Asian Cardiovasc Thorac Ann 1998;6:104-107
© 1998 Asia Publishing EXchange Pte Ltd
Role of Lymphocyte Subsets in Pathogenesis of Chronic Rheumatic Heart Disease
Rajendar K Suri, MS,
Neerod K Jha, MCh,
Harpreet Vohra, PhD1,
Ratna S Manjari, MCh,
Rajam Venkateshwaran, MS2,
Madhulika Sharma, MSc1,
Shyam KS Thingnam, MCh,
Nirmal K Ganguly, PhD1
Department of Cardiovascular and Thoracic Surgery
1 Department of Experimental Medicine
2 Department of General Surgery Postgraduate Institute of Medical Education and Research Chandigarh, India
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For reprint information contact: Rajendar K Suri, MS Department of Cardiovascular and Thoracic Surgery Postgraduate Institute of Medical Education and Research Chandigarh 160012, India Tel: 91 172 54 1031 Ext. 302 Fax: 91 172 54 0401
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ABSTRACT
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Analyses of lymphocyte subsets using flow cytometry were conducted to determine the significance of these cells in the pathogenesis of chronic rheumatic heart disease. Lymphocytes (B cells, T cells, CD4 cells, CD8 suppressor or cytotoxic T cells, activated T cells, and natural killer cells) were measured in blood and left atrial appendage samples of 30 patients with rheumatic heart disease and 10 patients with acyanotic congenital heart disease. Monoclonal fluorescent-labeled antibodies were used to identify various cells by flow cytometry. There was a significant increase in CD4 cells and activated T cells with a significant decrease in B cells in the left atrial appendage tissue of patients with rheumatic heart disease compared to those in the control group. There was no significant difference between the two groups in the distribution pattern of T lymphocytes in peripheral blood. These changes in rheumatic heart disease reflect an abnormal immunoregulatory mechanism with an ongoing enhanced immunological process continuing into the chronic phase of the disease. In our opinion, this persistent T cell response may lead to fresh damage to the myocardium and deformation of the heart valves.
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INTRODUCTION
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Rheumatic heart disease (RHD) is a poststreptococcal nonsuppurative inflammatory disease with an abnormal cellular and humoral autoimmune response caused by the products of type A beta hemolytic streptococci. Type A beta hemolytic streptococci cross-react with connective tissues glycoproteins of various organs, with the greatest damage occurring in the heart.1 The incidence of rheumatic fever in India is 100 per 100,000 school children.2 Although the incidence of RHD has decreased in developed countries, it is the leading cause of death in the 5-year to 24-year age group in developing countries.3 Increased infiltration of T lymphocytes in heart valves has been well documented and these lymphocytes were found to be toxic to cultured heart cells.1 This cytotoxicity was not enhanced by plasma or serum, suggesting the nonparticipation of complement or antibodies.4 It has also been suggested that helper lymphocytes have a role in contracture and fibrosis of heart valves.1
Flow cytometry is a technique for measuring single particles or cells as they flow in a stream through a detection point transected by a laser beam.5 It is used increasingly to analyse T cell subsets with fluorescent-labeled monoclonal antibodies directed against cell surface antigenic glycoproteins involved in the immune response of RHD. Patients undergoing surgery for chronic rheumatic valvular disease may not show serological evidence of rheumatic activity (C-reactive protein, anti-streptolysin O) or histopathologic markers (Aschoff's bodies). The purpose of this study was to determine the significance of various T cell subsets in the pathogenesis of chronic RHD.
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MATERIAL AND METHODS
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Forty patients with heart disease who were admitted to the cardiothoracic surgery department of our institute were randomly selected and divided into 2 groups. The study group consisted of 30 patients with incapacitating chronic rheumatic valvular heart disease who required mitral valve surgery, while the control group comprised 10 patients with acyanotic congenital heart disease (atrial or ventricular septal defects) with no history of rheumatic fever or other inflammatory connective tissue disease. Peripheral venous blood (2 mL) and a tissue specimen of the left atrial appendage (LAA) were obtained from each of the 40 patients. Patients who received steroids and those with increased anti-streptolysin O and C-reactive protein titers suggestive of active RHD were not included in the study. Lymphocytes from LAA and blood samples were processed and labeled with monoclonal antibodies (Table 1
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CELL ANALYSIS OF LEFT ATRIAL APPENDAGE
The LAA specimen was collected in a plastic vial containing normal saline and transferred to the laboratory for immediate analysis. A modification of the method of Davies and Parrott6 was used for separation of the lymphocytes. The tissue was minced with a surgical knife and washed 3 times for 10 minutes each with Hank's basal salt solution pH 7.2. They were then washed once for 10 minutes with Hank's basal salt solution containing 5 mM ethylenediaminetetraacetic acid (EDTA). The washed tissues were incubated with 0.08% (w/v) collagenase in an incubator shaker at 37°C for 45 minutes. The tissue debris was discarded and the cell suspension was removed. The separated cells were washed and suspended in RPMI 1640 (Rossel Park Medical Institute medium no. 1640; Gibco-BRL Products, Grand Island, NY, USA) containing 10% (v/v) fetal calf serum. The cells were layered on a metrizoate gradient and centrifuged for 15 minutes at 1800 rpm. The cell monolayer was aspirated and washed. Viability of cells was checked by trypan blue exclusion. Cell concentration was adjusted to 1 x 106 per mL. Cell samples were stained and incubated with 6 types of specific monoclonal labeled antibodies (Table 1
). After incubation at 37°C for 30 minutes, the cells were washed in phosphate-buffered saline pH 7.2 and 0.5 mL of 0.5% (v/v) paraformaldehyde was added as a fixative before storing at 4°C. The control was gamma1 fluorescein isothiocynate gamma2 phycoerythrosin. Samples were analyzed with LYSUS II software (FACSCAN Immunocytometry System; Becton-Dickinson, San Jose, CA, USA).
PERIPHERAL BLOOD CELL ANALYSIS
The samples (2 mL) of venous blood collected from the 40 patients were transferred to the laboratory in fresh ethylenediaminetetraacetic acid vials. A sample (0.1 µL) of whole blood was added to each of 6 tubes containing specific monoclonal labeled antibodies, incubated at 37°C for 30 minutes, and washed with phosphate-buffered saline pH 7.2. The cells were lysed with a blood cell lysing solution (FACS lysing solution; Becton-Dickinson, San Jose, CA, USA), kept at room temperature for 10 minutes, and washed again with phosphate-buffered saline. They were fixed with 0.5 mL of 0.5% (v/v) paraformaldehyde and stored at 4°C. Analysis was performed with SIMULSET software (FACSCAN Immunocytometry System; Becton-Dickinson, San Jose, CA, USA).
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RESULTS
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The data were statistically analyzed using the unpaired Student t test and expressed as mean ± standard deviation. The mean age of patients in the study group was 25 ± 7 years. The male to female ratio was 1:2. In the control group, the mean age was 17.5 ± 5 years and the male to female ratio was 3:7.
There was an increase in total T cells in the LAA samples from the study group as shown in Table 2, although this was not significant (p > 0.05). There was a significant increase in the mean percentage of CD4 cells and activated T cells in the patients with RHD. The B cells and natural killer cells were decreased in the study group compared to the control group but in the case of natural killer cells this was not significant. The decrease in CD8 cells in RHD patients was also not significant. The CD4:CD8 ratio in the LAA samples of the study group was significantly higher than that of the control group. In the peripheral blood samples, there was no significant difference in the mean percentages of total T cells, CD4, or CD8 cells. However, the percentage of B cells and activated T cells was higher in the RHD patients who showed a decrease in natural killer cells (Table 2).
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Table 2. Comparison of Lymphocyte Subsets in Blood and Left Atrial Appendage Samples of Study Group (n = 30) and Control Group (n = 10)
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DISCUSSION
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The exact pathogenesis of chronic RHD remains unclear although the available literature suggests an aberrant immunologic process with antibodies of the streptococcal antigens cross-reacting with connective tissue of the target organs.1,2 The role of CD4 lymphocytes in the development of contracture and fibrosis of heart valves has been established, while that of other T cell subsets remains controversial.1 Automated flow cytometry, often referred to as fluorescent activated cell sorter analysis, is a specialized research tool being more frequently used because of the greater availability of monoclonal antibodies made possible by hybridoma technology.5
There was no significant difference in the mean percentage of T cells or T cell subsets in the peripheral blood samples from either group in our study, reflecting the chronic nature of the disease process in these RHD patients. Several studies have reported a decrease in T cells and CD4 cells and a relative increase in the CD8 and B lymphocytes in the peripheral blood of patients with acute RHD. This can be attributed to the acute nature of the disease process and to the effect of steroids used to treat these patients because these values generally returned to normal after the acute phase.7–9 However, the percentage of T cells, CD4, and helper T cells increased in the LAA samples of our study group. CD4 cells help in collagen deposition and act as an ancillary factor in the genesis of contracture and fibrosis in a deformed cardiac valve. Their increase in the chronic phase of RHD indicates an ongoing immunological process leading to fibrotic deformation of the cardiac valves, thus emphasizing the role of the helper T cells in the pathogenesis of chronic RHD, which corroborates other studies.1,3,10,11
The mean percentage of CD8 cells in the study group LAA samples was not significantly lower than in the controls but suggests a lack of immunoregluatory or suppressor control on the disease process and may be due to the presence of circulating immune complexes, antilymphocytic antibodies, or antigenic cross-reactivity between lymphocytes. Other reports have suggested that the decrease in CD8 cells may lead to unchecked helper cell (CD4) activity and tissue damage.1,11
In the LAA samples, the percentage of B cells in the RHD patients was significantly lower than that of the controls. This indicates that the B cells may not have a role in the pathogenesis of chronic RHD. Raizada and colleagues1 found that the interstitial infiltrates in heart valves were predominantly T cells. The activated T cells in the LAA samples from our patients with chronic RHD were significantly elevated. This increase in activated T cells possibly leads to production of interleukin-2, a lymphokine produced by activated T cells. The release of interleukin-2 triggers the accumulation of CD4 cells, resulting in an undamped T cell response.12
There was no age difference between the two groups of patients in our study. In our country, many patients present with congenital acyanotic heart disease in middle age, thus affording us the opportunity to select comparable groups and avoid any differences in T cell changes related to age. The left atrial appendage was selected as the sample site because biopsies of this tissue in patients with rheumatic heart disease have shown the persistence of focal inflammatory lesions in those who no longer have other evidence of rheumatic activity.13 In addition, samples of the LAA can be removed easily in patients undergoing cardiac surgery without increased risk of morbidity or mortality.
We postulate that the sequence of events responsible for cellular damage in chronic RHD might be stimulation of T lymphocytes by streptococcal antigens, leading to an increase in activated T cells with secretion of interleukin-2. This in turn results in increased natural killer cell cytotoxicity and the pathological changes leading to fibrosis of the cardiac valves and ultimately to incapacitating hemodynamic changes.
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REFERENCES
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Raizada V, Williams RC, Chopra C. Tissue distribution of lymphocytes in rheumatic heart valves as defined by monoclonal anti-T cell antibodies. Am J Med
1983;74:90–6.[Medline]
-
Kissane JM. Inflammatory heart diseases: rheumatic fever and rheumatic heart disease. In: Anderson's pathology. 9th ed. St. Louis: Mosby, 1990:639.
-
Braunwald E. Rheumatic fever and other rheumatic diseases of the heart. In: Braunwald E, editor. Heart disease: a text book of cardiovascular medicine. 4th ed. Philadelphia: Saunders, 1992:1721–41.
-
Ganguly NK, Vohra H. Immunology of streptococcal diseases. PGI Bull
1996;30:91–101.
-
Creamer P. Flow cytometry and rheumatic diseases: clinical review. Br J Rheumatol
1992;31:465–72.[Abstract/Free Full Text]
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Davies MD, Parrott DMV. Preparation and purification of lymphocytes from the epithelium and lamina propria of murine small intestine. Gut
1981;22:481–8.[Abstract/Free Full Text]
-
Sapru RP, Ganguly NK, Sharma S. Cellular reactions to groups A "ß" hemolytic streptococcal membrane antigen and its relation to complement levels in patients with rheumatic heart disease. Br Med J
1977;1:422.
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Ganguly NK, Anand IS, Khanna AK, Kohli RS, Wahi PL. T cells and T cell subsets in rheumatic heart disease. Indian J Med Res
1982;76:854–8.[Medline]
-
Bhatia R, Narula J, Reddy KS. Lymphocyte subsets in acute rheumatic fever and active rheumatic heart disease. Clin Cardiol
1989;2:34–8.
-
Marboe CC, Knowles DM, Weiss MB, Fenolgio JJ. Monoclonal antibody identification of mononuclear cells in endomyocardial biopsy specimens from a patient with rheumatic carditis. Hum Pathol
1985;16:332–8.[Medline]
-
Kemeny E, Grieve T, Marcus R, Sareli P, Zabriskie JB. Identification of mononuclear cells and T cell subsets in rheumatic valvulitis. Clin Immunol Immunopathol
1989; 52:225–37.[Medline]
-
Mogdy MZ, Farha A, Shennawy E, Aboubakr HM, Albasousy AM. Interleukin-2 in relation to T cell sub-populations in rheumatic heart disease. Arch Dis Child
1992;67:1373–5.[Abstract/Free Full Text]
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Virmani R, Robert WC. Aschoff bodies in operatively excised atrial appendages and in papillary muscles: frequency and clinical significance. Circulation
1977; 55:559.[Abstract/Free Full Text]
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ABSTRACT
INTRODUCTION
