.lournal o f Tongj i Medical University 14 (4) : 213-- 215, 1994 213

Phenotype Analysis of Mononuclear Cells in Endomyocardial Biopsy Specimens from Patients with Viral Myocarditis and Dilated Cardiomyopathy

LI Wu (~ ~ ) , YU Shu ( g~. ~ ), WANG Dao-wen ( L ~ . ) Department o f Internal Medicine, Tongji Hospital, Tongji Medical University, Wuhan

Summary: This study investigated the distribution and phenotype of mononuclear cells in endomyocardial biopsy (EMB) specimens from patients with vira ! myocarditis (VMC, 25 cases) and dilated cardiomyopathy (DCM, 10 cases). T lymphocytes, in- cluding T~/~ cells in VMC and To, cells in DCM, predominated in the infiltrating mononuclear cells" in these specimens. The morphological relationship between infil- tration of T lymphocytes and pathological changes of myoeardia was also observed and neither macrophages nor B lymphocytes were foilnd.

Key words : myocarditis, congestive cardiomyopathy, biopsy, myocardium, T lym- phocyte

Infiltration of mononuelear cell (MNC) was observed during pathological examina- tion in endomyocardial biopsy (EMB) speci- mens from patients with viral myocarditis (VMC) and some patients with dilated car- diomyopathy (DCM) t~. The in situ analy- sis on phenotypes and distribution of MNC was helpful to understand the pathogenesis of the cell-mediated immunological damages of human myocardia Iz3. In this study, by using monoclonal antibodies and immuno- histochemical techniques, in situ analysis on phenotypes of MNC was performed in EMB specimens from 25 patients with VMC and 10 patients with DCM.

MATERIALS A N D M E T H O D S

Patients Observed group 35 EMB specimens

were obtained in this study from 35 patients hospitalized in Tongji Hospital ( W u h a n ) , Yichang Hospital (Yichang) and Central Hospital (Yichang). The average age and

duration of symptoms of 17 males and 8 fe- males with VMC were 23 .34-6 .4 years and 9. 3 -4- 3.3 months respectively; the mean age and duration of symptoms were 3 3 . 2 + 5.5 years and 13 .4-4-5 .3 months with 7 males and 3 females with DCM respective- ly.

The Reference Criterion of Symposium on VMC (China, 1988) E3] and definition and classification of DCM of WHO/ISFC Task Force for DCM (Paris, 1980) E41 were used for criteria of clinical diagnosis for VMC and DCM.

Conventional examinations such as im- munoglobulin, C3, ASO, ESR, CK-MB, ANA, autoantibodies against smooth mus- cle and heart muscle, circulating immune complex and culture of throat swab were done during hospitalization. The patients also received the examinations of ECG, UCG, ambulatory ECG, and chest radiog- raphy etc. The neutralizing antibodies for viruses of Coxasckie B1- -6 and ECHO 11. 13, 15 were titrated in sera of the patients.

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Control group 10 patients with con- genital heart diseases (4 cases of atrial sep- tal defect and 6 cases of ventricular septal defect) and 5 patients who died of brain trauma were included.

S a m p l i n g and S ta in ing Sampling 6--8 pieces of EMB spec-

imens (0. 5 - - 1 . 5 mm s) from patients with VMC, DCM and congenital heart diseases were obtained from right ventricle with EMB fordeps and sheath (Cordis, USA). 5 autopsy specimens were collected from brain trauma patients at sites corresponding to those of patients with EMB forceps. The interval between sampling and death were less than 30 min. Samples were embedded in OCT (Mile, USA) and stored in liquid nitrogen for further processing. Some of the samples were fixed in 10 ~ formalin for HE and Van Gieson staining and conventional histological examination, and in 2 ~ glu- taraldehyde'for electron microscopic obser- vation. The pathological diagnosis was con- firmed by referring to Dallas criterion for VMC [s] and Wang's criterion for DCM [6].

Reagents The specificities of mono- clonal antibodies (Orthomune, USA) panel used in the study were OKTs (pan T ) , OKT, (T help/inducer, Th/i), OKTa (T cy- totoxic/suppressor, To/. ) , OKM~ (macrophage, NK and granulocyte) and OKB7 (B cells and granulocyte).

Staining 4--5"~m sequential frozen tissue sections were fixed in acetone for 10 min, dried in air, incubated with monoclon- al antibodies (dilution 1: 50) and stained by avidin-biotin-complex (ABC) immuno-per-

oxidase procedure ~73 with biotinylated horse antimouse IgG (1: 100, Vector, USA) and ABC reagent (1: 200, Becton-Dickson). The positive cells were identified by a "red- brown rim of cell surface staining. The frozen sections of human tonsil were used as positive control. The negative control slide' was obtained by replacement of first anti- body by healthy mouse serum and replace- ment of each antibody and ABC reagents by PBS.

Counting .The total numbers of positive cells of MNC, OKT3, OKT4, OKTs, OKM1 and OKB7 were counted in randomly selected 5"high power fields (mag- nification • 400).

Statist ics All data were subjected to Student t-

test.

RESULTS

All sections of human tonsil were posi- tive and all slides of negative control were negative.

Only few B cells and macrophages were detected in EMB specimens of VMC and DCM. T lymphocyte, including Th/i in VMC and To/. in DCM, predominated in in- filtrating mononuclear cells (table 1). The data of congenital heart diseases and brain trauma were combined with control groups for statistic analysis because there was no significant difference in amount of MNC and T lymphocyte subsets between these pa- tients.

Table 1. Mononuclear cells and subsets of T lymphocytes in EMB ( �9 + s , cel ls /0 .5 ram z)

VMC DCM CONTROL Celh

(nffi 25) (n~ I0) (n= 15)

MNC 175. 39+43 . 80" * 125.50• 27" " 75. 4 0 + 18:57

OKT3 35. 44:1::6. 65" " 20, 4 0 • 42 11- 5 0 • 2, 95

OKT4 11. 1 0 + 3 . 8 2 " * 6. 60 • 63 6 . 5 0 • 1r

OKTs 5. 72 + 1+ 49 1 1 . 1 0 • 5. 32 �9 5+ 0 0 • 1.41

OKTs/MNC 0. 31 • 0. I0 * 0. 17 • 0. 04 0. 16+ 0. 07

OKT4/OKTa 2. 21 • 32 * * 0. 64 S 0 . 2 1 " " 1 . 3 2 •

* P<0.05, * * P<0. 01, as compared with control group.

Journal o f Tongji Medical University 14 (4) : 213--215, 1994 215

DISCUSSION

Cell-mediated immunological damage plays an important role in pathogenesis of myocardial diseases, which involves many cell types including T lymphocyte, macrophage, killer or K cells and natural killer or NK cells. T lymphocytes are in- volved in a wide range of immunological functions such as antibody formation, cell- mediated cytotoxicity response and delayed hypersensitivity reactions [s].

T cells in human and mice carl be fur- ther classified into two subsets. Ts/~ lym- phocyte reacts specifically with infected car- diomyocytes (virus-specific cytolytic T lym- phocytes, VSCTL) and the amount and ac- tivity of VSCTL decrease rapidly after elim- ination of virus. Whereas the To/. lympho- cytes reacts mainly with uninfected heart cells (autoreaetive CTL, A C T L ) ; the le- sions in myocardia caused by ACTL were more extensive and necrotizing than that by VSCTL. Lysis of the uninfected myocyte could result from autoimmune CTL recog- nizing virus-modified eardiocyte antigens and cross-reactively lysing uninfected tar- get [~]. The results in this study also indicate that it is a different lymphocyte subset that damages myocytes during different stages of myocardial diseases. In contrast to the re- sults of this study and Deguchi's findings, some investigators reported that the de- crease of T~/. subset was observed in periph- eral blood. This difference may be related to different sampling timing. The EMB was often performed after improvement of car- diac {unction of patient, at that time the ra- tio of O K T J O K T ~ had decreased ~].

The analysis of mononuclear cells might be helpful in distinguishing intersti- tial cells which includes fibrocytes, occa- sionally mast cells, histocytes, pericytes, endothelial cells and lymphocytes. Peri- cytes, s nuclei in cross-section and

endothelial cells all may be mistaken for b;mphocytes in HE staining and light micro- scopic observation [~3.

In some patients, it was found that.the sum of OKT~ and OKTs cells was more than total number of OKT~, which indicates the presence of T cells with double markers. This phenomenon was not observed in my- ocardial diseases by other investigators but existed in viral hepatitis, which was be- lieved to be an abnormal expression of sur- face marker induced by abnormal differenti- ation of T lymphocytes after virus infec- tion ~o3.

REFERENCES

1 Kereiakes D J, Parmley W W. Myocarditis and cardiomyopathy. Am Heart J, 1984 ! 108 : 1319

2 Deguchi H, Hayashi T, Kotaka Metal. In situ analysis with monoclonal antibodies of lympho- cyte subsets in myocardial biopsies from pa- tients with dilated cardiomyopathy and idio- pathic (viral) myocarditis. Jpn Circ J, 1987! 51 : 1365

3 ~ ' ~ r 1 6 2 ~ t . ~ ; ~ . ~ ; ~ - ~ a r ~ . i ~ l ~ , ~ ~ & , 1987, 26: 597.

4 WHO/ISFC Task Force. Report of the WHO/ ISFC task force on the definition and classifica- tiort of cardiomyopathies. Br Heart J, 1980; 44:672

5 Aretz H T, Billingham M E, Edwards W D et al. Myocarditis: a histopathologic definition and classification. Am J Cardiovasc Pathol, 1987; 1 : 3

~ P - ~ . s 1986; 15:282

1990. 69-70

-~ ~ l ~ $ ~ f . I~$~,P~--~&, 1990; 7:10

= ~ t ~ - , 1991:89:92

~ f f * ~ , 1989; 18:79 (Received Nov. 24, 1993)