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European Journal of Heart Failure 2006 8(4):366-372; doi:10.1016/j.ejheart.2005.10.010
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© 2006 European Society of Cardiology

The anti-CD14 antibody IC14 suppresses ex vivo endotoxin stimulated tumor necrosis factor-alpha in patients with chronic heart failure

Sabine Genth-Zotza,b,*, Stephan von Haehlinga,c, Aidan P. Bolgera, Paul R. Kalraa, Roland Wensela, Andrew J.S. Coatsa, Hans-Dieter Volkd and Stefan D. Ankera,c

a Clinical Cardiology, National Heart and Lung Institute, Imperial College School of Medicine London, UK
b Department of Medicine II, Johannes Gutenberg-University Mainz, Germany
c Division of Applied Cachexia Research, Department of Cardiology, Charité Medical School, Humboldt University Berlin, Germany
d Institute of Medical Immunology, Charité Medical School, Humboldt-University Berlin, Germany

* Corresponding author. Department of Medicine II, Johannes Gutenberg-University, Langenbeckstraße 1, 55131 Mainz, Germany. Tel.: +49 6131 173747; fax: +49 6131 176613. E-mail address: genth{at}2-med.klinik.uni-mainz.de


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Activation of the endotoxin (LPS) receptor, CD14, leads to tumor necrosis factor-alpha (TNF) production. Plasma LPS activity is elevated in patients with severe chronic heart failure (CHF). An anti-CD14 antibody, IC14, blocks TNF production in healthy volunteers. It is not known whether IC14 prevents TNF production in CHF patients.

Methods and results: Blood from 20 CHF patients (age 64±2.1 years, NYHA class 2.2±0.1, LVEF 27±3%, mean±SEM) was pre-incubated with 0.5, 1.0, 5.0, 10 and 50 µg/mL IC14 for 1 h followed by incubation with 1 or 10 ng/mL LPS for 6 h. Fourteen subjects served as controls (58±2.4 years). LPS-stimulated TNF release was 76% and 60% greater at 1 and 10 ng/mL LPS, respectively, in CHF patients versus controls (p=0.07 and p=0.008). IC14 at concentrations of 5.0, 10 and 50 µg/mL substantially reduced TNF production in response to stimulation with LPS (all p<0.05). CD14 receptor density was similar in patients and controls. In controls, but not in CHF patients, there was a positive correlation between CD14 receptor density and TNF production (r=0.61, p=0.03).

Conclusion: IC14 suppresses LPS-stimulated whole blood TNF production in patients with CHF and in normal subjects and therefore may represent a novel therapeutic strategy for CHF patients with systemic immune activation.

Key Words: Immunactivation • CHF • CD14 antibody • TNF inhibition

Received November 29, 2004; Revised August 2, 2005; Accepted October 13, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The aetiology of inflammatory immune activation in chronic heart failure (CHF) is at present not fully understood. It has been proposed that endotoxin may be an important stimulus through its action on circulating peripheral mononuclear cells [1]. Inflammatory cytokines are thought to contribute to both the central and peripheral manifestations of CHF [2]. Recent studies have shown that circulating levels of tumor necrosis factor-alpha (TNF), soluble TNF receptors 1 and 2 (sTNFR1, 2) and Interleukin-6 (IL-6) are strong prognostic markers in patients with CHF [3,4]. Immunomodulatory therapy for patients with CHF is therefore of considerable clinical interest [5,6].

CD14 is an important recognition receptor for lipopolysaccharide (LPS). On the surface of monocytes and granulocytes, CD14 exists as a glycosylphosphatidylinositol-linked protein, whilst in the serum it is found in its soluble form (sCD14). Recognition and signalling through CD14-dependent pathways requires three proteins: LPS binding protein, CD14 and Toll-like receptors [7]. The interaction of gram negative bacterial LPS and CD14 results in signal transduction through Toll-like receptor-4 (TLR4) leading to cellular activation, production of inflammatory cytokines (including TNF and IL-6), chemokines and activation of inducible nitric oxide synthase [8-10].

In CHF increased plasma levels of sCD14 are found in patients with marked immune activation [1]. One might, therefore, anticipate that blocking the CD14 receptor would inhibit the inflammatory response to LPS. Animal studies have shown that anti-CD14 antibodies prevent the deleterious systemic responses that occur in association with LPS administration [11,12]. Furthermore, Verbon et al. [13] showed that treatment with an anti-CD14 antibody (IC14) inhibits the LPS-induced clinical syndrome of fever, chills, myalgia and reduces cytokine release in healthy subjects. Chronic heart failure is a complex syndrome with multi-system involvement and it is frequently seen as a state of chronic inflammation. As such it cannot be assumed that the results of studies in health are readily translated into patients with this condition.

We sought to establish whether an anti-CD14 antibody reduces LPS-stimulated TNF and IL-6 production ex vivo in whole blood from patients with CHF. We also aimed to determine the relationship between TNF production in whole blood and CD14 receptor density.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Study population
We studied 20 patients with stable CHF (17 males, age 64±2.1 years, New York Heart Association class 2.2±0.1, peak VO2 21.7±1.7 ml/kg/min, LVEF 27±3%, mean±SEM) and 14 healthy control subjects (9 males) of similar age (58±2.4 years, p=ns). The patients were recruited from the Royal Brompton Hospital heart failure clinic with the diagnosis of CHF being based on appropriate clinical signs and symptoms, together with objective evidence of reduced left ventricular function (ejection fraction <40%). CHF was due to coronary artery disease in 16 and to idiopathic dilated cardiomyopathy in 4 patients, respectively. All patients were on ACE inhibitors and diuretics, 55% patients received beta blockers and 80% low dose aspirin. Seven patients had cardiac cachexia, defined as non-intentional weight loss of >7.5% over at least 6 months [14]. No changes in medication had occurred in the 30 days prior to the study. No patients had signs of congestion at the time of assessment.

The investigation conforms with the principals outlined in the Declaration of Helsinki and all patients gave their written informed consent.

2.2. Whole blood studies and stimulation assays
Blood samples were taken after semi-supine rest for at least 15 min. Citrated venous blood was collected from the antecubital vein and 1 mL aliquots were placed in Eppendorf tubes. IC14 (ICOS Corporation, Bothell, WA, USA, received as a gift), a recombinant chimeric (murine/human) monoclonal antibody (mAb) recognising human CD14 was reconstituted in 0.9% NaCl. In total 12 aliquots were taken from each patient.

An initial series of studies was performed to determine (i) baseline TNF production (no IC14, no LPS), (ii) the effects of IC14 on TNF production (IC14, no LPS) and (iii) the effects of LPS alone (no IC14, LPS). Prior to LPS stimulation, whole blood was incubated with a range of IC14 doses (0.5, 1.0, 5.0, 10 and 50 µg/mL) for 1 h in a humidified atmosphere (37 °C, 5% CO2). LPS (E. coli, serotype 0111:B4, Sigma, UK) was then added to achieve final concentrations of either 1 or 10 ng/mL, and this was followed by a further incubation period of 6 h. We found that the peak TNF response was achieved after a 6-h incubation period (data not shown). After incubation, the samples were centrifuged at 1500 rpm for 5 min. The supernatants were removed and stored at –80 °C until final assessment. Previous studies in healthy controls (n=4) had demonstrated that spiking whole blood with 1 ng/mL LPS resulted in an LPS recovery of 0.08±0.07 EU/mL, whilst 10 ng/mL LPS yielded 2.73±0.26 EU/mL.

2.3. Measurements of cytokines
The concentrations of TNF and IL-6 in the supernatant were measured by enzyme-linked immunosorbent assay (ELISA, Duo Set, R&D, Minneapolis, USA). The lower limits of detection were 15 and 4 pg/mL, respectively.

2.4. Endotoxin measurement
Endotoxin-free tubes (Endo Tube ET, Chromogenix AB, Sweden) were used to collect blood. Plasma levels of LPS were determined using the Limulus Amebocyte Lysate assay (QCL-1000 test kit, Bio Whittaker Inc, Walkerswill, USA), the lower limit of detection being 0.05 EU/mL).

2.5. Serum cytokines
Further blood samples were taken for biochemical and cytokine measurements. After immediate centrifugation, serum samples were stored at –80 °C until final analysis. Serum levels of TNF were determined using the Quantiglo Human chemiluminescent immunoassay (R&D, Minneapolis, USA, lower limit of detection 0.7 pg/mL). Soluble TNF receptor 2 (sTNFR2) and sCD14 were measured using colorimetric sandwich ELISA (Quantikine, R&D Minneapolis USA, lower limit of detection 7.8 pg/mL for sTNFR2 and 250 pg/mL for sCD14).

2.6. Quantification of TNF mRNA levels
Total RNA was isolated using a commercially available reagent (QIAamp®, Qiagen Ltd, UK) following the manufacturers instructions. Determination and quantification of TNF mRNA was performed by RT-PCR, as described elsewhere [15]. An internal RNA control housekeeping gene (hypoxanthine-guanine phosphoribosyltransferase, HRPT) was examined with cDNA probe.

2.7. Cell preparation and flow cytometric analysis
The CD14 receptor expression on circulating monocytes was quantified in EDTA anticoagulated blood. Whole blood (100 µL) was incubated with 5 µg/mL of a fluorescein isothiocyanate-labelled (FITC) anti-human CD14 non-conjugated mAb or a respective isotype control IgG2a (both from Sigma, Saint Louis, USA) at 4 °C for 30 min. After lysing the erythrocytes, samples were centrifuged at 1500 rpm for 5 min and the remaining cells were washed twice in PBS supplemented with 2% v/v FCS and 0.01% w/v sodium azide. To quantify CD14 cell surface expression in whole blood at least 30,000 cells were analysed for each test using a FACScan flow cytometerTM (Becton Dickinson, Mountain View, CA). After scatter based monocyte gating, CD14 expression was calculated based on the mean fluorescence intensity (MFI). On average 6.5±1.0% of the measured cells per sample were positive for CD14.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Study population
There were no significant differences between patients and controls in terms of age (64±2.1 vs. 58±2.4 years), height (172±2 vs. 168±2 cm), weight (79±3 vs. 74±3 kg) and body mass index (25.3±0.5 vs. 25.4±1.0 kg/m2, all p values>0.05). When considering those with and without cachexia, body mass index was significantly lower in the cachectic group (22.2±0.5 vs. 24.5±0.7, p<0.002). There were no other significant differences between cachectic and non-cachectic patients.

3.2. Whole blood stimulation
Lipopolysaccharide-stimulated TNF release was 76% greater at 1 ng/mL LPS (274.6±72.4 vs. 66.9±21.9 pg/mL, p=0.07) and 60% greater at 10 ng/mL LPS (573.6±116.1 vs. 219.5±80.2 pg/mL, p=0.008) in CHF patients (including those with cardiac cachexia) in comparison to the control group. When considering CHF patients alone, those with cardiac cachexia had a significantly reduced capacity for TNF release as compared with non-cachectic CHF patients following stimulation with either 1 or 10 ng/mL LPS (35.7±11.8 vs. 281.4±88.7 pg/mL with 1 ng/mL; 150.8±53.3 vs. 554.3±141.2 pg/mL with 10 ng/mL, both p<0.01).

IC14 (50 µg/mL) did not suppress constitutive TNF production derived from either patients [n=20, 22±6.3 (no IC14, no LPS) versus 29±11 pg/mL (IC14, no LPS)] or controls [15±0.5 (no IC14, no LPS) versus 15±0.46 pg/mL (IC14, no LPS)]. In contrast, blocking the CD14 receptor with IC14 caused a significant inhibition in LPS-stimulated TNF production in patients and control subjects. Pre-incubation of whole blood with IC14 at 5.0, 10 and 50 µg/mL substantially reduced TNF release in patients and control subjects at both concentrations of LPS (Table 1). IC14 at a concentration of 5 µg/mL reduced TNF release in CHF patients by 70% (p=0.0001), at 10 µg/mL by 89% (p<0.0001) and at 50 µg/mL by 87% (p<0.0001) following stimulation with LPS at 1 ng/mL. The same concentrations of IC14 resulted in a reduction in TNF release by 70% (p<0.0005), 87% (p<0.0001) and 91% (p<0.0001), respectively, after stimulation with 10 ng/mL LPS. However, when IC14 was used at a concentration of 0.5 and 1 µg/mL, no inhibitory effect was observed.


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  		Table 1 Effects of IC14 on TNF production in CHF patients as a whole, divided in cachectic and non-cachectic patients and controls stimulated with LPS at 1 and 10 ng/mL

 
In healthy controls, IC14 concentrations of 5.0, 10 and 50 µg/mL resulted in a 48% (p=0.004), 78% (p=0.005) and 78% (p=0.004) reduction, respectively, in TNF release following LPS stimulation with 1 ng/mL. Using the same concentrations of IC14 and stimulating with 10 ng/mL LPS resulted in 91% (p=0.001), 93% (p=0.001) and 93% (p=0.001) decrease in TNF release.

In patients with cardiac cachexia, 1 ng/mL LPS induced a non-significant increase in TNF production (no LPS, 15±0. pg/mL vs. 1 ng/mL LPS, 35.7±11.8 pg/mL, p=0.10). However, when the higher concentration of LPS was used (10 ng/mL) a significant increase in TNF production was seen as compared with no stimulation (150.8±53.3 pg/mL, p=0.01). An inhibitory effect of IC14 was still apparent in these patients, but this only reached statistical significance with 50 µg/mL of IC14 (150.8±53.3 to 15±0 pg/mL, p=0.006).

Similar results were obtained for LPS induced IL-6 production in non-cachectic CHF patients (n=13). At a concentration of 5 µg/mL IC14 reduced IL-6 release by 98% (335.2±167.5 vs. 7.2±3.2 pg/nL, p≤0.0001), at 10 µg/mL by 99% (335.2±167.5 vs. 4±0 pg/m, p≤0.0001) and at 50 µg/mL by 99% (335.2±167.5 vs. 4±0 pg/nL, p≤0.0001) following stimulation with LPS at 1 ng/mL. Pre-incubation with 5 µg/mL IC14 followed by stimulation with 10 ng/mL LPS reduced IL-6 production by 94% ( 1393.2±206.5 vs. 66.7±27.2 pg/mL, p≤0.0028), 98% (1393.2±206.5 vs. 22±7 pg/mL, p≤0.0005) and 99% (1393.2±206.5 vs. 14.2±0 pg/mL, p≤0.0001), respectively. In control subjects LPS induced IL-6 production was also reduced following pre-incubation with IC 14 at 5.0, 10 and 50 µg/mL (p<0.05). No effects on IL-6 production were seen at IC14 concentrations of 0.5 and 1.0 µg/mL in CHF patients and control subjects.

To determine whether the activity of IC14 is maintained beyond 6 h a comparative series of studies was performed in 6 non-cachectic patients following 24 h of incubation. This confirmed that the inhibitory effect of IC14 on LPS-stimulated TNF and IL-6 production present after 24 h is similar to that seen after an incubation period of 6 h (Fig. 1A and B).


Figure 1
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  		Fig. 1 A and B. Effects of IC14 (10 µg/mL) following stimulation with 10 ng/mL LPS on TNF production and TNF mRNA. * vs. pre-treatment with IC14. One symbol p<0.05.

 
3.3. Quantification of TNF mRNA levels
Tumor necrosis factor mRNA was quantified in four non-cachectic CHF patients and seven control subjects. Stimulation with LPS 10 ng/mL resulted in a 30-fold increase in transcripts in CHF patients and a 31-fold increase in controls. The administration of IC14 10 µg/mL led to a 97% reduction in TNF mRNA levels in CHF patients (p=0.04), and a 93% reduction in control subjects (p=0.03).

3.4. Relation between CD14 density and TNF response
The CD14 receptor density was assessed in 13 non-cachectic CHF patients and 17 control subjects. There was no difference in mean fluorescence intensity (MFI) between patients and controls (267±20 versus 235±11, p=0.22). There was a positive correlation between the MFI of CD14 receptor density and TNF release in controls following stimulation with 1 ng/mL LPS (r=0.61, p=0.03, Fig. 2A). Following stimulation with 10 ng/mL LPS the correlation between MFI and TNF release almost reached significance (r=0.51, p=0.077). In contrast, in non-cachectic CHF patients this correlation was not apparent (r=0.22, p=0.48, Fig. 2B).


Figure 2
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  		Fig. 2 A and B. Linear regression between CD14 receptor density and TNF response in healthy control subjects and CHF patients following stimulation with 1 ng/mL LPS.

 
3.5. Cytokine and endotoxin levels
Serum levels of TNF and sCD14 were elevated in CHF patients as compared with control subjects (p<0.05). There was no relationship between sCD14 and TNF response. The sTNFR2 levels were not different between groups (p=0.38). Cytokine levels were similar in cachectic and non-cachectic patients. Whilst plasma endotoxin levels were 63% higher in patients as compared to control subjects, this just failed to reach statistical significance (p=0.07), Table 2.


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  		Table 2 Cytokine and endotoxin levels in CHF patients with and without cardiac cachexia (CC) and control subjects

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We have shown that IC14, an anti-CD14 recombinant monoclonal antibody, substantially reduces LPS induced ex vivo TNF and IL-6 production in patients with CHF. The antibody appears to exert its effect at a transcriptional level as demonstrated by the reduction in TNF-mRNA expression in response to LPS stimulation. The antibody alone did not affect the production of TNF and IL-6 in unstimulated blood. We also demonstrated that LPS responsiveness of whole blood was significantly higher in patients with non-cachectic CHF as compared to patients with cardiac cachexia and control subjects. The CD14 receptor density on circulating monocytes was similar in non-cachectic CHF patients and controls. Whilst a positive correlation was found between CD14 receptor density and TNF production in healthy control subjects, this was not the case in CHF patients.

No previous study has evaluated the in vivo or ex vivo effects of an anti-CD14 mAb in humans with CHF. Studies in models of sepsis have shown that absence of/or blocking the CD14 receptor in mice [16], rabbits [17] and monkeys [12] protects against the toxicity associated with LPS administration. The first study using IC14 in vivo in humans was published by Verbon et al. [13]. They demonstrated that blocking the CD14 receptor reduced LPS responsiveness, including cytokine release (TNF and IL-6) and granulocyte activation in healthy human subjects. A recent study published by Knuefermann et al. [18] highlights the importance of CD14 in a model of septic cardiomyopathy in CD14 deficient mice. They demonstrated that LPS induced a significant increase in myocardial TNF content and protein expression in wild type mice. In contrast TNF mRNA and protein levels were significantly blunted in CD14 deficient mice. These changes in myocardial TNF release were accompanied by depressed left ventricular fractional shortening and dP/dtmax in the wild type mice, whereas in CD14 deficient mice cardiac function remained normal. These results add credence to a clinical role for LPS and CD14 in acute heart failure. Yet it would be inappropriate to assume that results derived from studies with healthy young volunteers or animals are completely applicable to patients suffering from chronic heart failure. As an initial step we investigated the effects of blocking the CD14 receptor ex vivo in CHF patients and healthy controls of similar age. In an attempt to recreate an experimental ex vivo setting that may truly reflect the in vivo situation we used whole blood; various components of blood (e.g. mononuclear cells, lipopolysaccharide binding protein) may be important contributors to the immune activation in CHF and are likely to be required to obtain the maximal effect of exogenous LPS.

Inflammatory immune activation is present in CHF patients. Previous studies have shown that elevated levels of cytokines and cytokine receptors predict poor clinical outcome in CHF [3,4]. Inhibition of the detrimental effects of TNF has been attempted with the TNF receptor fusion protein etanercept as well as the monoclonal antibody against TNF, infliximab [19,20]. Results have, however, been disappointing. The reasons for this are not clear. An alternative approach would be to target mechanisms that are potentially involved in the initiation of cytokine production. The current study lends support to this strategy. As such, blockade of the CD14 receptor could represent a novel therapeutic approach in patients with CHF and systemic immune activation because it inhibits the production of at least two major inflammatory cytokines (TNF and IL-6). Blocking of alternative pathways may also be feasible. For example, Akashi et al. [21] demonstrated that blocking TLR4, inhibits ex vivo LPS induced TNF production from whole blood and isolated monocytes in healthy human subjects. An alternative approach would be to block protein kinases such as p38 mitogen-activated protein kinase (MAPK), since the production and action of many cytokines is dependent upon the activation of this kinase. Several specific p38 MAPK inhibitors have been developed. They have shown potent inhibitory effects on cytokine production in vivo and in vitro in a variety of models [22]. Besides blocking the above described pathways, inactivation of LPS itself could reduce immunactivation. Conraads et al. [23] elegantly demonstrated that eradication of the gram negative intestinal flora with antibiotics in patients with advanced CHF (NYHA class III and IV) attenuated monocyte CD14 expression and intracellular cytokine production (TNF, IL-6 and Il-1β). Of note, plasma endotoxin concentrations were unchanged. Endothelial function improved during treatment and returned to baseline after discontinuation of treatment.

Another interesting finding of our study is that patients with cardiac cachexia exhibited a reduced TNF response after LPS challenge. This appears to be similar to the findings of De Werra et al. [24], who described decreased TNF production in response to LPS (10 ng/mL) in patients with either cardiogenic or septic shock. Although we did not study patients in cardiogenic shock, patients with cardiac cachexia showed a similar pattern of whole blood cytokine activation after LPS stimulation. There was no difference between monocyte count in patients with cachectic and non-cachectic CHF (data not shown). Interestingly, it has been shown that catecholamines inhibit TNF production in LPS-stimulated whole blood [25]. Plasma levels of adrenaline and noradrenaline are significantly higher in cachectic compared with non-cachectic patients [26], and this might be one explanation for the reduced TNF response after LPS challenge. Furthermore, sCD14 might play a role in the blunted TNF response in cachectic patients [27]. In our study sCD14 levels were similar in cachectic versus non-cachectic patients, furthermore there was no correlation between sCD14 levels and LPS induced TNF response. Thus, sCD14 might influence the blunted TNF response in cachectic patients but other mechanisms appear to be important as well.

In control subjects we demonstrated a positive correlation between CD14 receptor density and TNF production following stimulation with 1 ng/mL LPS. This was not the case in CHF patients. The CD14 receptor density was similar in CHF patients and controls, which is consistent with data from de Werra and colleagues [23]. Nevertheless, enhanced TNF production in response to LPS is apparent in stable, non-cachectic CHF patients. This suggests that signalling via the CD14 receptor might be altered in CHF [28]. Blocking the CD14 receptor reduces TNF mRNA and TNF production, but additional CD14 independent pathways may also play a role.

In conclusion, our study indicates that blocking CD14 receptors with a monoclonal antibody reduces the LPS responsiveness of circulating monocytes ex vivo in patients with CHF and control subjects. Although several studies have not fully substantiated the benefit of immunomodulatory therapy in CHF [19,20] others have shown more promise [29,30]. An alternative approach whereby one stimulus for immune activation is targeted, such as the LPS receptor, may prove to be a more rewarding strategy. In this respect treatment with IC14 may represent a novel therapy for CHF patients with systemic immune activation, particularly at times of exacerbation (i.e. during decompensation or cardiogenic shock).


   Acknowledgements
 
SGZ is supported by the Deutsche Forschungsgemeinschaft (GE1172/1-1). SvH is supported by the German Heart Foundation, Frankfurt, Germany. APB, PRK and the Department of Cardiac Medicine are supported by the British Heart Foundation. AJSC was supported by the Viscount Royston Trust. SDA was supported by postgraduate fellowship of the Max-Delbrück-Centrum for Molecular Medicine, Berlin Germany and by a grant from Dr. Hubert Bailey.


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

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