Induction of autoimmune valvular heart disease by recombinant streptococcal m protein

Abstract

Rheumatic heart disease is an autoimmune sequela of group A streptococcal infection. Previous studies have established that streptococcal M protein is structurally and immunologically similar to cardiac myosin, a well-known mediator of inflammatory heart disease. In this study, we investigated the hypothesis that streptococcal M protein could produce inflammatory valvular heart lesions similar to those seen in rheumatic fever (RF). Fifty percent (3 of 6) of Lewis rats immunized with recombinant type 6 streptococcal M protein (rM6) developed valvulitis as well as focal lesions of myocarditis. Valvular lesions initiated at the valve surface endothelium spread into the valve. Anitschkow cells and verruca-like lesions were present. T cells from rM6-immunized rats proliferated in the presence of purified cardiac myosin, but not skeletal myosin. A T-cell line produced from rM6-treated rats proliferated in the presence of cardiac myosin and rM6 protein. The study demonstrates that the Lewis rat is a model of valvular heart disease and that streptococcal M protein can induce an autoimmune cell-mediated immune attack on the heart valve in an animal model. The data support the hypothesis that a bacterial antigen can break immune tolerance in vivo, an important concept in autoimmunity.

FIG. 1

Illustration of myocarditis in Lewis rat heart sections stained with hematoxylin and eosin. (A) Myocarditis is shown in rats immunized with streptococcal rM6 protein (magnification, ×400). Focal lesions were observed scattered throughout the rat myocardium, which contained interstitial accumulations of mononuclear cells intermixed with a lesser number of neutrophils. Myocyte necrosis (arrow) is noted in the central lesion. (B) Heart tissue sections from control rats immunized with PBS and adjuvants contained no lesions (magnification, ×400). Although not shown, lesions were not found in kidneys and livers of any of the animals.

FIG. 2

Illustration of valvulitis and cellular infiltration in hematoxylin- and eosin-stained mitral valves from Lewis rats immunized with streptococcal rM6 protein. (A and B) Valvulitis in the base of the mitral valve (V) adjacent to myocardium, which did not contain lesions. The lower magnification in panel A orients the valve (V) adjacent to the myocardium. The enlargement in panel B shows the presence of mononuclear and Anitschkow (arrows) cells. (C) Arrows indicate disruption of endocardial (endothelial) surface with infiltrating cells. (D) Verruca-like nodule (arrow) on the valve surface. (E) Anitschkow cells (arrows). (F) Hematoxylin- and eosin-stained normal heart valve tissue section from control rats immunized with PBS and adjuvants. Magnifications, ×200 and ×400.

FIG. 3

Lymphocytes from rM6-immunized rats proliferate in the presence of human cardiac myosin. Lymphocytes from inguinal and popliteal lymph nodes of rats immunized and boosted with 500 μg of rM6 were reacted with human cardiac myosin (HCM), rabbit skeletal myosin (RSM), and actin in the tritiated thymidine incorporation assay as described in Materials and Methods. Values represent the SI (mean of test cpm/mean of medium control cpm). Medium controls in the proliferation assays ranged from 2,000 to 5,000 cpm.

FIG. 4

Response of rat T-cell line M6.8 to rM6 protein and human cardiac myosin. The proliferative response of line M6.8 was measured with a tritiated [³H] thymidine uptake assay. T-cell line M6.8 was cultured as 5 × 10⁴ cells per well with 20 μg of antigen per ml. rM6, purified type 6 M protein; HCM, human cardiac myosin; Lyso, lysozyme plus 2.5 × 10⁵ mitomycin-treated spleen cells. Samples were tested in triplicate, and the results were recorded as cpm with the background subtracted. Error bars are shown.

FIG. 5

Human rheumatic heart valve section reacted with anti-CD4⁺ antibody shown for comparison with the rat valve sections. The human rheumatic valve section (A) illustrates the infiltration of CD4⁺ lymphocytes (stained with fast red) through the endothelium into the valve and the presence of a necrotic Aschoff body in the valve tissue. A rheumatic valve section was reacted with a control immunoglobulin G1 antibody and did not show any reactivity (B). Arrows point to CD4⁺ T lymphocytes entering the valve.