International Journal of Cardiology, 8 (1985) 113-123 Elsevier 113 1JC 00254 Humoral immunity and lymphocyte subpopulations in patients with dilated cardiomyopathy Roberto Franceschini ‘, Valeria Messina ‘, Aldo Petillo 2, Marco Corazza 2, Luigi Bottaro ’ and Renato Gianrossi ’ ’ Department of Internal Medicine, University of Genoa, Genoa and ’ II Division of Cardiology. Ospedale S. Martino. Genoa. Italy (Received 9 November 1984; revision accepted 12 December 1984) Franceschini R, Messina V, Petiilo A, Corazza M, Bottaro L, Gianrossi R. Humoral immunity and lymphocyte subpopulations in patients with dilated cardiomyopathy. Int J Cardiol 1985;8 : 113-123. The incidence of serum organ and non-organ specific autoantibodies, the peripheral blood lymphocyte blastogenic response to phytohaemagglutinin, concanavalin A and pokeweed mitogen, and the surface markers of peripheral T and B lymphocytes were studied in 15 patients affected by coronary artery disease and in 21 patients with dilated cardiomyopathy. In the latter group there was a significantly impaired blasto- genie response to concanavalin A with respect to both the normal control group and patients with coronary artery disease (P < 0.01). The percentage of peripheral blood lymphocytes with cytotoxic/suppressor activity was also reduced in patients with dilated cardiomyopathy in comparison to normal subjects and patients with coronary artery disease (P -c 0.01 and P < 0.05, respectively). These data may reflect an in vivo defect in suppressor cell function in patients with dilated cardiomyopathy. (Key words: serum autoantibodies; lymphocyte blastogenic response; T-cell subsets) Introduction The term dilated cardiomyopathy refers to diseases of unknown aetiology in which presenting signs and symptoms are mainly due to dysfunction of the myocardium. A number of factors have been implicated in the causation of cardiomyopathy including alcohol [l], nutritional deficiency [2], the postpartal state Correspondence to: Dott. Roberto Franceschini. Via Amarena 4/9. 16143 Geneva, Italy. 0167-5273/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
Transcript
International Journal of Cardiology, 8 (1985) 113-123
Elsevier 113
1JC 00254
Humoral immunity and lymphocyte subpopulations in patients with dilated cardiomyopathy
’ Department of Internal Medicine, University of Genoa, Genoa and ’ II Division of Cardiology. Ospedale S. Martino. Genoa. Italy
(Received 9 November 1984; revision accepted 12 December 1984)
Franceschini R, Messina V, Petiilo A, Corazza M, Bottaro L, Gianrossi R. Humoral immunity and lymphocyte subpopulations in patients with dilated cardiomyopathy. Int J Cardiol 1985;8 : 113-123.
The incidence of serum organ and non-organ specific autoantibodies, the peripheral blood lymphocyte blastogenic response to phytohaemagglutinin, concanavalin A and pokeweed mitogen, and the surface markers of peripheral T and B lymphocytes were studied in 15 patients affected by coronary artery disease and in 21 patients with dilated cardiomyopathy. In the latter group there was a significantly impaired blasto- genie response to concanavalin A with respect to both the normal control group and patients with coronary artery disease (P < 0.01). The percentage of peripheral blood lymphocytes with cytotoxic/suppressor activity was also reduced in patients with dilated cardiomyopathy in comparison to normal subjects and patients with coronary artery disease (P -c 0.01 and P < 0.05, respectively).
These data may reflect an in vivo defect in suppressor cell function in patients with dilated cardiomyopathy.
The term dilated cardiomyopathy refers to diseases of unknown aetiology in which presenting signs and symptoms are mainly due to dysfunction of the myocardium. A number of factors have been implicated in the causation of cardiomyopathy including alcohol [l], nutritional deficiency [2], the postpartal state
Correspondence to: Dott. Roberto Franceschini. Via Amarena 4/9. 16143 Geneva, Italy.
[3] and virus infections [4]. An underlying immunological disturbance has also been suggested as a pathogenic mechanism linked to the presence of endomyocardial fibrosis and sparse lymphocytic or other mononuclear cellular infiltrates [5]. Both immunoglobulins bound to myocardial tissue [6,7] and serum organ and non-organ
specific autoantibodies [8-111 have been reported in patients suffering from dilated cardiomyopathy, but only a few studies have investigated alterations in cell-media- ted immune mechanisms. Anderson et al. [12] showed an increase in the risk of lymphoma in patients who underwent cardiac allografts for dilated cardiomyopathy, and postulated a defect in mitogen induced mononuclear cell suppressor activity.
Fowles et al. [13] and Eckstein et al. [14] showed an in vitro defect in suppressor cell
function in patients with dilated cardiomyopathy.
In the present study we have evaluated some parameters of humoral and cellular
immunity in patients with dilated cardiomyopathy. Our aim was to investigate a possible underlying immunological disorder involved in the pathogenesis of the
disease.
Materials and Methods
Patients
Our study covered a period of 18 months and included 36 patients; 21 patients (14 males and 7 females, aged from 17 to 58 years) were suffering from congestive
heart failure unassociated with coronary artery disease (50% or greater narrowing of luminal diameter), familial cardiomyopathy, history of rheumatic fever, inordinate alcohol consumption, endocrine and metabolic disorders or collagen diseases. No
patient had a history of viral illness prior to the onset of cardiac symptoms, and at the time of the study the serological titres for cardiotropic virus directed antibodies
were always in the normal range. Fifteen patients (11 males and 4 females, aged from 35 to 68 years) with congestive heart failure secondary to coronary artery
disease were used as the “disease control” group. All patients were subsetted according to the New York Heart Association classification for heart failure. All
patients were treated with digoxin, diuretics and nitrates; no patient was receiving immunoactive drugs. All patients underwent electrocardiography, polycardiography and M-mode and cross-sectional echocardiography. Ten patients with coronary artery disease and 9 patients with dilated cardiomyopathy underwent cardiac catheterization, left ventricular angiography and coronary arteriography. In 2 pa- tients with dilated cardiomyopathy diagnosis was confirmed at necropsy. As noted above, in 10 patients the diagnosis was based either on detailed clinical histories, absence of pathologic findings of significant coronary disease or non-invasive examinations. Ten age-matched healthy subjects were used as the “normal control” group. Clinical findings are reported in Table 1.
Chemicals
Anti-human IgG-A-M fluorescein isothiocyanate-labelled rabbit serum and anti- mouse Ig fluorescein isothiocyanate-labelled goat serum were purchased from Behr-
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TABLE 1
Clinical characteristics of patients
Coronary artery Dilated
disease cardiomyopathy (n=15) (n =21)
NY HA Class
II
III
IV
Electrocardiography
Ischaemia
Old myocardial infarction
Left ventricular strain
Ventricular arrhythmias
Left bundle branch block
Phonocardiography
Normal
S3 gallop
S4 gallop
Systolic murmur
Echocardiography (M-mode)
Left atrium (mm)
Fractional shortening
IVS amplitude (cm)
PLV amplitude (cm)
Echocardiography (B-mode)
Segmental hypokinesia
Diffuse hypokinesia
Cardiac catheterization’
Hypocontractility
Coronary occlusion
Mean LVEDP (mm Hg)
7 12
5 5
3 4
15. _
9 _
6 16
8 15
3 5
I 3
5 9
2 11
4 R
42.7 + 6.5 46.lk 7.2
22.6 k 7.7 = 17.4 + 8.6 a
5.7kl.l a 3.5 ? 0.8 a
9.6k4.3 4.7 f 2.0
12 _
3 21
severe
severe
22
severe
not significant
23
’ Values calculated only in the absence of left bundle branch block.
’ Performed in 10 patients with coronary artery disease and in 9 patients with dilated cardiomyopathy.
NYHA = New York Heart Association; IVS amplitude = interventricular septum motion amplitude;
PLV amplitude = posterior left ventricular wall amplitude; LVEDP = left ventricular end diastolic
pressure.
ingwerke (Scoppito, L’Aquila, Italy). Monoclonal antibodies to T-cell subsets (OK T3-T4-T8) were purchased from Ortho (Milano. Italy); the characteristics of these have been previously reported [15,16]. The final concentrations of phytohaemag- glutinin (Wellcome), concanavalin A (Pharmacia AB, Uppsala) and pokeweed mitogen (Grand Island Biological Co., New York) were 10 pg/ml, 100 pg/ml and a 1 : 20 dilution, respectively; the same batches of mitogens were used for the entire study. Methyl[3H]thymidine (spec. act. 2 Ci/mmol) was purchased from Amersham (England). 2-Aminoethylisothiouronium bromide hydrobromide was purchased from Sigma Chemical Co., St Louis, MO.
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Detection of Serum Autoantibodies
Detection of serum autoantibodies by indirect immunofluorescence was per- formed using cryostatic sections of rat organs (heart, skeletal muscle, liver, kidney and stomach) as substrate according to Nairn [17]. All sera were diluted 1 : 10 before testing and incubated for 25 min in a moist chamber at room temperature. Slides were then incubated with anti-human IgA + IgG + IgM fluorescein isothiocyanate-
labelled rabbit serum for 25 min, washed in PBS, mounted in buffered glycerol and viewed under a Zeiss microscope, equipped with a fluorescent light. Negative and positive control serum samples were included in each batch of slides. The sections
were viewed independently by two observers. The intensity of fluorescence staining was graded from + 1 to + 4. Autoantibodies were classified according to Maisch et
al. [lo] as heart specific (anti-interfibrillary), muscle specific (antisarcolemmal and antifibrillary) and non-organ specific (antinuclear, anti-smooth muscle and anti-
gastric parietal cells). Forty normal sera were used as controls.
Isolation of Peripheral Blood Lymphocytes
Lymphocytes were separated from heparinized blood by centrifugation at 400 g for 20 min through a Ficoll-Urovison gradient (sp.g.t. 1077) and washed three times in Hank’s balanced salt solution (Eurobio, Labtek, Paris). All cell cultures were performed in triplicate in microtitre plates with U-bottomed wells (Linbro, Hamden, USA).
Lymphocyte Blastogenic Response
The test was performed according to a variation of the method described by Penhale et al. [18]. Peripheral blood lymphocytes were resuspended at a concentra-
tion of 2 X 106/ml in TC 199 medium buffered with Hepes and 7% bicarbonate (Eurobio, Labtek, Paris), supplemented with 1% r_-glutamine, penicillin 100 IU/ml,
streptomycin 100 pg/ml and 20% heat inactivated normal AB human serum. 100 ~1 of the cell suspension were added with 100 ~1 of solution containing TC 199 (control cultures), phytohaemagglutinin, concanavalin A or pokeweed mitogen. Plates were incubated at 37°C in a humidified atmosphere for 55 hr (phytohaemagglutinin and concanavalin A cultures) or 96 hr (pokeweed mitogen cultures). Methyl[3H]thymi- dine 0.5 PCi was added to each well. Seventeen to 20 h later, cultures were harvested onto glass fibre filters with a semiautomatic device (Sacrificator. Labtek). Filters were added to 2.5 ml of scintillation fluid and counted in a Beckmann scintillation spectrometer. Blastogenic response was expressed as counts per min (cpm).
Enumeration of Lymphocyte Subsets
Lymphocytes positive for membrane immunoglobulins were detected incubating 40 ~1 of 1 x lo7 PBL with polyclonal anti-human Ig fluorescein isothiocyanate-
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labelled rabbit serum for 25 min at 4°C in microtitre plates. Cells were washed and 200 cells were counted.
The E-rosette test was performed according to the method described by Madsen and Johnsen [19], using 2-aminoethylisothiouronium bromide hydrobromide treated fresh sheep red blood cells. Five ml of sheep red blood cells were washed three times with isotonic saline and mixed with 5 ml of a 0.14 M solution of 2-aminoethyliso-
thiouronium bromide hydrobromide (pH 9). The mixture was incubated at 37°C for 15 min, washed three times with saline, and the final concentration was adjusted to 1% in RPM1 1640 supplemented with 20% heat inactivated normal human AB
serum. 40 pl of a 1 x 10’ cell suspension were mixed with 40 ~1 of 1% 2-aminoethyl- isothiouronium bromide hydrobromide treated sheep red blood cells and, after
centrifugation at 200 g for 5 min, incubated at 4°C for at least 2 hr. The pellet was
gently resuspended and 200 lymphocytes were counted; lymphocytes with three or more erythrocytes were counted as rosettes.
T-cell subsets were investigated using monoclonal antibodies OKT3-T4-T8; 40 ,ul
of 1 X 10’ mononuclear cells were incubated at 4°C for 25 min with 5 ~1 of monoclonal antibodies. The cells were washed and 40 ~1 of fluorescein iso- thiocyanate-labelled anti-mouse Ig goat serum were added. After an additional 25 min incubation at 4°C the cells were washed and 200 cells were evaluated for fluorescent staining.
Statistical Analysis
Incidence of serum autoantibodies in the three groups was compared using the &i-square test. The results obtained in cellular studies were analyzed by the
two-tailed Student’s t-test for unpaired observations; correlations were performed by the linear regression test.
The incidence of autoantibodies in normal subjects, in the “control disease” group and in patients with dilated cardiomyopathy is reported in Table 2. The
Results
TABLE 2
Incidence of autoantibodies in the three study groups (actual frequencies).
ASA IFA AFA ANA SMA GPCA
NS(n=40) 2 nd 1 1 2 5
CAD (n =15) 4 3* 3 1 3 3
DC(n=21) 8 ** 4* 7 ** 3 2 7
In comparison with normal subjects: * P < 0.05; ** P < 0.01.
NS = normal subjects; CAD = coronary artery disease; DC = dilated cardiomyopathy; ASA = anti-
sarcolemmal; IFA = anti-interfibrillary; AFA = anti-fibrillary; ANA = anti-nuclear: SMA = anti-smooth
Lymphocyte blastogenic responses in the three study groups
PHA
Con A
PWM
Normal Coronary artery
subjects disease
(n =lO) (n =15)
47761.2 + 9940.4 51882.3 f 14687.6
28078.5 f 11064.9 32027.3 + 12039.5
23699.1 f 4614.3 29487.8+ 7093.1 *
Dilated
cardiomyopathy
(n = 21)
40957.8 k 16068.5
11823.9* 8091.6 **+
31520.5 + 10601.9 *
Values (mean + SD) are expressed as counts per min.
PHA and Con A cultures were harvested after 3 days; PWM cultures were harvested after 5 days, In
comparison with normal subjects: * P < 0.05; ** P < 0.01. In comparison with coronary artery disease:
t P i 0.01.
PHA = phytohaemagglutinin: Con A = concanavalin A; PWM = pokeweed mitogen.
incidence of heart specific autoantibodies was significantly higher both in patients with dilated cardiomyopathy and in patients with coronary artery disease in com-
parison to normal subjects (P < 0.05). The incidence of muscle specific antibodies
(antisarcolemmal and antifibrillary autoantibodies) was statistically higher in pa-
tients with dilated cardiomyopathy with respect to normal subjects (P -c 0.01). No statistical difference was observed in the incidence of autoantibodies between
patients with dilated cardiomyopathy and coronary artery disease. The frequency of non-organ specific autoantibodies was not statistically different among the three
study groups. The results of the lymphocyte studies are reported in Tables 3 and 4 and in Figs.
1 and 2. The concanavalin A induced blastogenic response of peripheral blood lymphocytes was reduced in patients with dilated cardiomyopathy when compared both with normal subjects and patients with coronary artery disease (P < 0.01). The response to pokeweed mitogen was significantly higher in patients with dilated
TABLE 4
Percentage of B and T cells in the three study groups.
Normal Coronary artery Dilated
subjects disease cardiomyopathy
(n=lO) (?r=15) (n = 21)
OKT3+ 73.9k11.2 73.7 f 6.3 68.9+ 9.8
OKT4+ 45.4+ 6.4 42.7 5 9.7 47.3 * 12.0
OK T8+ 28.4* 5.7 23.5 + 6.9 18.7? 4.7 **+
OK T4+/OKT8+ 1.6+ 0.2 2.0 + 0.6 2.7+ 0.8 **+
Mlg + 15.55 3.3 16.9 k 5.3 15.2* 6.5
E-RFC 79.0+ 6.7 81.5 + 5.6 77.1 + 10.6
Values are mean + SD. In comparison with normal subjects: ** P < 0.01. In comparison with coronary artery disease: + P c 0.05.
Fig. 3. Correlation between concanavalin A-induced blastogenic response and OKT4/OKT8 positive cell
ratio in patients with dilated cardiomyopathy.
cardiomyopathy than in normal subjects (P -c 0.05). whereas no statistical difference was observed with respect to patients with coronary artery disease. Furthermore, the pokeweed mitogen-induced blastogenic response was statistically higher in patients with coronary artery disease with respect to normal subjects (P < 0.05). The phyto- haemoagglutinin-induced blastogenic response of peripheral blood lymphocytes did not differ in the three groups.
The percentage of surface immmunoglobulin bearing lymphocytes, E-rosette forming cells, 0KT3 and 0KT4 positive cells was not statistically different in the three study groups, whereas the percentage of OKT8 positive cells was significantly lower in patients with dilated cardiomyopathy compared to both controls and
patients with coronary artery disease (P -c 0.01 and P < 0.05, respectively). In
patients with dilated cardiomyopathy the OKT4/OKTS positive cell ratio was significantly higher when compared to both control groups (P < 0.01 and P < 0.05 with respect to normal subjects and patients with coronary artery disease, respec- tively). No significant difference was observed between normal subjects and patients with coronary artery disease.
In patients with dilated cardiomyopathy, but not in normal subjects or patients with coronary artery disease, there was a significant correlation between concanava- lin A induced blastogenic response and OKT4/OKT8 positive cell ratio (P < 0.05) (Fig. 3).
Discussion
In the past years several authors have demonstrated the presence of serum autoantibodies [S-lo] and immunoglobulins bound to myocardial tissue in patients with dilated cardiomyopathy [6,7], but the physiopathological significance of these
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results is still unclear. Maisch et al. [20] reported a high incidence of anti-inter- fibrillary and anti-fibrillary antibodies which increased according to the severity of
the disease in sera from patients with primary dilated cardiomyopathy and with alcoholic cardiomyopathy, but not of patients with secondary cardiomyopathy.
Other authors [11,21] have suggested that anti-heart antibodies detected by im- munofluorescence probably reflect damage to cardiac muscle regardless of the cause. They suggest that binding of antibody to myocardial tissue is not specific and cannot
be considered the primary pathological event. Our data on the incidence of autoanti-
bodies support this view, as we were unable to detect circulating autoantibodies specific for dilated cardiomyopathy. The same patterns of immunofluorescence were observed in sera from patients with coronary artery disease. It is well known,
however, that the immune response is a balance of several immunological mecha- nisms. The presence of autoantibodies may be due to an alteration of cooperation between T and B lymphocytes resulting in activation of B cells or T helper or
depression of T suppressor cells. Pokeweed mitogen has been shown to induce a
proliferative response and antibody synthesis in peripheral human B cells (22,231. In our study, this mitogen induced an increased blastogenic response in patients with
dilated cardiomyopathy with respect to normal subjects, but no significant increase was observed compared to the response seen in patients with coronary artery disease. So it is possible that in patients with dilated cardiomyopathy or coronary
artery disease the higher response to pokeweed mitogen and the presence of autoantibodies are correlated. It is well known that the pokeweed mitogen driven lymphocyte response is dependent on precise interactions between functionally
distinct T cell subsets and B cells [24]. The blastogenic response to the T cell mitogen concanavalin A was significantly lower in patients with dilated cardiomyopathy with respect to both normal subjects and patients with coronary artery disease. Earlier
studies have shown that concanavalin A treated peripheral blood lymphocytes suppress the in vitro response of homologous lymphocytes to allogeneic or mitogenic stimulation [25,26]. This can be explained by a selective suppressor T cell activation
by this mitogen [27]. Moreover, in the present study the characterization of T cell subsets by monoclonal antibodies showed that in patients with dilated cardiomyopa-
thy there is a decreased percentage of peripheral OKT8 positive cells and a higher
OKT4/OKT8 positive cell ratio. The OKT8 monoclonal antibody recognizes mainly suppressor/cytotoxic T cells, whereas 0KT4 recognizes the T cells with helper/in- ducer activity. The OKT4/OKT8 ratio is considered an index of the balance
between the helper/suppressor activity of T cells [28]. Little information is available on cellular immune function in patients with
dilated cardiomyopathy. Fowles et al. [13] and Eckstein et al. [14] showed that concanavalin A treated peripheral blood lymphocytes from patients with dilated cardiomyopathy failed to inhibit in vitro immune responses. These authors pos- tulated a defect in T suppressor activity in this disease. The results of the present
study are in keeping with these observations, and clarify the possible immunological mechanisms of the immune disturbance in dilated cardiomyopathy. We have demon- strated that the presence of autoantibodies is an epiphenomenon, probably due to the cardiac muscle damage. The activation of B cells (demonstrated also by the
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increased blastogenic response to pokeweed mitogen) is related to a functional and quantitative defect of T cells (showed by the reduced response to concanavalin A) and particularly of lymphocyte subpopulations with suppressor/cytotoxic activity (OKT8 positive cells). Other authors have reported a lack of normal suppressor cell
function in active systemic lupus erythematosus [29] and in chronic active hepatitis [30]. Fudenberg [31] suggested that all disease with autoimmune or immunologically aberrant characteristics may have deficient suppressor cell activity. Implication of the impaired T suppressor function in patients with dilated cardiomyopathy as a component of the pathogenesis of the disease is speculative.
Among the possible causes of dilated cardiomyopathy, many authors [32,33] have
pointed out the relevance of a preceding acute viral illness (“flu-like syndrome”) before the onset of cardiac symptoms. But the presence of high levels of anti-cardio-
tropic virus antibody titres is confined only to those patients with a short history of
dilated cardiomyopathy [32]. The patients involved in this study underwent immuno-
logical evaluation at least one year after the onset of cardiac symptoms and anti-cardiotropic virus antibody titres were always in the normal range. The dis-
turbance of T suppressor/cytotoxic cells, found in patients with dilated cardiomyop-
athy, may be due to an acute viral illness, responsible, at the same time, for the cardiac disease.
In conclusion, our results show that the decreased concanavalin A induced blastogenic response and changes in T cell subpopulations distinguish dilated from ischemic cardiomyopathy. Further studies are needed to determine what role these
immunological alterations play in the pathogenesis of dilated cardiomyopathy.
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