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European Journal of Heart Failure 2003 5(4):469-479; doi:10.1016/S1388-9842(03)00037-0
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© 2003 European Society of Cardiology

Cytotoxic perforin+ and TIA-1+ infiltrates are associated with cell adhesion molecule expression in dilated cardiomyopathy

Michel Noutsias*, Matthias Pauschinger, Heinz-Peter Schultheiss and Uwe Kühl

Department of Cardiology and Pneumonoly, University Hospital Benjamin Franklin Free University of Berlin, Hindenburgdamm 30 Berlin D-12200, Germany

* Corresponding author. Tel.: +49-30-8445-2344; fax: +49-30-8445-3565 E-mail address: noutsias{at}zedat.fu-berlin.de


   Abstract
Top
 Abstract
1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 References
 
Objective: To phenotypically characterize cytotoxic T-lymphocytes (CTLs: Perforin+ and TIA-1+ phenotypes) and to study the interactions with cell adhesion molecules (CAMs) in dilated cardiomyopathy (DCM).

Background: DCM is linked to intramyocardial inflammation, being characterized by T-lymphocytic infiltration and CAMs abundance. However, the pathogenic significance of increased CD3+ lymphocytes remains obscure as these do not correlate with CTLs (perforin+ and TIA1+ phenotypes). CAMs participate in the phenotypic repertoire and effector pathways of CTLs.

Methods: CAMs-expression (ICAM-1, VCAM-1, LFA-3, CD29, CD62E and CD62P and β2-integrins), CD3+ (T-lymphocytes), CD57+ (NK-cells) and adhesion related (CD18+, CD11a+, CD11b+, CDw49d+) phenotyped infilitrates were investigated in endomyocardial biopsies (EMBs) from 89 DCM patients (33 female; LVEF<40%) using immunohistochemisty. The enteroviral genome was identified by nested RT-PCR.

Results: CAMs abundance was confirmed in 55 DCM patients (62%) and 29 EMBs (33%) were graded CTLs+ (>1.5 TIA-1+ and/or >2.0 perforin+ infiltrates/hpf). CTLs correlated with all endothelial CAMs-markers studied (P<0.01), the adhesion related phenotypes of infilitrates (LFA-1, VLA-4, CD18) and CD57+ NK-cells (P<0.02). There was no correlation of CTLs with CD3+ T-lymphocytes, CD11b+ macrophages, enteroviral infection (present in n=16/18%), clinical history and LVEF (P>0.05). Phenomena suggestive of CTLs mediated myocytolysis were observed in 10 patients (11%).

Conclusions: CTLs-infilitrates are associated with endothelial CAMs-abundance and co-express adhesion related (β2-integrins, VLA-4) and NK-cellular antigens (CD57) in DCM. Endothelial CAMs expression also reflects cytotoxic activation of intramyocardial infilitrates, which is not reflected by immunologically naïve CD3 T-lymphocytes.

Key Words: Abbreviations • DCM, dilated cardiomyopathy • InfCM, inflammatory cardiomyopathy • CAMs, cell adhesion molecules • CTLs, cytotoxic T-lymphocytes • ICAM-1, intercellular cell adhesion molecule-1 • LFA, lymphocyte function antigen • VCAM-1, vascular cell adhesion molecule-1 • VLA-4, very late activation antigen-4 • HPF, high power field (0.28 mm2) • RT-PCR, reverse transcriptase polymerase chain reaction

Received September 10, 2002; Revised October 24, 2002; Accepted January 23, 2003


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients and methods
 3. Results
 4. Discussion
 References
 
Intramyocardial inflammation evoked by viral infection is not primarily detrimental to the heart, but teleologically protective, by producing viral elimination. However, once autoimmunity has been established (e.g. by molecular mimicry), the anti-cardiac immune response continues unabated despite viral elimination, and results in detrimental immune-mediated cardiomyocyte loss, leading to dilated cardiomyopathy (DCM). The therapeutic implications of these pathogenic processes, is that immunosuppression might abrogate the chronic anti-cardiac autoimmune process in DCM [1]. Inflammatory cardiomyopathy (InfCM) is characterised by idiopathic heart failure with evidence of intramyocardial inflammation [2]. The Dallas criteria proved to be most suitable for the diagnosis of the acute stage of myocarditis [3], but lack sensitivity and specificity for a reliable diagnosis of InfCM [4–6]. Using more sensitive and specific immunohistologic techniques, InfCM shows approximately 50% of DCM patients [5]. These immunohistologic criteria for InfCM (>2.0 CD3+ lymphocytes/high power field [hpf]), are associated with concurrent abundance of ≥3 endothelial cell adhesion molecules (CAMs) in DCM [7]. However, the pathogenic significance of isolated increase in CD3+ infiltrates remains unclear, as this parameter does not necessarily specify cytotoxic T-lymphocytes (CTLs), which are significantly increased in DCM biopsies when compared to controls [8]. Finally, it is uncertain whether CAMs assessment provides any additional information when compared with the InfCM criteria [7]. Importantly, the first successful randomised immunosuppressive study in DCM were based on endothelial HLA abundance [9], providing evidence that CAMs, in contrast to the histologic Dallas criteria [10], are a sensitive and specific diagnostic tool, enabling selection of the DCM-patients who will benefit from immunosuppression [11].

The hypothetical pathogenic mechanism involved in InfCM is CTLs induced apoptosis of cardiomyocytes by cytotoxic mediators, such as perforin and granzymes (e.g. TIA-1), leading to contractile impairment [1]. The generation of CTLs and their mechanism of action is a complex process comprising: (a) the specific priming of LFA-1+ large granular lymphocytes and CD57+ NK-cells, by the transduction of target epitopes, (b) transendothelial migration of these cells into the target organ, and (c) the chemotactic attraction of these CTLs towards the target cells (e.g. cardiomyocytes expressing viral proteins or cryptic self-proteins), adhesion to the cell surface and secretion of cytotoxic signals inducing the irreversible cascade of apoptosis [12]. It has been suggested that CTLs can be subdivided into adhesive and secretory types [13]. Animal experiments on LFA–/– mice, however, proved that CTLs essentially need both adhesive and secretory properties to confer their ‘kiss of death’ [14].

We therefore investigated the diverse phenotypes of intramyocardial infiltrates, both immunologically naive CD3+ T-lymphocytes and phenotypes participating in the repertoire of CTLs (perforin+, TIA-1, β2-integrins+, CD57+ NK-cells [12,15]), endothelial CAMs expression (immunoglobulin-superfamily, selectins, and β1-integrins) and the presence of enteroviral genome, in EMBs from DCM patients. The aim was to define the possible diagnostic and pathogenic significance of the three immunohistologic diagnostic criteria (InfCM+ [5], CAMs+ [7], and CTLs+ [8]) for intramyocardial inflammation in DCM.


   2. Patients and methods
Top
 Abstract
 1. Introduction
 2. Patients and methods
3. Results
 4. Discussion
 References
 
Study group: We enrolled 89 consecutive patients (33 female; aged: 47±16 years), clinically presenting with DCM (LVEF <40%) referred to our hospital for invasive evaluation from June 1999 to March 2000. Patients with excessive alcohol consumption, a family history of DCM or heart failure, or peripartum cardiomyopathy were excluded. Seven patients (8%) were in NYHA functional class I, 39 patients (44%) in NYHA class II and 43 patients (48%) in NYHA class III, at the time EMBs were obtained. The duration of symptoms was greater than 6 months for all patients (range: 8 months to 7 years). The main symptom was dyspnea on exertion in 74 patients (83%). Atrial fibrillation with left ventricular enlargement, assessed by echocardiography, was the leading diagnosis in 12 patients who were (14%) referred to our hospital for invasive evaluation of the underlying etiology. Three patients (3%) were referred due to sustained ventricular tachycardia detected on Holter monitoring with left ventricular dilatation assessed by echocardiography. All patients underwent electrocardiography, echocardiography, left heart catheterisation (coronary angiography and assessment of LVEF). Secondary etiopathogenesis (systemic hypertension, metabolic disorders, ischaemic, valvular and congenital heart diseases) were excluded. This investigation conformed to the principles outlined in the Declaration of Helsinki [16]; all patients gave written informed consent.

EMB-preparation: Three EMBs from the right ventricular septum were obtained by percutaneous transvenous right femoral approach, using a Cordis bioptome (Cordis, Haan, Germany). For histologic evaluation using the Dallas criteria, one EMB was fixed in 10% formalin. For immunohistologic evaluation, the second EMB was embedded in Tissue Tec® (SLEE, Mainz, Germany), immediately snap-frozen in liquid nitrogen and cut serially into cryosections of 5 µm thickness, which were placed on 10% poly-L-lysine precoated slides. Six sections were analysed for each antibody per patient. The third EMB was subjected to RNA extraction for RT-PCR detection of the enteroviral genome.

Histologic and immunohistologic EMB evaluation: The histologic evaluation of active and borderline myocarditis was based on the Dallas criteria, using hematoxylin-eosin stained, formalin fixed EMBs [3]. The immunohistochemical staining techniques and evaluation procedures have been published elsewhere [5,7,8]. EMBs with greater than 2.0 CD3+ lymphocytes/hpf ({approx}7.0 infiltrates/mm2) were graded InfCM+ [5]. Samples with at least three different CAMs-markers were classified as CAMs+ [7]. Specimens with ≥1.5 TIA-1+ and/or ≥2.0 perforin+ infiltrates/hpf were classified as positive for cytotoxic T-lymphocytes (CTLs+) [8].

Nested RT-PCR of enteroviral genome: Total RNA was extracted from the EMBs by a modified RNAzol method. cDNA was synthesised by MMLV reverse transcriptase and subjected to nested RT-PCR simultaneously, with positive (cloned cDNA of coxsackievirus B3) and negative control reactions (devoid of RNA template) as published in detail elsewhere [17].

Statistical analysis: Statistical analysis was performed using JMP Statistical Discovery Software, Version 4.02 (SAS Institute, Inc., Cary, NC, USA). As normal distribution was excluded for all parameters using the Shapiro-Wilk W Test (P<0.05), exclusively non-parametric tests were performed. Quantitative data were correlated using the Spearman Rho analysis, quantitative and qualitative data were compared using the Wilcoxon/Kruskal–Wallis-Test on rank sums, and qualitative data were compared using the chi-squared test. A probability value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
4. Discussion
 References
 
According to the Dallas criteria, 3 DCM patients (3%) were diagnosed as borderline myocarditis, and no EMB demonstrated myocytolysis/active myocarditis. A substantially higher frequency of intramyocardial inflammation was diagnosed by immunohistology; 42 EMBs (47%) were classified as InfCM+, 55 (62%) as CAMs+ and 29 (33%) were graded CTLs+, respectively. In detail, ICAM-1 abundance was observed in 56 (63%) patients, VCAM-1/CD106 in 55 (62%), LFA-3/CD58 in 52 (58%), E-selectin/CD62E in 39 (44%), P-selectin/CD62P in 14 (16%), and CD29/β1-integrins in 61 (69%) patients, respectively. In agreement with previous reports [7,18], expression of ICAM-1 and CD29 was distributed homogeneously within all serial sections obtained from a single subject and within each section, irrespective of the immunoreactivity grade (Fig. 1). This is in contrast to the other CAMs-markers, which were predominantly induced on single vascular endothelia [7]. For CTLs, 29 (33%) of the biopsies exceeded the statistical limit of perforin+ lymphocytes/hpf, and 24 (27%) were graded positive for significantly increased TIA-1+ infiltrates. Interestingly, the CTLs staining pattern was not restricted to the cytoplasm, as typically seen in other phenotypes of infiltrates, but was of granular nature and apparently released onto adjacent cardiomyocytes (Fig. 2a) [8]. Clusters of CTLs encircling and apparently invading cardiomyocytes, suggesting myocytolysis (Fig. 2b) were observed with respect to perforin+ and TIA-1+ phenotypes in 10 cases (11%).


Figure 1
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  		Fig. 1 Expression of ICAM-1 in DCM: Panel (a) Baseline ICAM-1 expression in DCM-biopsy classified CAMs- (original magnificationx200). Panel (b) ICAM-1 abundance, being homogeneously distributed within the entire section, in a specimen graded CAMs+ (original magnificationx200).

 


Figure 2
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  		Fig. 2 Perforin+ infiltrates in DCM: Panel (a) Perforin is not restricted to the cytoplasm, but released onto the adjacent cardiomyocytes (white arrows, original magnificationx1.000). Panel (b) Perforin+ infiltrates encircling and entering (white arrows) a cross-sectioned cardiomyocyte suggesting myocytolysis (original magnificationx630).

 
It is interesting to note that all the three biopsies which were diagnosed as borderline myocarditis according to the Dallas criteria,were also graded as InfCM+, CAMs+ and CTLs+. Interestingly, although CAMs+ correlated significantly, with both InfCM+ (P=0.0002) and CTLs+ (P<0.0001), the diagnostic classifications of InfCM+ and CTLs+ were not related significantly (P=0.5515). In detail, whereas all biopsies graded CTLs+ were also assessed CAMs+, only 15 (52%) of the CTLs+ biopsies were also InfCM+, and the remaining n=14 (48%) CTLs+ specimens were InfCM-. CTLs+ grading was reported in 30% of the InfCM- patients compared to that of 36% of the InfCM+ patients, which was not statistically significant (Table 1 Scheme 1). In clear contrast to the InfCM evaluation based on CD3+ infiltrates, perforin+ (P<0.0001) correlated significantly with the CAMs evaluation of the DCM specimens (Table 2 Scheme 2). This finding was also true for TIA-1+ infiltrates: TIA-1+ infiltrates were significantly (P=0.0008) lower in EMBs classified CAMs– (median: 0.55/hpf, interquartile range: 0.30–0.73) compared with CAMs+ EMBs (1.0/hpf, interquartile range: 0.60–1.50). However, there was no significant correlation with the InfCM-classification (P=0.7921). All studied CAMs-markers correlated with perforin+ infiltrates significantly (Table 3 with respect to TIA-1 also P<0.01, data not shown).


Figure 3
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  		Scheme 1
 


Figure 4
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  		Scheme 2
 


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  		Table 3 Perforin+ CTLs and CAMs expression in DCM

 
Multivariate analysis of the investigated infiltrate phenotypes revealed that in contrast to CD3+ T-lymphocytes and Mac-1/CD11b+ macrophages, CD18+ pan-leukocytes, CD57+ NK-cells and adhesion related (LFA-1/CD11a+ and CD49d/VLA-4+) subsets correlated significantly (P<0.02) with perforin+ and TIA-1+ CTLs (Table 4 Scheme 3). Although all β2-leukocyte integrins (CD18, CD11a/LFA-1 and CD11b/Mac-1) were interrelated (P<0.0001) [7], CD11a/LFA-1+ infiltrates correlated with CTLs as opposed to CD11b/Mac-1+ macrophages. The most significant associations (P<0.0001) were calculated within the functionally related phenotype groups: adhesion related infiltrates (CD18, CD11a/LFA-1, CD11b/Mac-1 and CDw49d/VLA-4) and CTLs (perforin and TIA-1). CD57+ NK-cells correlated with CD11a/LFA-1+, CDw49d/VLA-4+ and CTLs (both perforin and TIA-1), but not with CD3+ T-lymphocytes and Mac-1+ macrophages.


Figure 5
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  		Scheme 3
 
Enteroviral genome was confirmed by RT-PCR in 16 (18%) of the DCM patients. Enteroviral infection was not associated with the histologic diagnosis of borderline myocarditis (P=0.41), nor the immunohistologic classifications of InfCM (P=0.42), CAMs (0.53) or CTLs (P=0.48).

The (immuno-)histological EMB-findings failed to correlate with LVEF (Table 5). Moreover, there was no significant difference regarding the clinical presentation of the DCM patients, classified according to the immunohistological criteria of InfCM, CAMs or CTLs positivity (NYHA staging, duration of symptoms, leading symptom or main referral diagnosis; P>0.05).


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  		Table 5 LVEF and immunohistological EMB-findings

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
References
 
4.1. Diagnostic approaches for intramyocardial inflammation in DCM: InfCM, CAMs and CTLs
In the immunohistologic diagnosis of InfCM, attention to date has focussed primarily on the mean density of T-lymphocytes, without specifying on the phenotypic characterisation and actual inflammatory activation of infiltrates. In this respect, it is not surprising that a diagnostic process based solely on the mean density of immunologically naive T-lymphocytes, might fail to give the same information as endothelial CAMs induction [7,18,19] and the functional competence of infiltrates [8]. In this analysis, EMBs from DCM patients, 47% had the criteria for InfCM (>7.0 CD3+ T-lymphocytes/hpf), 62% were CAMs+ and 33% CTLs+ positive, respectively. There was no association with enteroviral infection, LVEF or clinical presentation of the patients. Using the Dallas criteria, borderline myocarditis was diagnosed in 3% of the cases. These results are consistent with previous publications [5,7,8] and confirm that:
  1. intramyocardial inflammation is significantly involved in the pathogenesis of DCM, which is apparently unrelated to enteroviral persistence and the patients’ clinical parameters;
  2. the Dallas criteria are insensitive and unspecific when compared with the immunohistologic diagnostic approach;
  3. the three diagnostic concepts InfCM, CAMs and CTLs apparently do not provide redundant information.

4.1.1. CTLs and phenotypic characterisation of intramyocardial infiltrates in DCM
Both CTLs markers (perforin and TIA-1) correlated with CD18+ pan leukocytes, CD57+ NK-cells and adhesion related LFA-1/CD11a+ and VLA-4+ infiltrates, but not with the immunologically naive CD3+ lymphocytes and CD11b/Mac-1+ macrophages. This is in accordance with the phenotypic pattern of CTLs comprising large granular lymphocytes (CD11a/LFA-1, CDw49d/VLA-4) and NK-cells (CD57) [15]. Our data are consistent with novel experimental studies, which have repealed the formerly hypothesised dichotomy of secretory and cytotoxic phenotypes of CTLs and have provided proof of the need for both adhesive and secretory properties, in order to exert the lethal attack on target cells [14]. CTLs, co-expressing adhesion molecules such as CD11a/LFA-1 and CDw49d/VLA-4, may readily migrate into the myocardium, presenting an abundance of the respective endothelial counter-receptors such as ICAM-1/CD54 and VCAM-1/CD106 [7], thereby, exerting their lethal attack on cardiomyocytes in DCM-hearts with endothelial CAMs-abundance. The multivariate correlation analysis of infiltrates shows that the most significant associations (P<0.0001) were within the functionally related phenotypic groups: T-lymphocytes, adhesion related infiltrates and CTLs. Interestingly, despite the fact that all β2-leukocyte integrins were significantly interrelated, CD11a/LFA-1+ phenotypes correlated with CTLs, in contrast to CD11b/Mac-1+ infiltrates, which is conclusive, as macrophages do not participate in the CTLs repertoire. CD57+ NK-cells were differentiated when compared to T-lymphocytes and further, immunocompetent cells correlate with phenotypes and is known to be co-expressed by NK-cells: CD11a/LFA-1+, CDw49d/VLA-4+ infiltrates and CTLs (both perforin and TIA-1) [12,15,20]. This discrepancy (significant correlations between adhesion related phenotypes with CTLs in the lack of correlation of CTLs with CD3+ T-lymphocytes, the latter on the other hand were related to the adhesion related phenotypes) might be explained by the fact that both these adhesion related and CTLs phenotypes participate in the NK-cells repertoire, which are CD3 negative [12,15]. In other words, endothelial CAMs abundance in DCM mediates infiltration by counter-receptor+ cells (lymphocytes, NK-cells and macrophages), and apart from the fraction of immunologically naive CD3+ T-lymphocytes, also variable fraction of CTLs and NK-cells belong to this repertoire, which in turn are partly CD3-negative.

These findings support the notion that phenotypic characterisation and exact quantification of intramyocardial infiltrates, rather than semiquantitative assessment [21], will give a more profound understanding of the actual inflammatory process in DCM [5,6,22,23]. The lack of correlation between these immunohistologic findings and clinical parameters, such as LVEF and NYHA functional class in the present study and in previous observations [7,8,18], however, does not necessarily infer that the investigated immunocompetent factors are of negligible clinical importance, since longitudinal rather than cross-sectional studies have shown that intramyocardial inflammation has an adverse prognostic impact in DCM [24]. Follow-up studies are now required to evaluate the prognostic significance of these diagnostic approaches.

Ultrastructural studies have shown that the CTLs released perforin, forms pores on the target membranes up to 20 {nu}m in diameter, which permit the entry of granzymes and synergistically induce the irreversible apoptotic pathways [25]. Such circular membrane lesions have been shown in murine myocarditis [26] and the critical role of perforin and granzymes has been demonstrated in cardiac transplant rejection before the onset of histologically identifiable damage [27]. The typical pattern of perforin and TIA-1, not being spatially restricted to the cytoplasm of the mononuclear cells, indicates that these cytotoxic factors are released onto the adjacent cardiomyocytes [8,28]. Phenomena suggestive of myocytolysis, namely, clusters of infiltrates adjacent to and partly invading cardiomyocytes (Fig. 2) were observed in perforin and TIA-1 immunostainings in 11% of our DCM patients. Further studies are now warranted to show that focal accumulation of CTLs released cytotoxic factors is related to active myocytolysis and/or CTLs mediated apoptosis of cardiomyocytes.

4.1.2. CTLs and endothelial CAMs in DCM
CTLs correlated with endothelial CAMs expression with respect to virtually all investigated CAMs markers. Most importantly, the CTLs-classification correlated highly significantly with the CAMs evaluation, as opposed to the InfCM-criteria, which failed to discriminate between the DCM-biopsies graded CTLs+ and CTLs-. Significant correlations between CTLs and endothelial HLA-expression have been previously reported in DCM-biopsies [8], and HLA expression in DCM is related to the expression of a number of other CAMs [7]. These data support the notion that endothelial CAMs abundance may reliably reflect intramyocardial inflammatory activity [7,18,19]. Moreover, our data are in clear contrast to reports by Devaux et al. showing equally enhanced CAMs expression in all heart failure patients with interindividual variations [21]. Thus, conceptually evaluated abundance of several endothelial CAMs markers reflects specifically cytotoxic T-lymphocytes, which are not reflected by the mean density of immunologically naive CD3+ T-lymphocytes.

The crucial importance of perforin+ infiltrates has been demonstrated in the murine model of coxsackievirus induced myocarditis. Perforin knockout mice developed only a mild pattern of myocarditis and scarce fibrotic lesions in chronic follow-up, whereas the perforin +/+ littermates demonstrated extensive intramyocardial infiltration in the acute phase after viral infection and severe fibrotic lesions in the chronic stage, respectively [29]. Association between interstitial fibrosis and CTLs has been confirmed in human DCM biopsies [8]. In this setting, cytokines such as MIP-1 were barely detectable in the Coxsackievirus infected hearts of perforin knockout mice in contrast to their perforin +/+ littermates, in which MIP-1 was readily detectable at any point of time after viral infection [29]. Hence, cytokines may be the common factor in our reported correlation between CAMs abundance and CTLs in DCM. Proinflammatory cytokines induce CAMs expressions both on endothelial cells and leukocytes [30], ultimately mediating transendothelial migration of activated immune effector cells into the impaired myocardium and generation of CTLs.

Noticeably, the first successful randomised immunosuppressive study in DCM used HLA abundance as the diagnostic target [9], and showed long-term hemodynamic effects lasting for the period of two years. This highlights the diagnostic sensitivity and specificity of CAMs abundance in identifying those DCM-patients, who will benefit from immunosuppression, in contrast to the histologic Dallas criteria [10,11]. Staudt et al. showed that the hemodynamic improvement observed in DCM patients subjected to immunoadsorption is accompanied by suppression of intramyocardial CAMs (HLA) abundance [31]. In the light of our data, close functional relationship between CAMs abundance and CTLs+ phenotypes co-expressing the specific counter-receptors of endothelial CAMs, immunoadsorption mediated down-regulation of CAMs abundance might also have inhibited CTLs infiltration. The beneficial outcome of immunosuppression in DCM patients with CAMs abundance might also be due to concomitant CTLs infiltration. This hypothesis deserves special attention, since CTLs are held predominantly responsible for the progressive myocyte loss in DCM [1], and CAMs abundance represents the condition, which facilitates the transendothelial migration of that counter-receptor+ CTLs to the target organ.

4.1.3. CTLs and enteroviral infection
Enteroviral infection has been reported in approximately 20% of myocarditis and DCM patients by PCR amplification in EMBs [1]. The cardiotropism of enteroviruses has also been confirmed in animal models and by meta-analysis in human myocarditis and DCM [32]. Enteroviral replication and late persisting expression of the capsid protein VP-1 [33,34] suggests that a chronic enteroviral antigenic stimulus in DCM might evoke CTLs generation against viral proteins. Gebhard et al. showed that perforin does not play any detectable role in the clearance of coxsackieviruses in murine myocarditis [29]. Thus, perforin mediated myocytolysis, evoked by viral infection, does not assist in controlling enteroviral replication in murine myocarditis. The lack of correlation between CTLs and enteroviral infection in our study appears concordant with these experimental data. However, given the hypothesised indistinctness of transitional periods which lead to DCM and the potential for reinfection in the course of disease, especially from the new cardiotropic viruses identified recently (of which the pathogenic significance for human DCM is less well established than for enteroviruses, e.g. adenovirus, hepatitis C virus, parvovirus B19) [22], the functional importance of CTLs for elimination and infection of all potential cardiotropic viruses cannot be ruled out completely, so far.

4.1.4. Limitations of the study
The sampling error is a considerable issue in the histological diagnosis of myocarditis according to the Dallas criteria [35]. However, sampling error is anticipated to be significantly lower in the immunohistologic evaluation of intramyocardial inflammation in DCM, especially, for the homogeneously distributed CAMs [7,9]. Sampling error has not been studied systematically in the immunohistologic assessment of EMBs and enteroviral genome detection by PCR. Thus, sampling error might have influenced our observations to some extent, especially in the case of the focally clustered CTLs infiltrates suggestive of myocytolysis.

Despite the reported statistical significance of T-lymphocytic infiltrates, CAMs abundance and CTLs in DCM when compared with control non-cardiac deaths [7,8], the exclusivity of our criteria for intramyocardial inflammation is still an issue of debate [21], possibly due to methodological discrepancies (quantitative vs. semiquantitative evaluation) [22,23]. Several reports have shown the exclusivity of intramyocardial inflammation in DCM when compared with non-DCM. Immunohistological quantification of T-lymphocytes and macrophages revealed low infiltration densities in alcoholic cardiomyopathy, which further supports the hypothesis that intramyocardial inflammation is not a common finding in all failing hearts, irrespective of the underlying pathogenesis [36]. EMBs from patients with ischaemic heart disease demonstrated a low mean CD3+ count of 0 (standard deviation up to 2.6) per mm2 and significantly lower ICAM-1 and HLA DR expression when compared with DCM EMBs [37]. However, since these ischaemic hearts did not display left ventricular dysfunction, this investigation does not rule out lymphocytic infiltration or CAMs abundance in ischaemic cardiomyopathy.


   Acknowledgements
 
Michel Noutsias was supported by the Konrad-Adenauer-Foundation with a scholarship for his doctoral thesis under the supervision of Professor Schultheiss and Dr. Kühl.


   References
Top
 Abstract
 1. Introduction
 2. Patients and methods
 3. Results
 4. Discussion
 References
 

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