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The European Society of Cardiology

Immune activation in severe heart failure

Does etiology play a role?

Laura Agnolettia, Salvatore Curellob, Fabio Malacarnec, Paolo Airòc, Anna Cargnonia, Marco Valgimiglid and Roberto Ferrarid,*,1

a Cardiovascular Pathophysiology Research Center, S. Maugeri Foundation IRCCS, Gussago, Brescia, Italy
b Chair of Cardiology, Spedali Civili, Brescia, Italy
c Immunology Department, Spedali Civili, Brescia, Italy
d Chair of Cardiology, Cardiovascular Pathophysiology Research Center, S. Maugeri Foundation IRCCS and University of Ferrara, Via Pinidolo 23, 25064 Gussago, Brescia, Italy

* Correspondence: Tel.: +39 30 252 8391; fax: +39 30 252 2362 (E-mail: fri{at}dns.unife.it).

Abstract

AIM: Immune activation in severe congestive heart failure (CHF) due to idiopathic conditions is a well-known phenomenon. The question remains: is it confined to a specific aetiology or is a disease-dependent derangement? We investigated immune alterations in ischaemic compared to idiopathic patients with CHF and healthy subjects.

METHODS AND RESULTS: We evaluated alterations of immune activation studying both in vivo and in vitro parameters. In both idiopathic and ischaemic CHF patients: (a) lymphocyte count was reduced (P<0.01); (b) lymphocyte subsets were altered (CD4/CD8 ratio increased vs. controls: 2.53±0.8 and 3.3±2.1% in idiopathic and ischaemic patients, vs. 1.8±0.6% in controls; P<0.01); (c) lymphocytes from patients underwent a higher apoptosis (27.1±4.3% in idiopathic CHF and 25.4±3.5% in ischaemic CHF vs. 4.5±1.4%; P<0.0001); (d) TNF-α production from lymphocytes and monocytes of patients was higher than controls. A positive correlation was observed between TNF-α from monocytes of patients and the relevant serum levels (r=0.58; P<0.01); (e) conditioned media of lymphocytes and monocytes from patients significantly increased rate of endothelial apoptosis.

CONCLUSIONS: In severe CHF, irrespectively from aetiology, significant activation of immune system occurs: several pro-inflammatory cytokines and soluble factors are spontaneously released in serum, possibly contributing to disease progression, as they induce apoptosis.

Keywords Congestive heart failure; Immune system activation; TNF-α

Introduction

Immune activation, characterised by T-cell, cytokine and auto-reactive activation, as well as by autoantibody formation,1 has recently been hypothesised to be involved in the pathogenesis of severe congestive heart failure (CHF) due to idiopathic cardiomyopathy.2 In this condition, association with a specific human leukocyte antigen of the major compatibility complex class II has been described3 as well as a modification in circulating lymphocyte subtypes 4. Moreover, in biopsies of hearts from patients with dilative cardiomyopathies, the activation of the adhesion molecules at endothelial level has been observed, this representing the primum movens for the activation of the lympho-monocytes and the trans-endothelial migration.5,6

Alterations of immune system activation in CHF due to non-idiopathic conditions, such as ischaemic heart disease, have scarcely been investigated so far. Therefore, it remains to be determined whether immune activation is confined to a specific (idiopathic) aetiology or is a disease — rather than aetiology-dependent derangement. This would possibly translate into pathophysiological and therapeutic implications in patients with advanced CHF.

The aim of the current study is to investigate immune system alterations in ischaemic compared with idiopathic patients with CHF and healthy subjects (controls).

To this end, the following in vivo parameters have been studied:

(1) neurohormone and pro-inflammatory cytokine levels in peripheral blood;
(2) quantification and characterization of the mononuclear population as T cell subsets — helper CD4 and cytotoxic CD8 — and their ratio; and as monocyte cells — CD14 population. Peripheral blood mononuclear cells (PBMCs) were also isolated for the following in vitro determinations:
(3) lymphocyte and monocyte activation in terms of:
(a) their own apoptotic activity (rate of apoptosis, Fas and Bcl-2 protein expression) and
(b) in vitro tumour necrosis factor-α (TNF-α) production;

(4) assess PBMCs ability — from patients and controls — to induce endothelial cell apoptosis.

Methods

Populations studied
Sixteen male patients (49±6 years) with class IV New York Heart Association (NYHA) due to coronary artery disease (CAD) and 18 male patients (43±5 years) with NYHA class IV due to dilated cardiomyopathy (DCM), according to accepted criteria7 were consecutively enrolled in the study.

Haemodynamic parameters were measured in the post-absorptive state with a Swan-Ganz catheter. Cardiac output was determined by thermodilution with a Gould model SP 1445 cardiac output computer. All patients were under treatment with angiotensin-converting enzyme (ACE) inhibitors, diuretics, and aldactone; eight and 10 patients in CAD and DCM group were also assuming β-blockers, respectively.

Exclusion criteria were: clinical and electrocardiographic signs of recurrent ischaemia, infections, renal failure, pulmonary, thyroid and collagen vascular diseases, and malignancy.

Fifteen healthy male control subjects (45±3 years) participated in the study. None of them had any clinical sign of acute or chronic illnesses, or any symptoms related to the cardiovascular system.

All patients and controls gave informed consent and the local Ethics Committees approval was obtained.

Blood analyses
After 30 min of supine rest, venous blood was withdrawn from each participating subject/patient to measure:

(1) Neurohormone and cytokine

Serum electrolytes, norepinephrine (NE), aldosterone, plasma renin activity (RA), atrial natriuretic peptide (ANP), TNF-α, sTNF-RI and sTNF-RII measurements were performed as previously described.8

(2) Cellular quantification and characterisation

Measurement of CD3, CD4, CD8 was performed in the blood by flow cytometry (Cytoron Absolute, Ortho Diagnostic System) using the following monoclonal Fluoresceine Isothiocyanate (FITC)-conjugated antibodies: FITC-OKT3 (CD3), FITC-OKT4 (CD4), FITC-OKT8 (CD8) from Ortho.

PBMC analyses
PBMCs, prepared from heparinised whole blood by density gradient centrifugation, were cultured under standard conditions for 24 h. PBMCs were resuspended in a phosphate buffered saline (PBS) pH 7.2, supplemented with 0.5% bovine serum albumin and 2 mM EDTA. CD 14 microbeads were added to PBMCs and incubated for 15 min at 6 °C. CD14-FITC was added and incubated for additional 10 min to evaluate the efficiency of the magnetic separation. Thereafter, the cells were separated on a specific column (Miltenyi) placed in a magnetic field. CD14+ cells are retained in the column while CD14– cells run through. After removal of the column from the magnetic field, the retained CD14+ cells can be eluted. Negative fraction was constituted by lymphocytes.

(3) PMBC activation:

(a) Lymphocyte apoptotic activity
Rate of apoptosis. Detection of subdiploid population was performed by flow cytometry using a FITC-Annexin V/propidium iodide (PI) double staining. Briefly, 1x106 PBMCs were incubated with 1 μ g/ml FITC-conjugated Annexin V (Bender Medical System) and 1 μ g/ml of Propidium iodide (Sigma) in Hepes/NaOH 10 mM buffer pH 7.4, 10 mM Ca2+, 140 mM NaCl, 2.5 mM CaCl2 as described by Vermes.9 Fas expression. Evaluation of lymphocyte Fas expression was performed on heparinised whole blood with the anti Fas monoclonal antibody FITC-conjugated from Pharmingen. Samples were analysed with an argon-ion laser (488 nm). Non-specific staining was assessed using FITC-conjugated non-immune mouse IgG (Coulter).
Bcl-2 expression. Lymphocytes were fixed and permeabilised prior to incubation with 1 μg/ml FITC-conjugated monoclonal antibody anti human Bcl-2 (Ancell) or irrelevant FITC-conjugated antibody. Thereafter, the cells were washed and analysed by flow cytometry. Quantification of positivity for Bcl-2 was performed using fluorochrome-coupled beads (QuickCal Flow Cytometry Standards Corp.) and results were expressed as specific MESFs (molecule equivalents of soluble fluorochrome).10 For western blot analysis, PBMCs were resuspended in lysis buffer solution (10 mM Tris—HCl pH 7.4, 1% sodium dodecyl sulfate (SDS), 1 mM sodium vanadate) containing proteinase inhibitors (10 μM pepstatin, 13 μM leupeptin, 1 mM phenylmethylsulfonylfluoride) and sonicated. After assessing total protein concentration by Lowry method,11 samples were resolved on a 12% SDS polyacrilamide gel. The electrophoresed proteins were transferred to a PVDF membrane (Bio Rad). For Bcl-2 detection the immunoblotting was incubated with mice monoclonal antibody (1:66) (Calbiochem). Then, the membrane was incubated with a horseradish peroxidase-conjugated anti-mouse antibody diluted 1:2000 (Dako). The blots were developed by the chemioluminescence detection system (ECL+PLUS, Amersham) and exposed to X-ray film.

(b) TNF-{alpha} Release from PBMCs
In order to evaluate TNF-{alpha} release, lymphocytes and monocytes were cultured at concentration of 1x106 cells/ml per 24 h. Thereafter, the conditioned medium was used for the immunoenzymatic dosages of TNF-{alpha}.

(4) Rate of HUVEC Apoptosis

Isolation and culture of HUVECs. HUVECs were isolated from umbilical cords according to Jaffe 12. No additional growth factor was added to propagate the culture. HUVECs were characterised. At the 3rd passage, cells were incubated for 48 h with the conditioned medium (CM) of control and CHF PBMCs cultured for 24 h. In order to evaluate the origin of TNF-{alpha}, monocytes were separated with magnetic coated beads with an anti-CD 14 thus allowing to selectively measuring TNF-{alpha} released from monocytes and lymphocytes.

Detection of HUVEC Apoptosis. HUVEC apoptosis was quantified by flow cytometry using the modified method of Nicoletti as previously described.13 In order to test the specific role of TNF-{alpha} in the HUVEC apoptosis, we repeated the above-described experiments in presence or not of an anti-TNF-{alpha} monoclonal antibody (1 μg/ml, R&D Systems).

Statistical analysis
Data are reported as mean±S.E. Comparisons between two groups were performed with the Student's t-test or Mann—Whitney test in the case of non-parametric variables. Comparisons between more than two groups were performed by 2-tailed ANOVA and post hoc comparisons by Turkey honest significance difference test. Pearson's test was used to assess correlations between variables. Probability was significant at a level of <0.05.

Results

Populations studied
Table 1 shows the clinical characteristics, the plasma hormones and TNF-{alpha} data of the studied populations.


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  		Table 1. Clinical, haemodynamic and neuroendocrine characteristics of the studied populations
 
Lymphocyte count in patients with CHF was significantly reduced (P<0.01) in keeping with data from the literature.14

In patients with CHF, cardiac index (CI) was severely reduced (2.1±0.7 and 1.9±0.7 l/min/m2 in ischaemic and idiopathic patients with CHF, respectively), right atrial and pulmonary pressures were high (13±4, 11±5 and 38±9, 35±8 mmHg as above), as well as the systemic vascular resistances (1933±495 and 2164±676 dyn/s/cm–5) and serum level of sodium lowered (133±5 and 132±4 mmol/l).

Mean values for RA and aldosterone were 38±14, 44±12 ng/ml/h and 535±343, 618±275 pg/ml. Plasma concentrations of NE and ANP were 4.3 and 3.5 and 18.5 and 20.5 times greater than those of the controls.

The mean values of antigenic TNF-{alpha} in idiopathic and ischaemic patients with CHF were 47.1±10.2 and 55.0±14.3 vs. 20.2±3 pg/ml in controls (P<0.05). The same patterns were recorded for the soluble receptors of the cytokine (P<0.05 for both sTNF-RI and sTNF-RII).

Cellular quantification and characterisation
Fig. 1 shows the composition of the lymphocyte sub-population relevant to the helper (CD4) and cytotoxic (CD8) lymphocytes, as well as CD4/CD8 ratio. The proportion of CD4 component significantly increased in patients with CHF (53.2±4.7% and 62.4±5.5% in idiopathic and ischaemic patients with CHF, respectively, vs. 40.8±5.1% in controls; P<0.0001). CD8 sub-population shows a trend to reduction in patients without reaching a statistical significance. Consequently, CD4/CD8 ratio increased in patients vs. controls (2.53±0.8 and 3.3±2.1% in idiopathic and ischaemic patients with CHF, respectively vs. 1.8±0.6% in controls; P<0.01).



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  		Fig. 1 Levels of CD4 and CD8 and CD4/CD8 ratio. Distribution of the lymphocyte sub-populations relevant to the helper (CD4) and cytotoxic (CD8) lymphocytes, as well as CD4/CD8 ratio in controls and idiopathic and ischaemic patients with CHF.

 
Lymphocyte apoptotic activity
Fig. 2 shows typical dot-plots of lymphocyte distribution of patients with CHF and controls. The apoptotic cells are positively coloured by Annexin V, but not by PI. Mean data clearly show that lymphocytes from patients with CHF, independently from aetiology, underwent a significantly higher percentage of apoptosis, vs. controls (27.1±4.3% in idiopathic CHF and 25.4±3.5% in ischaemic CHF vs. 4.5±1.4% in controls; P<0.0001).



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  		Fig. 2 Lymphocyte apoptosis. Cytofluorimetric analysis of lymphocytes from controls (a) and idiopathic (b) and ischaemic (c) patients with CHF using a FITC-Annexin V/propidium iodide double staining. The figure shows a representative histogram for each class population.

 
Bcl-2 expression. Fig. 3a shows intracytoplasmatic Bcl-2 protein expressed in lymphoid cells of patients with CHF and controls evaluated by flow cytometry and quantified by MESFs. This expression was significantly reduced in patients as compared with controls (15493±8542 and 13652±9152 in idiopathic and ischaemic patients with CHF, respectively, vs. 39757±6314 in controls; P<0.0001). The down-regulation of the protein in patients was also confirmed by the results of the western blot analysis (Fig. 3b).



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  		Fig. 3 Bcl-2 expression. Mean data of cytofluorimetric analysis of Bcl-2 expression in lymphocytes in controls and idiopathic and ischaemic CHF patients (a). Representative western blot of Bcl-2 expression (b).

 
Fas Expression. Fig. 4 shows lymphocyte Fas expression. The figure reports three histograms each typical for a different class population. Each class analysed is related to an own blank; the right-shift of the fluorescence indicates an increase of the Fas receptors in the individual lymphocytes.



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  		Fig. 4 Fas expression. Cytofluorimetric analysis of Fas expression in lymphocytes in controls (a) and idiopathic (b) and ischaemic (c) patients with CHF. The figure shows a representative histogram for each class population. Every class is referred to relative blank.

 
The mean data show an increase in Fas expression in patients with CHF vs. controls (from 43.6±6.2% in controls to 60.6±9.4% and 66.8±10.4% in idiopathic and ischaemic patients with CHF, respectively; P<0.0001).

TNF-{alpha} release from PBMCs
In order to better understand the pathological significance of PBMC activation, we have also selectively studied TNF-{alpha} release from lymphocytes and monocytes isolated from controls or patients with CHF and cultured for 24 h. The results are shown in Fig. 5. Lymphocyte TNF-{alpha} release from patients with CHF, both idiopathic and ischaemic, compared to the controls, is higher (89.3±22.1 and 95.8±30.3 vs. 65.6±25.1 pg/ml; P<0.01) but negligible if compared to the TNF-{alpha} released by monocytes from the same classes and markedly higher, if compared to the amount released by the monocytes from healthy subjects (3420.8±508.6 and 4127.2±655.0 vs. 338.8±51.3 pg/ml; P<0.0001).



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  		Fig. 5 TNF-{alpha} release from PBMCs from either controls and idiopathic and ischaemic patients with CHF after 24 h of culture.

 
Interestingly, a positive correlation was observed between TNF-{alpha} released from isolated monocytes from patients and the respective serum levels (r=0.58; P<0.01).

Rate of HUVEC apoptosis
Fig. 6 shows the rate of apoptosis of HUVECs incubated for 48 h with or without the CM of lymphocytes and monocytes incubated for 24 h.



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  		Fig. 6 Rate of HUVEC apoptosis. The mean data are referred to a cytofluorimetric analysis of HUVEC apoptosis, after 48 h incubation with CM from controls, idiopathic and ischaemic CHF lymphocytes and monocytes.

 
The lymphocyte and monocyte CM from patients with CHF significantly increased the rate of HUVEC apoptosis, if compared to the control CM (from 8.2±2.4 to 13.6±3.0 and 12.5±2.4%, in presence of lymphocyte CM from idiopathic and ischaemic patients with CHF, respectively; P< 0.0001; from 10.4±2.9% to 15.9±4.7% and 17.7±6.3% in presence of monocyte CM from idiopathic and ischaemic patients with CHF, respectively; P<0.0005).

The results always show the statistical significance vs. controls since there were no statistically significant differences between the two groups of patients.

Discussion

It is well recognised that severe CHF due to dilated cardiomyopathy is not only characterised by a haemodynamic and/or neurohormonal impairment, but also by an immune activation as supported by the following evidences: (a) cytokine increase;15 (b) auto-antibody elevation;16 (c) alteration of the lymphocyte pattern.17

The results of our study demonstrate, for the first time, that, during the advanced stages of CHF due to ischaemic cardiomyopathy, marked functional alterations of PBMCs occur, as shown by the chronic activation of both lymphocytes and monocytes. In particular, we found a comparable degree of disorder in the investigated immunological parameters in patients with ischaemic versus idiopathic CHF, strongly suggesting that immune activation is not confined to a specific (idiopathic) aetiology but it may reflect a disease — rather than aetiology-dependent derangement.

In terms of the quantitative impairment of the lymphocytes, our data confirm the presence of lymphocytopenia, which has recently been reported as a negative prognostic marker for CHF.14

Possible mechanisms for peripheral blood lymphocytopenia in the setting of CHF are: (a) redistribution of lymphocytes from peripheral blood to other sites; (b) decreased production; (c) accelerated cell death due to apoptosis.

Although there is no evidence for an impaired lymphocyte production as the cause of lymphocytopenia, the other two mechanisms might actually contribute to its occurrence.

In fact, the flow cytometry analysis of cells stained with Annexin V, has clearly indicated that lymphocytes from our patients were highly susceptible to in vitro "spontaneous" (i.e. not directly induced by activating stimuli) apoptosis.

The increased rate of spontaneous apoptosis is explained by the down-modulation of Bcl-2 gene product, which is considered the key molecule in the protection from this phenomenon,18 whereas Fas does not seem to have a relevant role.19,20

Several mechanisms may account for down-modulation of Bcl-2, particularly relative to deprivation of growth factors, such as IL-2.18 Although IL-2 has been found to be increased in patients with CHF,3 it has been shown that chronic immune activation may be responsible for in vitro spontaneous lymphocyte apoptosis due to starvation from growth factors.21

An unbalance between growth and pro-apoptotic factors is a possible explanation for our observation.

Our data also show that the circulating lymphocytes of patients with severe CHF have an increased expression of the CD95/Fas antigen. This is a molecule belonging to the Tumour Necrosis Factor Receptor superfamily that can transduce death signals operating in the so-called process of activation-induced cell death (AICD),22,23 which is at least partially independent from Bcl-2 19,20. The increased expression of CD95/Fas receptor could therefore contribute in signalling transduction of lymphocyte apoptotic death. However, it should be noted that cross-linking of Fas is per se insufficient to induce cell death of freshly isolated T cells from healthy subjects24 while it results in apoptosis in sensitive cells.25—28 Moreover, it can be considered that the susceptibility to CD95-induced AICD is function of the state of activation of T cells, requiring repeated antigenic stimulation.29

It should be acknowledged that this functional state of lymphocytes (increased apoptosis, down-modulation of Bcl-2, high expression of Fas) is by no means specific for patients with CHF. On the contrary, it has been described in several conditions characterized by T-cell loss and dysfunctions due to chronic activation due to allo- or auto-antigenic stimulation, such as AIDS,30 Systemic Lupus Erythematosus,31 immune reconstitution after allogeneic bone marrow transplantation32 and primary immunodeficiencies like idiopathic CD4+ lymphocytopenia33 and Omenn's syndrome.34

Finally, a redistribution of T-cells from peripheral blood might be suggested by histological data demonstrating an infiltration in the hearts of patients with CHF and a great percentage of heart-tissue T cells expressing activation markers, e.g. CD45RO 17. An expression of costimulatory, especially B7-1 that may make cardiac myocytes the target cells for the infiltrating killer lymphocytes,35 has been demonstrated as well. Moreover, a migration of peripheral blood lymphocytes in the interstitium might also be hypothesised considering the endothelial damage and the consequent extravasation of cells. These mechanisms might therefore contribute with accelerated cell death to the genesis of lymphocytopenia in our patients.

Indeed, the functional status of the lymphocyte patterns affects the disease in a more complex way. The pathological increase of one lymphocyte population (CD4 or CD8) results in an abnormal immunological reaction that eventually can determine either an auto-immune or an allergic disease.36 This hypothesis has been recently re-proposed to explain the autoimmune myocarditis that occurs after viral infections.37

Our data show that the lymphocyte pattern resembles that of an autoimmune disease, as confirmed by the relative increase of the CD4 population. A similar pattern has been so far observed only in patients with idiopathic dilative cardiomyopathy or with dilative cardiomyopathy due to myocardities, although with different distribution of the lymphocyte populations.38

The putative mechanisms responsible for lymphocytopenia and altered pattern of lymphocyte subpopulations in CHF are still elusive. Since peripheral blood lymphocytopenia is a well-known effect of TNF-{alpha} infusion it is speculative to correlate the lymphocytopenia with the increased serum levels of TNF-{alpha}.8

Indeed, we observed an overproduction of this pro-inflammatory cytokine by monocytes. Actually, an in vitro marked "spontaneous" production of TNF-{alpha} by monocytes in contrast to the much lower release of TNF-{alpha} from lymphocytes has occurred. This finding, together with the positive correlation between TNF-{alpha} levels in serum and that released by monocytes in vitro, suggests a key role of the monocytes as TNF-{alpha} source in patients with CHF. The production of TNF-{alpha} by monocytes also supports the hypothesis that mesenteric venous congestion, resulting in a bacteria translocation and endotoxin release, could be among the main causes of the immunological activation in CHF, as suggested by the increase of circulating levels of soluble endotoxin receptor, sCD14.39

In our patient population, we have also observed that TNF-{alpha}, as well as other cytokines released by monocytes, does impair the life/death equilibrium of the endothelium. In fact, the conditioned medium by the in vitro incubation with the monocytes of our patients shows a significant pro-apoptotic effect on HUVECs. Moreover, we have previously shown that serum from patients with severe CHF does have the same effect.8 Altogether, these and other data,40 suggest that PBMCs can spontaneously release both in serum and in vitro soluble factors that are able to trigger cell apoptosis; this phenomenon, when occurring in patients with CHF, might contribute to the disease progression.

Our data do not provide any evidence that immune system activation in CHF has a pathogenetic role. Whether immune activation is among the possible causes, or simply the consequence of CHF awaits further elucidations.

In addition, it should be acknowledged that our findings specifically apply only to advanced CHF. Therefore, it would be intriguing to investigate in future studies whether the degree of immune system activation directly correlates to functional class in CHF.

In conclusion, we have found that in patients with severe CHF, irrespectively from the aetiology, a significant activation of the immune system occurs both in vivo and in vitro in respect to controls. As a result, several pro-inflammatory cytokines and soluble factors are spontaneously released in serum, possibly contributing to disease decompensation and progression, as reflected by their capability to induce human cell apoptosis.

We believe that the lack of clinical benefit with the use of selective anti-TNF-{alpha} therapy in patients with CHF41 should not induce to reject the role of immune activation as a possible mechanism of left ventricular dysfunction. Future studies, aimed at evaluating alternative42 and new ways to modulate the immune system in CHF would be of great interest.

Acknowledgments

This work was supported by Grant No. ICS 030.4/RF.99.102 "Esplorazione fisiopatologica e validazione clinica di nuovi percorsi diagnostici e terapeutici nello scompenso cardiaco e nell'ictus ischemico acuto " from the Italian Ministry of Health. The authors thank Dr. Alessandro Bettini for editorial assistance.

Footnotes

1 Present address: Cardiologia, Arcispedale Sant' Anna, Universitá di Ferrara, Corso Giovecca 204, 44100 Ferrara, Italy. Tel.: +39 532202143; fax: +39 532241885. Back

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