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European Journal of Heart Failure 2005 7(5):865-868; doi:10.1016/j.ejheart.2004.09.014
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© 2004 European Society of Cardiology

Cellular endotoxin desensitization in patients with severe chronic heart failure

Rakesh Sharmaa,1, Aidan P. Bolgera,1, Mathias Rauchhausa,b,1, Stephan von Haehlinga,1, Wolfram Doehnera,b, Ian M. Adcockc, Peter J. Barnesc, Philip A. Poole-Wilsona, Hans-Dieter Volkd, Andrew J.S. Coatsa, Sam Limc and Stefan D. Ankera,b,*

a Clinical Cardiology, NHLI Imperial College School of Medicine, London, UK
b Division of Applied Cachexia Research Department of Cardiology, Charité Medical School, Berlin, Germany
c Thoracic Medicine, NHLI Imperial College School of Medicine, London, UK
d Department of Clinical Immunology Charité Campus Mitte, Berlin, Germany

* Corresponding author. Department of Clinical Cardiology, National Heart and Lung Institute, Dovehouse Street, London, SW3 6LY, UK. Tel.: +44 20 7351 8203; fax: +44 20 7351 8733. E-mial address: s.anker{at}imperial.ac.uk


   Abstract
Top
 Notes
 Abstract
1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusion
 References
 
Background: Increased levels of bacterial lipopolysaccharide (LPS) have been demonstrated in chronic heart failure (CHF). LPS can induce cellular desensitization, with specific down-regulation of LPS-mediated cellular tumor necrosis factor (TNF-{alpha}) production which does not affect other cytokine parameters. It is not known if LPS desensitization occurs in CHF.

Methods and results: Mononuclear cells from 24 CHF patients (mean age 70±2 years, age range 58 to 78 years, NYHA class 3.0±0.2) and 11 healthy controls (mean age 53±3 years, age range 39 to 75 years) were separated from venous blood and cultured for 24 h with LPS (E. coli, 0–10 ng/mL). Culture supernatants were tested for TNF-{alpha} and interleukin-1 receptor antagonist (IL-1RA). Patients were subgrouped into mild (n=10), moderate (n=5), and severe (n=9) CHF. Independently of age, mononuclear cells from patients with severe heart failure produced less TNF-{alpha} than controls (p<0.05) and patients with mild (p<0.001) or moderate CHF (p<0.05). IL-1RA release was higher for CHF patients as a group, compared with controls (p<0.05). There was no significant difference in IL-1RA release between CHF patient subgroups.

Conclusions: Mononuclear cells from patients with severe heart failure produce significantly less TNF-{alpha} than healthy controls or patients with mild to moderate disease. Production of IL-1RA is not affected. This resembles a picture indicative of LPS desensitization occurring in patients with severe CHF.

Key Words: Heart failure • Lipopolysaccharide • Cytokines

Received January 5, 2004; Revised June 8, 2004; Accepted September 20, 2004


   1. Background
Top
 Notes
 Abstract
 1. Background
2. Aims
 3. Methods
 4. Results
 5. Conclusion
 References
 
Proinflammatory cytokines play a crucial role in patients with chronic heart failure (CHF) [1]. The precise stimulus for immune activation is unknown, but endotoxin (lipopolysaccharide, LPS) could be an important trigger for cytokine release [2,3]. Endotoxin can induce a transient state of hyporesponsiveness to subsequent challenges, a phenomenon known as LPS desensitization. Restimulation of desensitized monocytes results in reduced tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-1{alpha}, and interleukin-6 production. Synthesis of interleukin-1 receptor antagonist (IL-1RA) is maintained or increased [4]. The latter phenomenon is a specific feature of LPS desensitization. It is not known whether LPS desensitization occurs in CHF.


   2. Aims
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 Abstract
 1. Background
 2. Aims
3. Methods
 4. Results
 5. Conclusion
 References
 
Patients with severe CHF are likely to have had recent episodes of decompensation (and therefore of endotoxin exposure). We aimed to test the hypothesis that LPS desensitization is present in this group of patients.


   3. Methods
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 Abstract
 1. Background
 2. Aims
 3. Methods
4. Results
 5. Conclusion
 References
 
3.1. Study population
The cytokine generation capacities of isolated peripheral blood mononuclear cells (PBMC) from 24 stable patients with CHF and 11 healthy control subjects was assessed. Patients were subdivided into mild (NYHA class II, 10 patients), moderate (NYHA class III, 5 patients), or severe (NYHA class IV with edematous decompensation and/or cardiac cachexia, 9 patients) heart failure. Patients were on stable medication (standard medication for CHF) for at least 3 months. The local ethics committee approved the study.

3.2. Cytokine and endotoxin analysis
Citrated venous blood samples were used to assess the following cytokine parameters using enzyme-linked immunosorbent assays (ELISA): TNF-{alpha}, soluble TNF receptor 1 (sTNFR-1), IL-1RA (all from R&D Systems, Minneapolis, USA). Plasma LPS levels were assessed as described previously using a Limulus Amebocyte Lysate assay (Bio Whittaker, Walkerswill, USA) [3,5].

3.3. Cell separation and stimulation assays
PBMC were separated using Ficoll gradient centrifugation (Amersham Pharmacia Biotech AB, Uppsala, Sweden) as previously described [6,7] and resuspended at a concentration of 1x106/mL. One millilitre aliquots were seeded in 12-well plates. E. coli-derived LPS (serotype 0111:B4, Sigma-Aldrich) was added at 10 different final concentrations: 0, 0.001, 0.01, 0.03, 0.06, 0.1, 0.3, 0.6, 1, and 10 ng/mL. Supernatants were harvested following 24 h incubation in a humidified atmosphere. The supernatants were frozen immediately at –80 °C for later analysis.

3.4. Statistics
Results are reported as mean±S.E.M. Unpaired Student's t-tests, ANOVA with Fisher's post hoc test, ANOVA for repeated measures, MANOVA, and simple regression analysis were used as appropriate. The results for TNF-{alpha} and IL-1RA were square-root transformed to achieve normal distribution. A p-value <0.05 was considered significant.


   4. Results
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 1. Background
 2. Aims
 3. Methods
 4. Results
5. Conclusion
 References
 
We detected significant differences in LPS-stimulated TNF-{alpha} production between CHF patients with mild, moderate, or severe disease (Fig. 1). Mononuclear cells from patients with mild CHF released more TNF-{alpha} in response to LPS than controls (all p<0.05), patients with moderate CHF (all p<0.01, except at 10 ng/mL LPS), or severe CHF (all p<0.001). The mononuclear cells from patients with severe CHF produced less TNF-{alpha} than controls (p<0.05 for 0.6 ng/mL to 1 ng/mL LPS), and moderate CHF patients (p<0.05 for 1 to 10 ng/mL LPS; Fig. 1).


Figure 1
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  		Fig. 1 TNF-{alpha} release from LPS-stimulated mononuclear cells isolated from healthy controls and heart failure patients subdivided according to clinical severity. *: vs. control subjects; {varphi}: vs. patients with moderate CHF; §: vs. patients with severe CHF. One symbol: p<0.05; two symbols: p<0.01; three symbols: p<0.001; four symbols: p<0.0001.

 
There was a dose-dependent relationship for LPS and the amount of TNF-{alpha} release by mononuclear cells from controls and patients (Fig. 1). No such relationship was found for IL-1RA (Fig. 2). The release of IL-1RA from CHF patients was greater than from controls for all LPS concentrations (p<0.05). However, there was no significant difference in IL-1RA release between patient subgroups (ANOVA: p>0.1 at all concentrations). The relationship between CHF severity and TNF-{alpha} production compared to controls was independent of age at all LPS concentrations (p>0.2). There was no correlation between serum creatinine and basal or LPS-stimulated TNF-{alpha} production (all p>0.1; Table 1).


Figure 2
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  		Fig. 2 IL-1RA release from LPS-stimulated mononuclear cells isolated from healthy controls and heart failure patients subdivided according to clinical severity. *: vs. control subjects. One symbol: p<0.05; two symbols: p<0.01; three symbols: p<0.001; four symbols: p<0.0001.

 


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  		Table 1 Clinical characteristics of study participants

 
Circulating TNF-{alpha} and sTNFR-1 levels were significantly elevated in patients with severe CHF compared to controls (both p<0.01; Table 2). Serum cortisol levels were raised in patients with CHF compared to controls (441 vs. 258 nmol/L, p=0.003). However, no significant relationship was found to exist between serum cortisol levels and PBMC LPS responsiveness (all r<0.3, p>0.1).


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  		Table 2 Plasma cytokine and endotoxin levels in study subjects

 

   5. Conclusion
Top
 Notes
 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusion
References
 
Mononuclear cells isolated from patients with severe CHF produce significantly less TNF-{alpha} than healthy controls or patients with mild to moderate disease. Production of IL-1RA is not affected. Previous studies have attributed these features as characteristic for LPS desensitization [4,8]. We suggest that LPS desensitization is present in patients with severe CHF, who likely had a recent episode of pathophysiologically relevant exposure to LPS. However, due to our small patient number, our data have to be seen as preliminary.

The precise mechanism for LPS desensitization is not known. In this study, cortisol levels were raised in patients with CHF as compared to the control group, but cortisol levels and LPS-mediated TNF production did not correlate (all p>0.1). It therefore appears unlikely that cortisol plays an important role in the regulation of cytokine production following LPS exposure in CHF.

To date, few studies have investigated LPS-stimulated cellular cytokine release in CHF. Two independent studies found increased TNF production in LPS-stimulated whole blood from CHF patients as compared to healthy controls [9,10]. Zhao and Xu [11] found the TNF-{alpha} generation capacity of LPS-stimulated mononuclear cells to be higher in CHF patients as compared to controls. However, all previous studies used only one (high) concentration of LPS. These high concentrations are unlikely to mimic the in vivo setting. Studies should perhaps focus on the effect of low (pathophysiologically more relevant) concentrations of LPS.

There are physiological factors present in whole blood which may play a crucial role in the inflammatory response [12]. These factors are not taken into account when isolated mononuclear cells are being analysed. Serum lipoproteins may play an important role, and it has been suggested that lipoproteins (that when decreased predict poor prognosis in CHF [13]) could act in forming micells around LPS [14]. This is further highlighted by the results published in our paper on whole blood responsiveness to LPS in CHF in this issue of the journal [15].


   Acknowledgements
 
RS was supported by the Robert Luff Foundation. AB and the Department of Clinical Cardiology are supported by the British Heart Foundation. AJSC is supported by the Viscount Royston Trust Fund. SDA is supported by a Vondervell Fellowship and by a donation from Dr. Hubert Bailey. The Division of Applied Cachexia Research is supported by the Charité Medical School. SvH is supported by the German Heart Foundation, Frankfurt, Germany.


   Notes
Top
 Notes
Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusion
 References
 
1 Rakesh Sharma, Aidan P. Bolger, Mathias Rauchhaus and Stephan von Haehling contributed equally to the work in this manuscript. Back


   References
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 Abstract
 1. Background
 2. Aims
 3. Methods
 4. Results
 5. Conclusion
 References
 

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