Rheumatic heart disease: proinflammatory cytokines play a role in the progression and maintenance of valvular lesions

Abstract

Heart lesions of rheumatic heart disease (RHD) patients contain T-cell clones that recognize heart proteins and streptococcal M peptides. To functionally characterize heart-infiltrating T lymphocytes, we evaluated their cytokine profile, both directly in situ and in T-cell lines derived from the heart (HIL). Interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 expressions were characterized in 20 heart tissue infiltrates from 14 RHD patients by immunohistochemistry. IFN-gamma-, TNF-alpha-, and IL-10-positive cells were consistently predominant, whereas IL-4 was scarce in the valves. In agreement with these data, the in vitro experiments, in which 13 HILs derived from heart samples of eight patients were stimulated with M5 protein and the immunodominant M5 (81-96) peptide, IL-4 was detected in HIL derived from the atrium (three of six) but not from the valve (zero of seven). IFN-gamma and IL-10 production were detected in culture supernatants in 11 of 13 and 6 of 12 HILs, respectively. The predominant IFN-gamma and TNF-alpha expression in the heart suggests that Th1-type cytokines could mediate RHD. Unlike in reversible myocardium inflammation, the significantly lower IL-4 expression in the valvular tissue (P = 0.02) may contribute to the progression of the RHD leading to permanent valvular damage (relative risk, 4.3; odds ratio, 15.8). The lack of IL-4 in vitro production by valve-derived HIL also emphasizes the more severe tissue destruction in valves observed in RHD.

Figure 1

Cytokines produced by infiltrating mononuclear cells from heart tissue fragments of RHD patients. Peroxidase (DAB) and alkaline phosphatase (Fast Red) immunohistochemistry were used for IFN-γ, TNF-α, IL-4, and IL-10 determination. The results were indicated by the number of positive cells by field. A: Positive cells (++) (10 to 50%) producing IFN-γ (patient 5); B: positive cells (++) (10 to 50%) producing TNF-α (patient 10); C: >50% of positive cells (+++) producing IL-4 with focal distribution (patient 4); D: 10 to 50% of positive cells (++) producing IL-10 indicated by long arrows on the right and >50% of positive cells (+++) in blood vessels indicated by large and small arrows on the left (patient 6). E: LA from patient 4; Aschoff bodies are indicated by short arrows and mononuclear infiltrating cells by long arrows. F: Mitral valve from patient 4; vegetations are indicated by short arrows and neutrophilic infiltrations by long arrows. H&E stain. Original magnifications: ×160 (A–D); ×20 (E, F).

Figure 2

Summary of Th1 and Th2 cytokines detected in infiltrating mononuclear cells from heart tissue samples of RHD patients. IFN-γ, TNF-α, IL-4, and IL-10 were determined in heart tissue samples by immunohistochemistry as shown in Figure 1. The frequencies of each cytokine were determined based on data from Table 2. *, <10% of positive cells, valve versus myocardium, P = 0.02, RR = 4.3, OR = 15.8, indicates the probability of valvular RHD lesion progression; **, >10 of positive cells, valve versus myocardium, P = 0.02, RR = 0.2, OR = 0.06, indicates a regulatory effect of IL-4 secretion leading to myocarditis healing.

Figure 3

Cytokine expression at the RNA level in five HILs after stimulation with the M5(81-96) peptide. Reverse transcriptase-polymerase chain reaction detection of IFN-γ, IL-4, and IL-10 mRNA from LA- and mitral and aortic valve (Mi v and Ao v)-derived T-cell lines at 12 and 48 hours after M5(81-96) stimulation. A: Reverse transcriptase-polymerase chain reaction product of patient 1 as an example of the pattern of cytokine detection. B: Cytokines produced were indicated by plus signs; negative means that no cytokines were detected.