide solution in methanol for 5 min, and washed in 10 mM phosphate buffered saline (PBS, pH=7.5). Cytospin preparations were treated with universal blocking serum (Novocastain Super ABC Kit, Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) for 20 min at room temperature. Afterwards, PBL were incubated at room temperature for another 60 min in a wet chamber with a polyclonal rabbit anti-NF-{kappa}B IgG antibody (c-Rel subunit) (Chemicon International Inc., Temecula, CA, USA). After washing in PBS, cytospin preparations were incubated with a biotinylated secondary anti-rabbit antibody (Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) at room temperature for 30 min. This was followed by washing in PBS and an application of peroxidase-conjugated avidin (Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom) in a wet chamber at room temperature for 30 min. After washing in PBS, chromogen fast diaminobenzidine (DAB) was used for 2–10 min (Liquid DAB Substrate Kit for Peroxidase, Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom). Subsequently, preparations were counterstained in hematoxylin (Mayer's, DakoCytomation, Glostrup, Denmark) and finally washed with distilled water and mounted on medium (Novomount Permanent Slide Mounting Medium, Novocastra Laboratories Ltd., Newcastle upon Tyne, United Kingdom). Further, 2 independent blinded observers quantified cytoplasmatic or nuclear staining in PBL (100 PBL were counted on each slide). PBL expressing c-Rel in the nucleus were labelled as NF-{kappa}B(+)cells. Activation of the NF-{kappa}B system in PBL was expressed as the percentage of NF-{kappa}B(+)cells from all quantified PBL. The inter-observer variability coefficient for the percentage of NF-{kappa}B(+)cells was below 6%.

2.4. Stimulation of PBL with LPS
In this in vitro experiment, we evaluated the effect of stimulation with different concentrations of LPS on activation of the NF-{kappa}B system in PBL. PBL from healthy subjects were washed twice with RPMI 1640 (Institute of Immunology and Experimental Therapy, Wroclaw, Poland) supplemented with 2% heat-inactivated calf serum, 2 mM glutamine, 100 U penicillin and 100 µg streptomycin per milliliter (Sigma-Aldrich, Fine Chemicals, St. Luis, USA). Afterwards, PBL were resuspended in RPMI 1640 at a concentration of 1x106 cells/mL and cultured in a humidified atmosphere (37 °C, 5% carbon dioxide) for 24 h in 24-well culture plates (Costar, Cambridge, MA, USA) without the addition of LPS (control) or with the addition of LPS from E. coli (strain O111:B4) at the following concentrations: 0.1 ng/mL, 10 ng/mL, and 5 µg/mL (Sigma-Aldrich, Fine Chemicals, St. Luis, USA). This range of concentrations was selected to represent the low concentrations likely to mimic decompensated CHF up to concentrations resembling those found in sepsis [24,25]. The activation of the NF-{kappa}B system in stimulated PBL was assessed using the immunocytochemical staining method described previously.

2.5. Cardiopulmonary exercise testing
Cardiopulmonary exercise testing was performed to assess exercise capacity in CHF patients, with the following parameters being measured: peak oxygen consumption (peak VO2, mL/min/kg) and ventilatory response to exercise (VE-VCO2 slope, calculated as the regression slope relating minute ventilation [VE] to carbon dioxide output [CO2] during the whole exercise) [26,27]. Both parameters have strong and independent prognostic value in CHF patients [26,27].

2.6. Assessment of serum C-reactive protein level
Serum level of C-reactive protein (CRP), an unspecific inflammatory marker, was assessed using a high sensitivity method (immunonephelometry, Dade Behring Marburg GmbH, Germany).

2.7. Statistical analysis
Results were reported as means±standard errors. The inter-group differences were tested using ANOVA with Fisher's post hoc test, unpaired Student's t test or {chi}2 test, as appropriate. Univariate and multiple regression analyses were applied to assess factors predicting the activation of NF-{kappa}B system in PBL. P values<0.05 were considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Disscusion
 References
 
3.1. Activation pattern of the NF-{kappa}B system in PBL from CHF patients, CAD patients without CHF and healthy subjects
Patients with CHF and patients with CAD without CHF had increased activation of the NF-{kappa}B system in PBL compared to healthy controls. The mean percentage of NF-{kappa}B(+)PBL in CHF patients (37.1±1.5%) and in CAD patients without CHF (29.1±3.0%) was significantly greater than in healthy controls (12.6±1.5%) (p<0.0001 for both comparisons). A direct comparison, between the two groups of patients showed a significantly higher activation of the NF-{kappa}B system in PBL from CHF patients (p=0.01 vs. patients without CHF) (data from individual patients are presented in Fig. 1).


Figure 1
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  		Fig. 1 Activation of the NF-{kappa}B system in peripheral blood leukocytes from CHF patients versus CAD patients versus healthy subjects.

 
3.2. Clinical determinants of NF-{kappa}B system activation in PBL in CHF patients
3.2.1. Age
The mean age of our CHF population was 62±1 years, ranging from 42–81 years. Age did not correlate with the percentage of NF-{kappa}B(+)PBL (r=– 0.24, p=0.1).

3.2.2. Aetiology
CHF aetiology did not significantly influence activation of the NF-{kappa}B system in PBL. The mean percentage of NF-{kappa}B(+)PBL in CHF of ischaemic (n=38) and non-ischaemic (n=8) aetiology was 36.0±1.6% vs. 42.0±2.8%, respectively (p>0.1).

3.2.3. Indices of left ventricular function
CHF patients had severely impaired systolic function as evidenced by decreased LVEF (mean: 30±1%, range: 14–44%) and dilated left ventricular cavity as evidenced by enlarged left ventricular end diastolic dimension (LVEDD: 67±1 mm, range: 53–88 mm). Neither LVEF or LVEDD correlated with the percentage of NF-{kappa}B(+)PBL (r<0.25, p>0.1 for both correlations).

3.2.4. Severity of exercise intolerance
Patients with advanced CHF symptoms, classified as being in New York Heart Association (NYHA) class III–IV (n=26), demonstrated enhanced activation of the NF-{kappa}B system in PBL when compared with less symptomatic CHF patients in NYHA class I–II (n=20) (NF-{kappa}B(+)cells: 40.2±1.9% vs. 34.4±1.9%, p<0.05).

In 18 CHF patients (16 men, age: 61±2 years, NYHA class: 2.7±0.2) a symptom-limited cardiopulmonary exercise test was performed. The patients demonstrated a markedly impaired exercise capacity as evidenced by reduced peak VO2 (mean: 16.1±0.9 mL/min/kg, range 10.4–24.6 mL/min/kg) and augmented ventilatory response to exercise as evidenced by elevated VE-VCO2 slope (mean: 33.7±1.3, range 21–52.5). There was an inverse relationship between NF-{kappa}B activation in PBL and peak VO2 (r=– 0.53, p=0.025) (Fig. 2), and a trend towards higher NF-{kappa}B activation in PBL from CHF patients with excessive exercise ventilation (VE-VCO2 slope) (r=0.42, p=0.08).


Figure 2
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  		Fig. 2 Relationship between NF-{kappa}B activation in peripheral blood leukocytes and peak oxygen consumption (peak VO2) in CHF patients.

 
3.2.5. Inflammatory status and peripheral oedema
In the CHF patients, serum hsCRP level correlated with NF-{kappa}B activation in PBL (r=0.40, p=0.02) (Fig. 3).


Figure 3
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  		Fig. 3 Relationship between NF-{kappa}B activation in peripheral blood leukocytes and serum hsCRP levels in CHF patients.

 
Eighteen (39%) CHF patients had signs of peripheral oedema. The oedematous patients had more advanced symptoms (NYHA class: 3.1±0.2 vs. 2.4±0.1 for. non-oedematous patients, p<0.05), and a strong trend towards higher hsCRP levels (9.1±1.5 mg/L vs. 5.8±1.1 mg/L, oedematous vs. non-oedematous patients, respectively, p=0.07), there were no differences in other clinical parameters when compared with non-oedematous patients. Patients with peripheral oedema had a more active NF-{kappa}B system in PBL (NF-{kappa}B(+)cells: 41.6±2.2% vs. 34.2±1.8%, oedematous vs. non-oedematous CHF patients, respectively, p=0.01).

3.2.6. Multivariate analysis
The relationship between NF-{kappa}B activation in PBL, and the presence of peripheral oedema and elevated hsCRP levels remained statistically significant when controlled for NYHA class (p<0.05 for both analyses).

3.3. The effect of clinical stabilisation on NF-{kappa}B activation in PBL
In 5 CHF patients (all men, with ischaemic aetiology, mean age: 52±3 years, mean LVEF: 20±2%) acutely admitted to hospital with signs of clinical decompensation (pulmonary oedema accompanied by peripheral oedema and congestive hepatomegaly), activation of the NF-{kappa}B system in PBL was assessed at baseline and after clinical stabilisation (on the day of hospital discharge, after 9±2 days). In all cases, clinical stabilisation resulted in a significant decrease in activation of the NF-{kappa}B system in PBL (38.2±4.9% vs. 26.8±4.7%, p=0.02) (Fig. 4), which was accompanied by a significant decrease in serum levels of hsCRP (18.3±4.6 vs. 7.3±2.8 mg/L, p=0.02).


Figure 4
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  		Fig. 4 Changes in NF-{kappa}B activation in PBL in 5 CHF patients during haemodynamic decompensation and after clinical recovery.

 
3.4. The effects of in vitro LPS stimulation of PBL from healthy subjects on the activation of the NF-{kappa}B system
In PBL from healthy subjects, LPS stimulation at all tested concentrations resulted in an increase in NF-{kappa}B activation in PBL as evidenced by the translocation of c-Rel into the nucleus (at 0.1 ng/mL: 19.0% vs. 43.5%; at 10 ng/mL: 19.0% vs. 43.5%, at 5 µg/mL: 13.8% vs. 42.5%; the percentage of NF-{kappa}B(+)PBL after the control stimulation without LPS vs. after the stimulation with LPS at different concentrations, respectively) (Fig. 5). Following LPS stimulation of PBL from healthy controls, the pattern of PBL c-Rel staining was similar to that detected in CHF patients.


Figure 5
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  		Fig. 5 Changes in the NF-{kappa}B activation in PBL from 6 healthy subjects exposed in vitro to LPS at different concentrations.

 

   4. Disscusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Disscusion
References
 
In this study, we have demonstrated augmented activation of the NF-{kappa}B system in peripheral blood leukocytes in patients with CHF. We have shown that there are three main clinical determinants of NF-{kappa}B activation in PBL from CHF patients, namely: 1) the severity of exercise intolerance, 2) the presence of peripheral oedema, and 3) the degree of low-grade inflammation (as evidenced by serum hsCRP level). Moreover, data from healthy subjects indicate that stimulation with various concentrations of LPS results in a significant increase in NF-{kappa}B activity, which appears to resemble the pattern of activation detected in CHF patients.

In this study, we have observed that activation of the NF-{kappa}B system in PBL is augmented in CAD patients with preserved left ventricular function and without symptoms of heart failure. This confirms the results of earlier studies, which have shown the activation of immunocompetent cells in CAD and their direct involvement in inflammatory pro-atherogenic processes [28,29]. Interestingly, patients with CHF demonstrated a significantly higher activation of the NF-{kappa}B system in PBL when compared not only to young healthy subjects but also to CAD patients without CHF. In CHF patients, the percentage of PBL expressing c-Rel within the nucleus was on average 20% higher than in patients with CAD but without CHF. Only recently, Frantz et al. demonstrated increased activation of the NF-{kappa}B system in peripheral blood leukocytes from stable CHF patients [30].

Although CHF is recognized as a state of chronic immune activation, surprisingly few data are available on the function or dysfunction of immunocompetent cells in this disease. Several hypotheses have been suggested to explain the origin of immune activation [31], but the evidence is not yet conclusive. Augmented activation of NF-{kappa}B in PBL therefore constitutes an important part of our understanding of CHF pathophysiology. Indeed, this finding appears to underscore the endotoxin hypothesis, which suggests that bacterial translocation through the oedematous gut wall with subsequent release of endotoxin may pose the relevant stimulus to trigger proinflammatory responses in CHF [20].

Recent studies have emphasized the potential role of the NF-{kappa}B system as a central mediator of the immune response. Indeed, over activation of NF-{kappa}B in skeletal muscle from CHF patients [14] as well as in the failing myocardium has recently been reported [9]. This is in keeping with other recent findings which suggest that the transcription of genes that regulate the expression of members of the tumour necrosis factor (TNF) superfamily (e.g. APRIL, TRAIL, LIGHT, CD27-ligand, Fas-ligand, TNF-{alpha}) is increased in immunocompetent cells in CHF patients [17]. The authors hypothesize that the NF-{kappa}B system is a final common pathway that controls the transcription of these genes. These findings are also in line with reports of peripheral blood mononuclear cells from CHF patients producing more TNF-{alpha} when stimulated with LPS in vitro than cells from patients without CHF [16,18,32,33].

In our study the enhanced NF-{kappa}B activation in PBL was accompanied by the more intense inflammatory response, as evidenced by elevated serum hsCRP level, a simple and unspecific inflammatory marker.

NF-{kappa}B antagonism may provide a new therapeutic strategy to counterbalance immune activation in CHF. Indeed, direct antagonism of TNF-{alpha} with etanercept (a TNF-{alpha} receptor fusion protein [34]) or infliximab (a TNF-{alpha} neutralizing antibody [35]) was of no benefit in patients with CHF. Although there has been some debate about the design of these studies [36], it has recently been speculated that a broader approach than targeting single cytokines to treat immune dysfunction could be of benefit [31,37]. Our findings indicate that the NF-{kappa}B system could provide a target for such intervention. There are data suggesting the beneficial anti-inflammatory effects of inhibition of NF-{kappa}B signaling for example collagen-induced arthritis in mice [38] and type 2 diabetes mellitus [39], to name but a few.

We found that enhanced NF-{kappa}B activation in PBL correlated with CHF severity, expressed as higher NYHA class and reduced peak VO2. We did not observe any correlation between indices of left ventricular function and NF-{kappa}B activation in CHF patients. Furthermore, activation of this system appeared to be irrespective of underlying CHF aetiology.

We have also shown that the presence of peripheral oedema was related to higher NF-{kappa}B activation in PBL. It is well established that the immune response is markedly augmented in oedematous CHF patients, who demonstrate both excessive plasma endotoxin activity and elevated levels of proinflammatory cytokines [19]. This is confirmed by our study. It may well be that LPS present in peripheral blood stimulates the NF-{kappa}B system in PBL and simultaneously triggers a cascade of low-grade proinflammatory reactions.

LPS is a very strong stimulus known to activate the NF-{kappa}B system in immunocompetent cells in a receptor-mediated pathway [7,8,11]. LPS stimulates TNF-{alpha} production by peripheral blood mononuclear cells in patients with and without CHF in vitro [15,33]. We observed that in vitro LPS stimulation of PBL from healthy subjects resulted in significant up-regulation of NF-{kappa}B activity. Interestingly, the pattern of NF-{kappa}B activation in PBL from healthy subjects after LPS stimulation was similar to that observed in PBL from CHF patients without LPS stimulation. This effect was present over a wide range of LPS concentrations. We have confirmed the results of other authors, that high LPS concentrations (similar to those found in vivo during endotoxaemic shock) are able to activate the NF-{kappa}B system in PBL [11,40,41]. But, what seems to have an intriguing clinical significance in the context of CHF pathophysiology, is the finding that low LPS concentrations (similar to those found in vivo in CHF patients) stimulate the NF-{kappa}B system in PBL. This suggests that an analogous mechanism may occur in vivo in decompensated CHF patients, when PBL are exposed to and stimulated by low concentration of LPS present in peripheral blood during decompensation.

Our study has some limitations. We studied a control group of healthy subjects who were much younger than the patients with and without CHF. However, healthy subjects of younger age were deliberately chosen to ensure they had no significant medical history, in particular underlying atherosclerosis, hypertension, other cardiovascular diseases, chronic/acute infections or any immune disturbances. Additionally, since we did not measure proinflammatory cytokines and LPS concentrations in peripheral blood, we are not able to link an overactivated NF-{kappa}B system in PBL with elevated levels of cytokines, or to confirm our hypothesis based on in vitro experiments on the potential role of LPS in the activation of NF-{kappa}B system in PBL in patients with CHF.

In conclusion, we have confirmed the enhanced activation of the NF-{kappa}B system in immunocompetent cells in CHF patients. The overactive NF-{kappa}B system in PBL is linked to the presence of peripheral oedema and low-grade inflammation. Low LPS concentrations are able to increase NF-{kappa}B activity in PBL from healthy subjects, with a pattern of activation similar to that detected in CHF. Broad therapeutic approaches, that target more than single cytokines could be beneficial in treating CHF. Therapies inhibiting the signal NF-{kappa}B transduction could be such an approach.


   Acknowledgements
 
This study was financially supported by National Research Committee in Poland (Grant No 2P05B 02426). EAJ was supported by British Council (British–Polish Young Scientists Programme). SvH was supported by the German Heart Foundation, Frankfurt, Germany.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Disscusion
 References
 

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