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(Circulation. 1995;92:2876-2885.)
© 1995 American Heart Association, Inc.

Articles

Phenotypic Patterns of Mononuclear Cells in Dilated Cardiomyopathy

Christoph Holzinger, MD; Andrea Schöllhammer, MD; Martin Imhof, MD; Christian Reinwald, MD; Gero Kramer, MD; Andreas Zuckermann, MD; Ernst Wolner, MD; Georg Steiner, Director

From the Second Department of Surgery (C.H., M.I., A.Z., E.W.) and Department of Urology (A.S., C.R., G.K., G.S.), University of Vienna, Austria.

Correspondence to Dr Christoph Holzinger, Allgemeines Krankenhaus Wien, Abteilung Herz- und Thoraxchirurgie, Währinger Gürtel 18-20, A-1090 Vienna, Austria.

	Abstract

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Background Immunological factors in the pathogenesis of idiopathic dilated cardiomyopathy (IDC) were suggested previously on the basis of the demonstration of mononuclear cell infiltrates and autoantibodies against the myocardium. The present study investigated whether tissue leukocyte subpopulations isolated from hearts with IDC (n=6) differ in phenotype from those of tissues without IDC (n=7).

Methods and Results Leukocytes were quantified as reactive cells per square millimeter in perivascular, interstitial, and parenchymal tissue sections. Freshly isolated heart-tissue T cells and peripheral-blood T cells from the same patients were analyzed by triple staining and flow cytometry to identify T-cell subpopulations as well as their states of differentiation (expression of CD45RA and Leu-8 versus CD45RO) and activation (IL-2R, IL-7R, very late antigen–1, HLA-DR). All types of infiltrating cells (T cells, B cells, macrophages, granulocytes) are increased in hearts with IDC compared with normal hearts, but only CD8⁺ T cells and macrophages are increased relative to the other leukocyte subpopulations. CD45RO⁺/CD45RA^-/Leu-8^- cells constitute the majority of heart-tissue T cells in both normal hearts and hearts with IDC. Strikingly, hearts with IDC are infiltrated by eightfold greater numbers of perivascularly located IL-2R⁺– (26% of all T cells) and CD45RO⁺–activated memory T cells; moreover, in contrast to normal heart, 40% of both CD4⁺ and CD8⁺ heart-tissue T cells express activation markers.

Conclusions Both normal hearts and hearts with IDC are populated by leukocytes. The quantitative increase in IDC, associated with a dramatically altered activation status of heart-tissue T cells, suggests a direct role of infiltrating leukocytes in the pathogenesis of IDC.

Key Words: cardiomyopathy • lymphocytes • genes • cells

	Introduction

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Idiopathic dilated cardiomyopathy (IDC) is a collective term for diseases of unknown cause in which signs and symptoms are due primarily to systolic dysfunction and cardiac dilatation leading to low-output states. The condition was defined by the World Health Organization as an often severe dilatation of one or both ventricles with impaired systolic function and invariably accompanied by hypertrophy. A number of factors have been implicated in the causation of IDC, including alcohol,¹ nutritional deficiency,² postpartal state,³ and viral infections.⁴ Its potentially pluricausal nature and the fact that its identification often requires a diagnosis of exclusion lead to difficulties in studying IDC. IDC patients were shown to carry both immunoglobulins bound to myocardial tissue^{5 6} and serum autoantibodies with and without organ specificity.^{7 8 9} On the other hand, only a few studies with conflicting results have dealt with alterations in cell-mediated immune mechanisms. Whereas some investigators observed a reduction of CD8⁺/CD11^- cytotoxic and an increase of CD4⁺/2H4⁺ suppressor/inducer peripheral-blood T cells in patients with IDC,¹⁰ others have demonstrated a defect in suppressor cell function in vitro.^{11 12} In another report, significantly increased numbers of both T-cell subpopulations were shown to be HLA-DR⁺ and thus activated.¹³ Sachs et al¹⁴ reported an elevated percentage of T suppressor cells, whereas Anderson et al¹⁵ observed no different frequencies of T-cell subpopulations. Also, it has been proposed that viral myocarditis can progress to IDC as a consequence of an autoimmune reaction.^{16 17 18} This assumption is supported by the identification of coxsackie virus B–specific RNA sequences and infiltrating lymphocytes among damaged myocytes in heart muscle biopsy specimens from some IDC patients.^{17 18 19 20}

Despite fundamental disagreement on many points, all of the cited reports share the observation that IDC is associated with an immunological process. Although this evidence essentially rests on analysis of peripheral-blood cells and largely unspecific investigations of the local infiltrate, the present study for the first time elaborates on the distribution and composition of cardiac tissue leukocytes, with the emphasis on T cells and their state of activation.

To determine which, if any, leukocyte subpopulations might have a pathogenic role, we evaluated the differential distribution of CD3⁺ T cells, their CD4⁺ and CD8⁺ subsets, CD20⁺ granulocytes, CD67⁺ B cells, and CD14⁺ macrophages in normal hearts (n=4), hearts with IDC (n=6), and hearts with ischemic congestive heart disease (n=3) by use of a number of recently developed monoclonal antibodies. In the next step, we addressed the state of differentiation and activation of tissue T cells derived from normal hearts compared with tissue T cells from hearts with IDC and the corresponding peripheral-blood T cells from the same patients. It was therefore necessary to develop a preparation technique for heart tissue–derived lymphocytes that allowed triple staining and analysis by flow cytometry. Subsequent screening for differential CD45RO (memory T cells), CD45RA (naive T cells), and Leu-8 (lymph node–homing receptor) expression enabled clear phenotypic differentiation between myocardium-specific tissue T cells and incidental contaminating or passenger peripheral-blood T cells; and triple staining for IL-2R -chain (CD25) and ß-chain, IL-7R, VLA-1, and HLA-DR was performed to evaluate their state of activation. By comparison with the phenotype of peripheral-blood T cells of the same patients, we analyzed whether the infiltrates were due to unspecific influx or specific migration and whether T-cell activation was local or systemic. Last, we addressed the question of whether infiltration is acute or chronic.

	Methods

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Specimen Collection
Myocardial tissues were derived from hearts excised for orthotopic heart allograft transplantation of patients in New York Heart Association functional class IV with end-stage IDC (n=6; 5 men, 1 woman; ages, 48 to 56 years) or ischemic congestive heart disease (n=3; 2 men, 1 woman; ages, 45 to 54 years). Corresponding tissues from normal individuals served as controls (n=4). Furthermore, 30 mL of heparinized peripheral blood was obtained from each patient at the time of the operation. All patients gave their informed consent.

Clinical Features
Criteria for diagnosing IDC were duration of symptomatology, gross enlargement of the heart relative to sex and age, congestive heart failure (in the absence of significant coronary artery sclerosis or valvular disease), and the microscopic findings of myocyte hypertrophy, nuclear hypertrophy, myocyte degeneration with myofibrillar attenuation, and interstitial and perivascular fibroses. All patients were in New York Heart Association functional class IV. Echocardiography, chest radiograph, gated radionuclide scan, and bilateral catheterization were performed in all cases. All IDC patients had highly dilated ventricles, as determined by left ventricular diastolic dimension, a cardiothoracic ratio >0.55, a severely depressed ejection fraction <15%, extremely poor contractility, and a normal coronary angiogram. Myocarditis was excluded on the basis of the Dallas criteria²¹ by histopathological evaluation of the excised organs. The postmortem examinations showed dilatation and enlargement of all chambers of the hearts. Valves and coronary arteries were normal. The clinical history of disease had lasted at least 8 months (mean, 13.4±7 months), and all patients had been on the transplantation list for at least 4 months. All patients were followed up on an outpatient basis but were advised to stay in the hospital for cardiac recompensation. All patients showed a gradual deterioration of myocardial function without signs of acute myocardial degradation. All patients met the World Health Organization criteria²² for the classification of IDC. Exclusion criteria at histopathological examination were hypertension, irradiation, pregnancy, endocrine disease, familial cardiomyopathy, exposure to toxic agents, and history of alcohol abuse or valvular disease. Patients with ischemic congestive heart disease had survived at least one myocardial infarction, had a severely reduced left ventricular function, intractability of heart ischemia due to surgical revascularization, or percutaneous transluminal coronary angioplasty as determined by coronary angiography. Control tissues from clinically normal hearts were harvested from multiorgan donors whose hearts were refused because of incipient coronary plaque formation of the left descending coronary artery (n=2), a bicuspid aortic valve (n=1), or logistic problems in the transplantation hospital (n=1).

Tissue Preparation
Heart specimens were frozen rapidly in isopentane in a liquid nitrogen bath and stored in liquid nitrogen. Frozen sections were cut into serial sections 4 µm thick, air-dried for 1 hour, and fixed in acetone for 10 minutes at 4°C.

Immunoperoxidase Technique
Controls for each staining procedure included irrelevant immunoglobulins of the appropriate isotype and/or second-step reagent controls without first antibody. Acetone-fixed cryocut sections were preincubated in 10% normal goat serum (Gibco BRL Life Technologies) for 30 minutes at room temperature, then incubated in the first antibody (Table 1; References 23 through 33) either overnight at 4°C or 1 hour at room temperature, followed by a PBS wash and incubation with biotinylated rabbit anti-mouse Ig (Dakopatts A/S) and Strept-ABComplex/HRP (Dakopatts) developed in 3,3-diaminobenzidine. Counterstaining was performed with hematoxylin, then sections were coverslipped in Aquamount (BDH Ltd).

View this table: [in this window] [in a new window]   Table 1. List of Monoclonal Antibodies Used in This Study

Quantitative Analysis of Tissue T-Cell Subpopulations
For enumeration of the various cell subpopulations, immunolabeled serial sections were analyzed with a rectangular ocular grid with a x40 oil objective. Approximately 100 fields within heart specimens were randomly chosen, and the density of positive cells was determined and expressed as the number of cells (±SD) per square millimeter of cryocut sections 4 µm thick. Microscopic tissue sections were reviewed with the sole purpose of characterizing the inflammatory cell infiltrates as follows: (1) inflammatory cell type: characterized and categorized by expression of various membrane antigens recognized by monoclonal antibodies and (2) location: perivascular (<30 µm from the endothelium), parenchymal (defined as the presence of inflammatory cells between myofibers without intervening fibrosis), and interstitial (regions of coarse, patchy, confluent areas of fibrosis in areas of fine interstitial fibrosis). Counted cells were quantified as cells per square millimeter. Intravascular cells were also analyzed, but because of the mechanical processes involved in the various washing steps, their presence was eventually purely random and the data inconclusive. Therefore, they were not included in the study.

Cell Preparation and Enrichment of Heart-Tissue T Cells
IDC tissue samples (50 to 100 g) were kept and minced into 1- to 2-mm³ fragments in PBS/heparin (Novo Nordisk A/S) and EDTA (Sigma Chemical Co) to prevent blood coagulation. Small pieces were then extensively washed by repeated intensive vortex treatment and short sedimentation in PBS/heparin until tissue fragments became white (light pink). Enzymatic dissociation and cell separation as previously described³⁴ were modified to create optimum conditions for tissue T-cell enrichment. Heart fragments were incubated with a solution of 200 U/mL type I collagenase (Sigma) and 100 µg/mL of DNAse type I (Sigma) in RPMI 1640 medium plus 10% FCS (complete medium). Digestion overnight revealed a homogeneous cell suspension without undigested larger particles, suggesting that all heart-tissue T cells were present in the suspension. Further enrichment, basically designed to remove major and minor debris, was performed by filtration through nylon wool and a 20% Percoll (Pharmacia) gradient centrifugation. Cells from the pellet were then adjusted to 1x10⁷/mL and layered on top of a discontinuous Percoll gradient containing 20%, 15%, 7.5%, and 5% Percoll solutions, 2 mL for each fraction, in a 15-mL tube and centrifuged for 20 minutes at 500g. Each Percoll fraction was harvested separately, and high-density fractions generated by 15% and 20% Percoll interfaces plus the pellet were subjected to osmotic shock treatment in 10 mL distilled water at 4°C for 2 minutes, followed by washing in complete medium. Killed cells were removed by density centrifugation over a Lymphoprep gradient (Nyco Med AS) at 37°C. Interphase cells were collected and are referred to as enriched tissue T cells.

Three-Color Staining of Tissue and Peripheral-Blood T Cells
Lymphoprep gradient–derived cells were washed twice in PBS containing 0.2% NaN₃ and 1% FCS. Aliquots of cell suspension (100 µL) containing 1x10⁵ cells were incubated for 30 minutes at 4°C with FITC-, phycoerythrin-, and peridinin-chlorophyll protein–conjugated monoclonal antibodies as well as appropriate nonsense controls in all possible combinations. Two- and three-color analyses were performed by flow cytometry (FACScan, Becton, Dickinson & Co). Cellular debris and nuclei were excluded by threshold and the conservative use of forward and side scatters without altering the view of enriched lymphocytes and their heterogeneity. Discontinuous Percoll gradient and osmotic shock treatment were performed because fluorescence-activated cell sorter (FACS) analysis of the nonenriched population revealed intense autofluorescence due to muscle and stromal cells. By using multicolor systems (LYSIS Software, Becton Dickinson), we were able to confirm that further enrichment had no effect on the composition or the antigen expression pattern of heart-tissue T cells. Osmotic shock treatment was used to kill nonlymphoid cells, which, because of their inflexible cytoskeleton compared with T cells, are unable to expand their surface effectively enough to survive.

Statistical Analysis
All values are expressed as mean±SD. Significance of differences was calculated with Student's t test. A value of P<.05 was considered significant.

	Results

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Differential Distribution Patterns of Peripheral-Blood T Cells and Heart-Tissue T Cells in Normal Heart, IDC, and Ischemic Congestive Heart Disease
No significant differences were observable between peripheral-blood T cells from normal heart and those from tissues with IDC or ischemic congestive heart disease, nor did tissue T cells from normal heart and from ischemic congestive heart disease differ significantly. Therefore, only the alterations of tissue T cells from hearts with IDC compared with peripheral-blood T cells from patients with IDC and tissue T cells from normal hearts will be discussed in detail.

Establishment of an Enrichment Method for Heart-Tissue T Cells
Although immunohistochemical staining demonstrates whether a certain antigen is expressed on the membrane of individual cells, it is unsuited to analyze the percentage distribution of various cell subpopulations, the presence of additional activation markers on the same cell, or the intensity of antigen expression. Since the definition of various cell subsets, and particularly any conclusion about their state of activation, strictly depends on demonstrating more than one antigen on the same cell, we established methods of cell separation enabling analysis of cardiac T cells by three-color flow cytometry.^{34 35}

Our enrichment technique for heart-tissue T cells was described in detail in the "Methods" section. The crucial point was to consistently produce fragments small enough to allow extensive washing until the red color was gone. To validate our technique regarding peripheral-blood contamination, freshly prepared tissue and peripheral-blood T cells of the same patients were tested for Leu-8, CD45RA (characteristic of peripheral-blood T cells), and CD45RO (present predominantly on tissue T cells) expression.

One representative triple-staining experiment is depicted in Fig 1, and Table 2 summarizes the results for all 5 IDC patients. Fig 1 shows the histograms of CD45RO, CD45RA, and Leu-8 expression by CD4⁺ and CD8⁺ tissue T cells and peripheral-blood T cells in IDC. The purity of tissue T-cell suspension is best documented by comparing the number of CD45RA⁺ and Leu-8⁺ cells of both origins. Whereas heart-tissue T cells predominantly belong to the group of Leu-8^-/CD45RA^-/CD45R0⁺ memory T-cell subset, most peripheral-blood T lymphocytes express Leu-8 and CD45RA, and only a minor percentage CD45RO. This phenotypic difference clearly demonstrates that contamination of the tissue T-cell suspensions by peripheral-blood T cells amounted to <10%.

View larger version (109K): [in this window] [in a new window]   Figure 1. Graphs showing comparison of heart-tissue T cells (top) and peripheral-blood T cells (bottom) regarding CD45RA, CD45RO, and Leu-8 expression on all CD3+ T cells. Triple staining using anti–CD8-PerCp or anti–CD4-FITC (y axis) in combination with anti–CD45RA-PE, anti–CD45RO-PE, anti–Leu-8-PE (x axis) and either anti–CD3-FITC or anti–CD3-PerCp (data not shown). The percentage of reactive cells is calculated on the basis of either CD3+/CD4+ or CD3+/CD8+ cells.

View this table: [in this window] [in a new window]   Table 2. Memory and Naive T Cells in Hearts With Idiopathic Cardiomyopathy Compared With Peripheral Blood of the Same Patients

Expression of Markers for Naive (CD45RA) Versus Memory (CD45RO) T Cells and for Lymph Node–Homing Receptor (Leu-8) by Peripheral-Blood and Heart-Tissue T Cells of IDC Patients
The above experiments (Fig 1, Table 2) were also performed to obtain data on the type of tissue T cells infiltrating IDC tissue. It emerged that they belong predominantly to the group of primed T cells known to constitute the "normal" tissue T-cell type in nonlymphatic organs, which are also present in normal heart tissue, albeit in significantly lower numbers. The demonstration of significantly more CD4⁺/CD45RO⁺ helper/memory than CD8⁺/CD45RO⁺ cytotoxic/suppressor memory T cells among tissue T cells, which is in contrast to the ratio seen in the peripheral-blood T-cell population of the same patients, suggests a selection of newly incoming peripheral T cells (Table 2). The overlapping percentages of CD45RA⁺/CD4⁺ (27.5%) and CD45RO⁺/CD4⁺ (96%) indicate that a population of about 20% of CD4⁺ tissue T cells coexpress both differentiation antigens and, thus, reflect a transient state of primary T-cell activation.

Whereas CD45RO expression was demonstrated on almost all CD4⁺ (96±7.2%) and most CD8⁺ (76.1±6.1%) tissue T cells in IDC, indicating that they bear predominantly the memory T-cell phenotype, it was observed on only a few CD4⁺ (27.8±2.2%) and CD8⁺ (33.2±4.7%) peripheral-blood T cells in IDC. Conversely, peripheral-blood T cells were found to include more CD4⁺/CD45RA⁺ (71±5.3%) and CD8⁺/CD45RA⁺ (94.5±6.7%) cells, compared with 27.5±4.3% and 28.2±4.7% tissue T cells, respectively (Table 2). Leu-8 (Mel-14) expression composed 11.1±3.7% of CD4⁺ tissue T cells, 12.4±1.2% of CD8⁺ tissue T cells, 93.3±7.8% of CD4⁺ peripheral-blood T cells, and 78.6±6.6% of CD8⁺ peripheral-blood T cells (Table 2). The relatively high percentages of Leu-8⁺ (mean, 23.5%) and CD45RA⁺ (mean, 55.7%) T cells in tissue T-cell suspensions, which were also confirmed in IDC tissue, suggest a frequent ongoing process of lymphocytic infiltration in IDC tissue.

Expression of T-Cell Activation Markers by CD4⁺ T-Helper Cells and CD8⁺ Suppressor/Cytotoxic Heart-Tissue T Cells and Peripheral-Blood T Cells in IDC
T cells can support or suppress B cells and other lymphocytes, as well as destroy target cells. To perform any of these functions, they have to be activated. Therefore, we investigated the expression of the T-cell activation markers IL-2R -chain and ß-chain, IL-7R, HLA-DR, and VLA-1. Intracellular signal transduction induced by IL-2 is transmitted via high- or intermediate-affinity receptors, indicating that the IL-2R ß-chain has a role in driving the intracellular signal transduction pathway. VLA-1 is the latest-emerging activation antigen currently known^{30 36} and binds to connective tissue compartments. IL-7 is a potent stimulator of T-cell proliferation and, in contrast to IL-2, is independent of any additional stimuli such as antigen presentation.^{37 38} Reexpression of functional IL-7R on tissue T cells was found to be associated with intense T-cell infiltration.³⁹ HLA-DR is an established T-cell activation marker expressed by all activated T cells.³²

Our experiments revealed that all immunohistologically analyzed IDC specimens distinctly differ from normal heart in both the intensity and distribution of activation marker expression. Fig 2 shows a representative experiment obtained with tissue and peripheral-blood T cells from IDC patients. Activation marker expression was found to be significantly increased on both CD4⁺ and CD8⁺ tissue T cells (Fig 2) compared with peripheral-blood T cells. HLA-DR expression was increased 4 times, VLA-1 >10 times, and IL-2R ß-chain 3 times (Table 3). Low-affinity IL-2R -chain was expressed primarily by the CD4⁺ subset, without a significant difference between tissue and peripheral-blood T cells (data not shown), suggesting partial high-affinity IL-2R (heterodimer) expression by tissue T cells. Similar results were obtained with an anti-CD29 antibody recognizing all members of the integrin-ß1 family (CD29^high expression by CD4⁺ tissue T cells, 82.3%; by CD8⁺ tissue T cells, 87.3%; by CD4⁺ peripheral-blood T cells, 50.2%; and by CD8⁺ peripheral-blood T cells, 45.7%). Interestingly, IL-7R, usually a marker for peripheral-blood T cells and found on tissue T cells only in cases of extreme infiltration, as in transplant rejection, was expressed by both CD4⁺ (17.3±3.2%) and CD8⁺ (9.1±2.7%) tissue T cells.

View larger version (96K): [in this window] [in a new window]   Figure 2. Graphs showing comparison of heart-tissue T cells (top) and peripheral-blood T cells (bottom) regarding activation marker expression on all CD3+ T cells. Triple staining using anti–CD8-PerCp or anti–CD4-FITC (y axis) in combination with anti–IL-7R-PE, anti–IL-2Rß-PE, or anti–VLA-1-PE (x axis) and either anti–CD3-FITC or anti–CD3-PerCp (data not shown). The amount of antigen expression is given as mean intensity of the ungated CD4+/CD3+ or CD8+/CD3+ populations. The values for the appropriate negative controls were 5.79 for CD8+ and 6.11 for CD4+ heart-tissue T cells and 2.29 for CD8+ and 2.32 for CD4+ peripheral-blood T cells.

View this table: [in this window] [in a new window]   Table 3. Activation Markers on CD4+ or CD8+ Tissue T Cells and Peripheral-Blood T Cells in Hearts With Idiopathic Cardiomyopathy

Patterns of Leukocyte Subpopulations in Normal Heart Versus IDC Tissue
The normal specimens exhibited small numbers of cells (17±2.7/mm²) bearing the CD45 antigen, a panhematopoietic cell surface marker, that were evenly distributed throughout the organ (Table 4).³¹ By comparison, all CD45⁺ leukocyte subsets in all IDC tissues analyzed (57.7±6.1 cells/mm²) were massively increased, accompanied by an altered percentage distribution: Whereas the increase of CD4⁺ T cells and CD20⁺ B cells was in accordance with the overall increase in CD45⁺ cells, the CD14 (monocytes/macrophages) and CD8 frequencies showed additional fivefold and fourfold increases, respectively. This is reflected in a shift of the T-cell ratio of CD4⁺ to CD8⁺ (T-helper cells to cytotoxic/suppressor T cells) from 4:1 in normal heart to 2.5:1 in IDC tissue. CD67⁺ granulocytes were increased in absolute numbers but decreased relative to the other leukocyte subpopulations (Table 4).

View this table: [in this window] [in a new window]   Table 4. Leukocyte Populations in Tissues Derived From Normal Hearts and Hearts With IDC as Determined by Immunohistochemical Staining

	Demonstration of Significantly Increased CD45RO+ Memory T Cells in Various Compartments of IDC Tissue

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All T cells initially express CD45RA and, after activation, lose this membrane antigen, acquiring CD45RO instead. These findings, together with the observation that CD45RO⁺ cells, in contrast to CD45RA⁺ cells,^{31 40} respond to soluble antigens maximally, indicate that acquisition of CD45RO identifies primed T cells.^{41 42} Since CD45RO⁺ T cells are characterized by prior exposure to antigen, they possess a much higher degree of cytotoxicity⁴³ and are inducers of increased immunoglobulin-producing B cells⁴⁴ ; they proliferate vigorously when restimulated with antigen^{44 45} and produce large amounts of growth factors such as interferon gamma.⁴⁶ Leu-8 is a lymphocyte adhesion molecule enabling these cells to localize in or home to certain secondary lymphoid organs such as peripheral lymph nodes or high endothelial vessels.

The number of cells expressing the above-mentioned markers increased from the perivascular through the parenchymal to the interstitial tissue areas (definitions in "Methods" under "Quantitative Analysis of Tissue T-Cell Subpopulations"). In normal hearts, CD45RA (naive T cells) was expressed by 0.18±0.1 and Leu-8 by 0.35±0.2 cells/mm² perivascularly, increasing through parenchymal to interstitial (4.5±1.1 and 7.6±2.1 cells/mm², respectively). CD45RO (memory T cells) was expressed by 0.4±0.2 perivascular cells/mm² and mostly by interstitial cells (21±5.3 cells/mm²). If we follow the relative percentages of both populations through the various tissue segments, there was a shift away from CD45RA⁺/Leu-8⁺ cells toward CD45RO⁺ cells from perivascular (1:1) through interstitial (3:1) to parenchymal (12:1) (Table 5). By comparison, IDC tissues overall showed dramatically greater numbers of T cells, ranging from 3.1±0.9 perivascular to 134±27 interstitial CD45RO⁺ cells/mm². Despite this sharp increase, the ratio of CD45RO⁺ memory to CD45RA⁺ and Leu-8⁺ naive tissue T cells in the three tissue compartments was more consistent, the former outnumbering the latter by about six times, suggesting a markedly intensified lymphocytic influx compared with normal tissue (Table 5). Interestingly, the IDC samples showed only twice as many CD45RO⁺ cells parenchymally as the normal hearts, whereas the two other tissue areas exhibited a sixfold increase of memory T cells compared with normal heart.

View this table: [in this window] [in a new window]   Table 5. Naive and Memory T Cells in Various Tissue Compartments of Normal Hearts and Hearts With IDC

Expression of IL-2R and IL-7R in Various Compartments of Normal and IDC Tissues
In normal tissues, expression of both IL-2R and IL-7R was extremely low, constituting only 3.2% (0.47±0.12 cells/mm²) and 4.7% (0.66±0.13 cells/mm²), respectively, of CD3⁺ T cells. In IDC tissues, IL-2R was expressed by 18.4% and IL-7R by 8.6% of CD3⁺ cells. In normal tissues, no IL-2R⁺ or IL-7R⁺ cells were found perivascularly, and only small numbers in both the interstitial and parenchymal compartments (0.3 and 0.17 cells/mm², respectively) (Table 6). By comparison, the number of perivascular CD3⁺ cells in IDC was four times higher, and 20% or 26% (0.2 or 0.3 cells/mm²) thereof reacted with anti–IL-7R and anti–IL-2R, respectively, which confirms the finding that lymphocytic traffic is intensified in IDC tissue (Table 6). Interstitial areas in IDC showed the most significant increase in CD3⁺ cells, associated with strongly elevated lymphokine-receptor expression (Table 6). Predictably, the percentage of IL-7R⁺ cells decreased from perivascular (20%) through interstitial (16%) to parenchymal (5%), indicating the pathway of lymphocytic traffic (Table 6).

View this table: [in this window] [in a new window]   Table 6. Expression of Activation Markers on CD3+ Cells in Various Tissue Compartments of Normal Hearts and Hearts With IDC

	Discussion

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The information available on the state of the immune system in normal hearts and in hearts with IDC is conflicting. Considerable attention has been focused on the concept of immune-mediated damage as a possible causal factor in IDC, especially after acute myocarditis. An enteroviral genome can be demonstrated in 29% of patients with end-stage IDC.⁴⁷ None of the investigations of cellular events in IDC have compared peripheral-blood cells with infiltrating cells of the same patients, and the number of T-cell markers included in these studies is very limited. It has been an open issue so far whether the T cells observed in hearts with IDC are representative of a specifically disease-related, ongoing, local immune response or, alternatively, are inactive residues of an earlier immune response not directly related to IDC.^{48 49 50} Since monitoring of peripheral-blood lymphocytes is inadequate even for diagnosing transplant rejection, an immunologically much more dramatic event than IDC,²⁵ we concluded that more in-depth information can be obtained by analyzing the phenotype of the leukocytic infiltrate within the myocardium, using a number of recently developed monoclonal antibodies that for the first time allow discrimination between the various T-cell subsets and their potential functional states.

The present study demonstrates that the overwhelming majority of heart-tissue T cells are CD45RO⁺/CD45RA^-/Leu-8^- primed memory T cells. In the perivascular tissue compartment (ie, where the phenotype of tissue T cells may be expected to come closest to that of newly incoming T cells) of normal heart, only about half of the CD3⁺ cells were CD45RO⁺ (Table 5). Compared with the phenotype of peripheral-blood T cells of the same patients, the conclusion suggests itself that these CD45RA- and/or Leu-8–expressing perivascular cells are part of the physiological lymphocyte turnover and not immediately related to the localized tissue T cells. These patrolling CD45RA⁺/Leu-8⁺ T cells advance as far as the interstitium but are hardly demonstrable in the parenchyma. On the other hand, the average influx of CD45RO⁺ cells into the perivascular region is eight times as high in IDC (3.1 cells/mm²) as in normal heart (0.4 cells/mm²), whereas the numbers of perivascular CD45RA⁺ and Leu-8⁺ cells remain almost constant. Hence, this massively stepped-up migration must be regarded as a selective process and, in addition to the possibility of local T-cell proliferation, is one possible explanation for the total T-cell increase in IDC tissue. Together with the striking finding that a large number of perivascular cells in IDC express the activation marker IL-2R, this means that hearts with IDC, unlike normal hearts, are heavily infiltrated by peripheral CD45RO⁺/IL-2R⁺–activated memory T cells. Flow cytometry revealed that this cell pool includes both CD4⁺/CD45RO⁺/IL-2R⁺ and CD8⁺/CD45RO⁺/IL-2R⁺ cells in equal proportions.

The present study clearly demonstrates that the number of T cells is significantly increased in all tissue compartments of hearts with IDC and, even more importantly, that in IDC a great percentage of heart-tissue T cells express activation markers. The findings of increased numbers of CD45RO⁺, and especially of activated IL-2R⁺, tissue T cells in IDC compared with normal heart argue in favor of an ongoing immune response. A similar picture was obtained in a variety of human diseases, such as multiple sclerosis or rheumatoid arthritis,^{52 53 54} in which the majority of T cells are CD45RO⁺ and CD45RA^-. Also, the interpretation that increased CD45RO expression in hearts with IDC reflects increased conversion from the naive to the memory state as a result of an ongoing immunologic response is fully consistent with patterns of tissue inflammation in leprosy⁵⁵ : Whereas in tuberculoid leprosy there is an effective immune response associated with both accumulation of CD45RO⁺ memory T cells and strong antigen reactivity, lepromatous leprosy, which is associated with T-cell inflammation primarily of the CD45RA⁺ phenotype, is characterized by an inadequate immune response. A recent report showed that mild heart allograft rejection was associated with infiltration by predominantly CD45RA⁺ T cells, whereas in the event of moderate rejection, significantly more CD45RO⁺ infiltrating T cells were observed.⁵⁶

The result that, with the exception of IL-2R -chain on CD4⁺ peripheral-blood T cells, all T-cell activation markers used in the present study were (1) more frequently observed on heart-tissue T cells than on peripheral-blood T cells of the same patients and (2) expressed in a similarly strong manner by both CD4⁺ and CD8⁺ tissue T cells shows that the predominant CD45RO⁺ memory T-cell phenotype demonstrated in IDC tissue reflects ongoing activation rather than a resting state after previous priming in situ. This conclusion is further underlined by 43.55% of CD4⁺ and 15.1% of CD8⁺ tissue T cells expressing IL-2R -chain and (50% of both tissue T-cell subsets expressing IL-2R ß-chain, in the complete absence of any ß-chain expression by peripheral-blood T cells. Similarly, both tissue T-cell subsets reacted intensely with anti–VLA-1 (46% and 41%, respectively) in the absence of any significant amounts of VLA-1⁺ peripheral-blood T cells. Since the integrin-ß1 family members are receptors responsible for the interaction with matrix compounds, it would appear likely that in IDC, upregulated VLA-1 and CD29⁴¹ expression (data not shown) reflects activation and increased attachment of tissue T cells to the myocardium,^{36 41} although extracellular matrix components have also been shown to modulate lymphocyte behavior.

To summarize, our findings (1) support the hypothesis that cell-mediated immunity has a role in myocyte degeneration, or destruction, in IDC and (2) argue in favor of an ongoing, chronic immune process in its pathogenesis. This is confirmed by the fact that the distribution of peripheral-blood T-cell subsets was not significantly different from that of tissue T cells, suggesting that tissue T cells from normal heart are identical to those involved in initiating and maintaining this process.

	Acknowledgments

 
This study was supported by the Bundesministerium für Wissenschaft und Forschung and the Ludwig Boltzmann Institut für Herzchirurgische Forschung.

Received March 2, 1994; revision received May 17, 1995; accepted June 13, 1995.

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