Antibodies against Trypanosoma cruzi ribosomal P proteins induce apoptosis on HL-1 cardiac cells

LEVY GV; LEVIN MJ; GOMEZ KA

Mar del Plata, Argentina

Congreso; XLIII Reunión Anual de la SAIB; 2007

Sociedad Argentina de Investigación en

Chronic Chagas Heart Disease (cChHD), a chronic manifestation of Trypanosoma cruzi (T. cruzi) infection, is characterized by high antibody (Ab) levels mainly toward parasite intracellular proteins. Among these, the acidic C-terminal region of the ribosomal P proteins (i.e. epitope R13 of TcP2b protein) is considered to be highly immunogenic and bears similarity to the second extracellular loop (H26R) of the b1-adrenergic receptor (b1-AR). Our previous results demonstrated that anti-R13 Abs from patients with cChHD recognize and activate b1-AR and M2-cholinergic receptors (M2-ChR), as seen by immunocytochemistry and cAMP accumulation on stably transfected cells. The aim of this work was to evaluate the long term effect of anti-R13 Ab stimulation on cardiac cells. Thus, adult murine cardiac HL-1 cells were treated with: a) mAb anti-R13, named 17.2, b) IgG fractions from cChHD patients and c) non related mAb, named 40.14. Programmed cell death parameters were evaluated with dual staining with Annexin-V-FITC and Propidium Iodide (PI) by flow cytometry, terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity (TUNEL) combined with DAPI staining, and Bax/BclXL mRNA levels by Quantitative Real-time PCR. These results showed that mAb 17.2 induced 6.37% ± 2.29 of apoptosis on cardiac HL-1 cells by TUNEL, while non related Ab produced only a 0.72% ± 0.92 of apoptosis cells. This long term induction was completely abolished by preincubation with propranolol, a b-AR antagonist. Moreover, mAb 17.2 increased the ratio between proapoptotic Bax and antiapoptotic BclXL mRNA levels. Late apoptosis changes on cardiac cells induced by mAb 17.2 were further confirmed using Annexin V-PI dual staining via flow cytometry. Likewise, IgG fractions from cChHD patients with an exclusive b1-AR activity also induced apoptosis as seen by the ratio Bax/BclXL, but those IgG fractions with both b1-AR/M2-ChR stimulating effects only produced apoptosis when cells were incubated with a muscarinic antagonist (atropine). Although another mechanism may be involved in heart functional alterations observed in patients with Chagas disease, these results support the hypothesis that b-adrenergic chronic stimulation by anti-R13 Abs could result in cardiotoxic effects similar to those known to be produced by agonists. This may have important clinical significance in the use of immunosuppression or immunoadsoption approaches to avoid the cross-reactive immune response produced by anti-R13 Abs without interfering with antiparasite immunity in order to improve the symptoms and prevent the development of cardiomyopathy.