Immunologic studies of peripheral blood from patients with idiopathic dilated cardiomyopathy

Immunologic studies of peripheral blood from patients with idiopathic dilated cardiomyopathy Immune function, T-lymphocyte subsets, serum quantitative immunogiobulin levels, serum lysoryme levels, and circulating immune complex levels were analyzed in patients with idiopathic dilated cardiomyopathy (IDCM). The percentage of helper/inducer T ceils (OKT4) was higher and the percentage of suppressor/cytotoxic T cells (OKT8) was lower in IDCM patients than in healthy controls and in patients with ischemic heart disease. IDCM patients, in addition, have higher 5/9+ T ceils, a T-cell subset known to give maximal helper activity in B-ceil differentiatfon assays. Peripheral blood mononuclear cells (PBMC) from IDCM patients demonstrated a statistically greater ability to induce B-cell differentiation (helper T-cell function) into plasma cells and a hypofunctioning suppressor T-ceil population in an in vitro pokeweed nitrogen (PWN)-driven B-ceil differentiation assay. Serum immunogiobulin IgM levels were higher in IDCM patients, but serum lysozyme levels and serum immune complex levels in IDCM patients were normal. These data verify that an immunoregulatory defect exists in IDCM. (AM HEART J 112:350, 1988.)

Roberto Gerli, M.D., Pietro Rambotti, M.D., Fabrizio Spinozzi, M.D., Alberto Bertotto, M.D., Vincenzo Chiodini, M.D., Pasquale Solinas, M.D., Ivano Gernini, B.Sc., and Stephen Davis, M.D.* Perugia, Italy

Idiopathic dilated cardiomyopathy (IDCM) is a diagnosis characterized by biventricular dysfunction and congestive heart failure. Patients with IDCM, by definition, do not have hypertension, coronary atherosclerosis, valvular lesions, toxin exposure, or metabolic disorders. The frequent presentation of IDCM after resolution of an apparently common viral illness,’ suggests that an abnormal immune response to viral-induced antigens may be involved in perpetuating myocardial damage. While this hypothesis is supported by the analysis of myocardial biopsies that may demonstrate mononuclear cell infiltration and immunoglobulin deposition on sarcolemmal membranes, functional studies of the immune system are sparse and controversial.2-4 Because abnormalities of the immune system may be involved in the pathogenesis of IDCM, we studied immunoregulatory mononuclear cell populations and their functional capacities in patients suffering

From lstituto di Clinica University; and the Divisione Received accepted Reprint Universita *Professore

350

for publication Dec. 22, 1985.

Medica I B di di Cardiologia, July

19, 1985;

requests: Pietro Rambotti, di Perugia, 06100 Perugia, a Contratto,

Istituto

revision

M.D., Italy.

di Clinica

Clinica Pediatric& Ospedale Regionale.

lstituto Medica.

received

Nov.

di Clinica

Perugia 15, 1985; Medica

1,

from IDCM. Our data support the hypothesis T-cell dysfunction is present in this disease.

that

METHODS Patients. The clinical features of the study groups are shown in Table I. The first group (cardiac control) comprised 18 subjects with ischemic heart disease without clinical signs of dilated cardiomyopathy. The second group (cardiac control) included eight patients with dilated cardiomyopathy associated with coronary artery disease and/or myocardial infarction. The third group included eight patients suffering from IDCM. In the last group, each patient had no history of alcohol abuse, hypertension, irradiation, pregnancy, endocrine disease, familial cardiomyopathy, exposure to toxic agents, or history of valvular disease. Healthy volunteer subjects (age-matched to the IDCM group) were used as healthy controls for all experiments. Lymphocyte preparation. Peripheral blood mononuclear cells (PBMC) were separated from blood samples by centrifugation over a Ficoll-Hypaque density gradient (Lymphoprep, Nyegaard, Oslo, Norway) at 400 x g and were then suspended in RPMI-1640 (Gibco, Grand Island, N.Y.) containing 10% fetal calf serum (FCS, Gibco) at 2 X lo6 ml and depleted of mononuclear adherent cells by incubation in tissue culture flasks for 90 minutes at 87O C in a 5% carbon dioxide atmosphere. The harvested nonadherent cells were treated by latex ingestion in order to evaluate the phagocytizing capacity. The monocyte con-

4

Volume

112

Number

2

Table

I. Clinical characteristics of the three patient groups disease

Ischemic Without

DCM

Sex

13 males 7 females

Age Cardiac symptom onset Therapy Physical examination

39-66 years 2 ms-27 yr ago Digoxin, furosemide -

Chest x-ray ECG

Normal Ischemic pattern, previous myocardial infarction (12/18) -

Echocardiogram Stint. (:O’Tl)

DCM

!I)( ‘A4

males 2 females 47-69 years 2-32 yr ago Digoxin, furosemide S3(7/8), S4{2/8), mitral (6/8) and tricuspid (l/S) valvular regurgitation Cardiac enlargement Ischemic pattern; previous myocardial infarction

4 males 4 females 25-42 year\ 2 ms-6 yr ago Digoxin. furosemide S3 (8/8), S4 iNX), mitral valvular regur&.ation

6

Hypoperfusion areas Signs of coronary artery obstruction

Coronary arteriography

----With

(5/8)

Cardiac enlargement No ischemic pattern

(4/8)

Left ventricular Hypoperfusion

dilatation

Left ventricular dilatation. hypokinesia, small pericardial effusion (2/8) Hypokinesia, le!‘t ventricular dilatation Absence of coronary artery obstruction !:i,%i

areas

Signs of coronary artery obstruction (81%)

(6/18)

Myocardial

biopsy

Not done

Not done

No fibrosis and inflammat,ory infiltration cpts 1.2.3,4’1

Normal healthy controls were free of disease and were not ingesting medications known to affect immune group. IDCM = idiopathic dilated cardiomyopathy; DCM = dilated cardiomyopathy; Stint = scintigraphy: ““‘TI

tamination were lessthan 2 % in all resultant lymphocyte suspensions.T cells were enriched to greater than 90% purity by E-rosette formation with neuraminidase-treated (Sigma, St. Louis, MO.) sheep erythrocytes (nSRBC), as previously described.6 lrnmunofluorescence studies. T-Lymphocyte surface phenotypes were determined by meansof murine antihuman mature T-cell (OKT3), antihuman mature inducer/ helper T-cell (OKT4), and antihuman mature suppressor/ cytotoxic T-cell (OKT8) monoclonal antibodies (Ortho, Raritan, N.J.).’ To identify a newly described restricted T-cell subsetwith maximal helper activity to induce B-cell differentiation in vitro, 519 monoclonal antibody was employed. The percentageof surface membrane immunoglobulin positive (SIg+) PBMC wasevaluated by a direct immunofluorescenceassay. Activated T-lymphocytes in enriched T-cell populations were assessed by membrane staining with monoclonal antibodies against the IL-2 receptor (anti-TAC)? against HLA-DR antigen (OKIa,)‘O and against the HLA-DC/IX antigen.(LeulO).” Pokeweed tion assay.

mitogen

(PWM)

driven

B-cell

differentia-

For culture experiments, 2 x 106/ml PBMC were suspendedin RPMI-1640 containing 10% FCS, 2 gm/L sodiumbicarbonate, 25mM/L Hepesbuffer (Gibco), 2mM/L L-glutamine (Gibco), 50 U penicillin, and 50 I.rg streptomicin/ml at pH 7.2. Cell suspensionwasplaced in a

Limbro

function.

_ ...__..^ They were age. rx;ntrhed

to the II)CM

= thallium-3~l.

24 flat-bottom

well

plate

(Flow

Laboratories,

Springfield, Va., U.S.A.) and cultured with and without pokeweedmitogen (PWN, Gibco) dilution of 1: 100, at 37” C in a humidified incubator containing FjC, CO,. After 7 days culturing, B-cell differentiation into secretory plasma cells was determined using acetic ethanol-fixed cytocentrifuge cell preparations stained with FITC-labelled goat antihuman Ig antibodies (Kallestad). Plasma cells containing brightly staining cytoplasmic Ig per 1000 nucleated cells were counted on a Leitz Ortholux microscopeequipped with an Osram 200 mercury arc lamp. Functional PWM-driven

tests B-cell

for help and differentiation

suppression of the assay. The helper

function of controls and IDCM patient T-lymphocytes was tested by adding 1 X 10” enriched T-cells to equal amounts

of control

or IDCM

patient

PBMC,

in duplicate

flat-bottom culture wells (Flow Laborat.ories). The suppressorfunction of T-cells was assayedby adding 1 x 10” irradiated (1000rads) enriched T-cells to PBMC. PWM at a final dilution of 1: 100 was added to each culture well. After 7 days, B-cell differentiation into secretory plasma cells was determined as mentioned above. Serum immunoglobulins. IgG, IgA, and IgM serum levels were determined by radial immunodiffusion method (NOR-Partigen plates, Boehring, West Germany).‘:! Values are expressedas milligrams per deciliter.

352

Gerli

Table

II. B- and T-cell subsets,as identified by SmIg and anti-T

Mean Ischemic Ischemic

et al.

American

IDCM patients

OKT3 (‘Ls)

1

72 80 76 83 92 70 79

2 3 4 5 6 7 8 + SEM with without

DCM

Normal healthy controls (N = 20) (age-matched to IDCM) At~breviations

78.8 + 2.4

nd nd nd 28 36 27 31 16 27.6 + 3.3

80.7 + 1.8

53.4 -+ 2.3

nd

as in Table

81.3 k 1.3 79.8 +- 1.6

OKT8 (‘h)

519 (“bI

58 62 59 62 83 68 77 48 64.6 + 3.9

79 DCM

OKT4 (27)

0.005 50.8 k 1.3 J 0.02 52.6 f 1.9 J

SmIg C’cI

16 17 19 22 14 24 15

5 5 8 7 4 4 13 12 7.2 -+ 1.2 ___7.6 ? 1.1

15.5 t 1.2 16.6 * 1.4

August. 1966 Heart Journal

6.6 i- 0.6 0.01 d

29.6 Jo 1.9

0.005 J

6.9 + 0.7

I.

Circulating immune complexes. ClqB-ELISA wasused for estimating the levels of circulating immune complexes, as previously described.‘:’ Statistics. The unpaired Student’s t test was adopted for analyzing samples.Values below p < 0.05 were considered significant. RESULTS Surface

monoclonal antibodies) in 8% (range: 0% to 13%) and 5 % (range: 0% to 6 % ) of cells, respectively. Only lymphocytes from patients No. 1, 3, and 7 had activated T cells demonstrated; however, activated T cells were neuer detected in preparations from cardiac control patients or in healthy controls.

marker analysis. No significant differences were seen in the total lymphocyte count of IDCM,

Serum immunoglobulins and circulating immune complexes (CIC). The increase of serum immunoglobulins

cardiac control and healthy patients (data not shown). As shown in Table II, B-cell (7.6 +- 1.1% vs 6.6 + 0.6%), total T-cell (80.7 + 1.8% vs 81.3 f 1.3%), and T-cell subset percentages (OKT4: 53.4 f 2.3 % vs 50.8 + 1.3 % ; OKTB: 29.2 + 2.8% vs 30.6 f 1.1%) did not differ between the two groups of cardiac control patients. Despite the fact that Band T-cell proportion in IDCM patients was similar to cardiac controls, we observed in IDCM patients a high OKT4+ (64.6 + 3.9%) and a low OKT8+ (19.9 + 2.1% ) cell percentage. Healthy volunteers were similar to cardiac controls. The high percentage of circulating OKT4+ phenotypic helper T cells was associated with an increased proportion of 5/9+ helper T cells (27.6 + 3.3% vs 15.15 -t 1.2%). Monoclonal antibody analysis carried out to reveal the presence of activated T cells in enriched T-lymphocyte preparations, indicated the presence of IL-2 membrane receptor (as detected by anti-TAC monoclonal antibody) in 3.5% of cells (range: 0% to 5%) and HLA-DR and HLA-DC/DS surface antigens (as identified by OKIa and LeulO

in IDCM patients with respect to the cardiac controls was due to a significant rise of IgM levels (IDCM = 310.4 +- 41.5 mg/dl; ischemic DCM = 165.7 f 17.8 mg/dl; ischemic = 183.4 + 30.6 mg/dl). Circulating immune complex levels in IDCM patients and in the two cardiac groups were in the normal range (data not shown). Lymphocyte culture experiments. Unstimulated IDCM lymphocytes had transformed into a slightly greater number of plasma cells per 100 nucleated cells after a 7-day culture than normal controls, but we observed a remarkable increase in plasma cell formation after a PWM-induced mitogenic stimulation in six out of eight IDCM patients with respect to normal controls Cp < 0.01, Fig. 1; note logarithmic scale). Healthy control and cardiac patient lymphocyte responses were similar, so only healthy controls are represented in Fig. 1 for simplicity. Assay

to test

helper

and suppressor

function

of T

cells. As shown in Fig. 2, a, in the six IDCM patients with exuberant plasma cell production following PWM stimulation, the addition to their enriched T

Volume Number

112 ‘2

cells of normal PBMC led to a considerable increase in plasma cell generation compared to that obtained with normal T-PBMC or cardiac T-PBMC cultures, supporting the hypothesis that an activated helper T-cell subpopulation exists in IDCM lymphocyte suspensions. Furthermore, enhanced plasma cell transformation of normal PBMC, observed following the addition of control irradiated T (TX) cells, was attenuated when IDCM patient TX lymphocytes were added to the culture, suggesting that IDCM lymphocyte suspensions contain a reduced number or a reduction in functional radiosensitive Tsuppressor cells (Fig. 2, a). Healthy control and cardiac control samples yielded identical results, so only healthy controls are shown for simplicity. Plasma cell generation declined when normal control or ischemic T lymphocytes were added to IDCM patient PBMC (Fig. 2, b), but rose again after control or cardiac-irradiated T cells were added, supporting the suggestion that the enhanced plasma cell generation in IDCM lymphocyte cultures is due to activated T cells. DISCUSSION

Immunoregulatory dysfunction in IDCM has been suggested by the findings of lymphoid myocardial infiltration and immunoglobulin deposition in myocardial tissue.14 Fowles et a1.2 reported defective in vitro suppressor T cell function in IDCM; and reduced suppressor T cell activity was subsequently confirmed by Eckstein et a1.3 On the other hand, Anderson et al.‘” could not find any immunoregulatory defect in IDCM patients. It is therefore apparent that data on immune function in patients with IDCM are limited and conflicting. Our phenotypic data of PBMC showed a reduced percentage of suppressor T cells (OKT8+) in IDCM. Additionally, however, the high OKT4+/OKT8+ cell ratio that we found in IDCM patients was also due to an increase of the helper cell subset (OKT4+). These data confirmed the recently published findings of Sanderson et al.‘” These investigators found in African IDCM patients an increase in OKT4+ cells and a decrease in OKT8+ cells, i.e., increased OKT4/OKT8. A more detailed phenotypic analysis within the functionally heterogeneous OKT4+ subpopulation demonstrated an increase in the 5/9+ cell subset, suggesting that the numerical decrease of suppressor T lymphocytes is associated with an increase of a T-cell population with maximal helper effect on B-cell differentiation. Despite the fact that circulating immune complexes were not elevated, we found a slight B-cell

Lnst

PWbl’ NC

PT

Fig. 1. Plasma cell generation in cultures with and without pokeweed mitogen (PWM) of IDCM patient (PT) and normal control (NC) mononuclear cells. The two patients within the normal range are cases No. 3 and 8. Cardiac controls are excluded since data generated were identical to that from healthy normal controls.

polyclonal activation “in viva” in IDCM patients, as demonstrated by an elevated serum IgM level, and in vitro it was manifested by an increase in spontaneous plasma cell generation (Fig. 1). Elevated serum IgM levels have previously been reported in African patients with IDCM.” Our data and that of Sanderson et al.lg on T lymphocyte subsets support the hypothesis that altered T-cell regulation may be responsible for the activation of B cells in IDCM. However, in vitro analysis of T- and B-interaction would be required to support a conclusion of immune dysfunction in IDCM. This was performed with the PWM-driven B-cell differentiation assay. We have shown that plasma cell transformation after PWM-induced

354

Gerli

et al.

American

August, 1986 Heart Journal

Fig. 2. a, Plasmacell transformation from normal control PBMC + T cellsof eight IDCM patients (pt); and eight normal controls (nc). T-cell suppressoractivity wasabrogated by irradiation (1000 rads) (7’2. b, Plasmacell transformation from IDCM patient PBMC + patient- and normal control-T and patient- and normal control-Tx cells. Cardiac controls are excluded, since data generated were identical to that from healthy normal controls.

polyclonal activation was markedly higher in IDCM mononuclear cells than in normal healthy or cardiac control patients. The hypothesis that this finding may be due to an alteration in the regulatory function seen in IDCM lymphocytes compared to non IDCM T lymphocytes was proven by the fact that when IDCM PBMC were cocultured with healthy or cardiac control T lymphocytes, plasma cell generation failed to increase above levels obtained when IDCM PBMC were cultured with irradiated healthy or cardiac control T lymphocytes. Futhermore, cocultures of IDCM T lymphocytes with healthy or cardiac control PBMC led to a considerable rise in plasma cell transformation, confirming the suggestion that circulating activated T cells with a strong helper effect on B-cell differentiation are present in IDCM patients. Detection of activation markers, both HLA-DR and HLA-DC/ DS antigens and the IL-2 receptor, on the circulating T lymphocytes of several IDCM patients seems to argue additionally for this concept. The observation that plasma cell generation with IDCM PBMC was higher when cocultured with irradiated IDCM T cells, with respect to that observed with healthy or cardiac control irradiated T cells, also suggests that the described hyperfunction of helper T cells is associated with hypofunction of suppressor T cells. Although the immune system derangement we

describe may account for a self-perpetuating immunologic damage of the myocardium secondary to unknown triggering agents, it was not found in all IDCM patients. Moreover, as similar immunologic abnormalities are also characteristics of known autoimmune disease, we suggest that abnormal immunologic data in IDCM patients may be a nonspecific finding, but perhaps useful to identify a subset of IDCM patients. With the use of myocardial biopsy, histologic evidence of active inflammation is reported in a widely variable number of IDCM patients’?; recently, however, investigations have found that the shorter the duration of illness, the greater the likelihood of finding inflammation on biopsy.18 This latter observation suggests that a myocarditis is the initial event of an acute dilated myocardial disease without evidence of histologic inflammation; however, the same observation, coupled with the fact that clinical signs of active myocarditis and the degree of myocardial functional failure do not correlate with inflammatory bioptic findings,L8 may reduce the clinical importance of biopsy analysis in IDCM patients. Therefore, in spite of a lack of histologic signs of myocardial inflammation in four of our patients (50%). persistence of both B-and T-cell dysregulation may present evidence of a continuous involvement of the immune system in IDCM patients.

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112 2

Since immunosuppressive therapy has been proposed in IDCM,‘” and since a strict correlation between myocardial biopsy findings and immunotherapy improvement has not been found,ls we suggest that detailed phenotypic and functional studies of the immune system may be useful, both in selecting IDCM patients and in monitoring possible immunosuppressive therapy. In addition, further studies will require that lymphocyte phenotypes be determined on endomyocardial biopsies to corroborate our peripheral blood findings with tissue infiltration. REFERENCES

1. Cambridge G. MacArthur CGC, Waterson AP, Goodwin JF, Oakley (‘M: Antibodies to Coxsackie B viruses in congestive cardiomyopathy. Br Heart J 41:692, 1979. 2. Fowles RE, Bieber CP. Stinson EB: Defective in vitro suppressor cell function in idiopathic congestive cardiomyopat.hy. Circulation 59:483, 1979. :j. Erkstein R, Mempel W, Bolte HD: Reduced suppressor cell activity in congestive cardiomyopathy and in myocarditis. Circulation 65:1224, 1982. 4. Trueman 7’. Thompson RA, Cummins P, Littler WA: Heart antibodies in cardiomyopathies. Br Heart J 46:296, 1981. 5. Blackett K. Ngu .JI,: Immunological studies in congestive cardiomvopathv in Camewon. Br Heart J 38:605. 1976. 6. Velardl .A: Ha&hot.ti I’. Cernetti C, Spinozzi l?, Gerli R, Martelli MF. Davis S: Monoclonal antibody-defined T-cell phenotypes and phytohemagglutinin reactivity of E-rosetteformin:: circulating lymphocytes form untreated chronic myelocytic leukemia patients. Cancer 53:913, 1984. 7. Reinherz El., Schlossman SF: Regulation of the immune rrsponse~inducer and suppressor T-lymphocyte subsets in human beings. N Engl .J Med 303970, 1980. 8. Cortr (;, Mingari NC’, Moretta A, Bargellesi A: Human T cell subpop\dations defined hy a monoclonal antibody. I. A small subset is responsible for proliferation to allogeneic cells or to soluble antigens and for helper activity for B-cell differentiation. .I !mmtmol 128:lti. L9X2.

Immune

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in cardiom\

opath?~

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9. Uchiyama T, Nelson DL, Fleisher TA, Waidmann TA: A monoclonal antibody (anti-TAC) reactive with activated and functionally mature human T cells. II. Expression of ‘TAC antigen on activated cytotoxic killer cells, suppressor cells and on one of two types of helper T cells d Immunol 126:1398, 1981. 10. Reinherz EL, Kung PC, Pesando JM. Ritz J, Goldstein G, Schlossman SF: Ia determinants on human T-cell subsets defined by monoclonal antibody: Activation stimuli required for expression. J Exp Med 150:1472, 1979. 11. Chen YX, Evans RL, Pollack MS, Lanier l,L, Phillips ,JH, Rousso C, Warner NL. Brodskv FM: Characterization and expression of the HLA-DC antigens defined by anti Leu-10. Human Immunol 10:221, 1984. 12. Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235, 1965. 13. Spinozzi F. Velardi A, Rambotti P, Loslto A. Zampi I, Cernetti C, Gerli R, Martelli MF, Grignani F: Circulating immune complexes and serum lysozyme levels in untreated Hodgkin’s disease. Their relationship to immune function. ,I Clin Lab Immunol 12:87, 1983. 14. O’Connell JB, Fowles RE, Robinson JA, subramanian R, Henkin RE, Gunnar RM: Clinical and pathological findings of myocarditis in two families with dilated c,ardiomyopathy. AM HEART J 107:127, 1983. 15. Anderson JL, Greenwood JH, Kawanishi H: Elevation of suppressor immune regulatory function in idiopathic congestive cardiomyopathy and rheumatic heart disease. Rr Heart J 46:410, 1981. 16. Reinherz EL, Morimoto C, Fitzgerald KA.. I-lussey RE, Dales JF: Schlossman SF: Heterogeneity of human ‘l’4’ inducer 7 cells defined by a monoclonal antibody t.hat delineates two functional subpopulations. J Immunol 128:463, 1982. 17. Zee-Cheng CS, Tsai CC, Palmer DC, Codd JE, Pennington DG, Williams GA: High incidence of myocarditis by endomyocardial biopsy in patients with idiopathic congestive cardiomyopathy. J Am Co11 Cardiol 3:63, 1984. 18. Dee WG, Palacios IF, Fallon JT, Aretz HT. Mills J, Lee DC, Johnson RA: Active myocarditis in the spectrum of acute dilated cardiomypathies. N Engl J Med 312:885, 1985. 19. Sanderson JE, Koech D, Iha D, Ojiambo HP: T lymphocyte subsets in idiopathic dilated cardiomyopathy Am J (lardiol 55:755. 1985.