Original ArticleSelective upregulation of cardiac brain natriuretic peptide at the transcriptional and translational levels by pro-inflammatory cytokines and by conditioned medium derived from mixed lymphocyte reactions via p38 MAP kinase
Introduction
The neuroendocrine activation associated with heart failure is characterized by an increase in the cardiac production and circulating levels of the natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP). Orthotopic cardiac transplantation is at present the most effective and definitive treatment for end-stage heart failure in humans. Therefore, it stands to reason that replacement of the failing heart would result in the normalization of cardiac natriuretic peptide levels. However, natriuretic peptide levels remain elevated post-heart transplant [1], [2]. In previous studies, we reported that BNP plasma levels, unlike ANF, increased prior to and peaked during significant rejection episodes as diagnosed by endomyocardial biopsy [2]. The selective upregulation of BNP observed in cardiac allograft rejection episodes is a unique response pattern, because under in vivo or in vitro conditions of hemodynamic overload or neurohumoral activation there is an increase in both ANF and BNP gene expression [3], [4].
A hypothesis regarding the events underlying the selective upregulation of BNP during acute cardiac allograft rejection may be formulated from clinical observations of patients who have undergone a heart transplant. Successful treatment of the rejection episodes with various immunosuppressive agents including OKT-3, a monoclonal antibody that inhibits T-lymphocyte activation, results in a decrease of BNP plasma levels together with an improvement of the histopathological grade [2] suggesting that activated T-lymphocyte secretion products during a rejection episode may modulate cardiocyte BNP secretion. Cytokines play central roles in the allo-specific components of graft rejection. They are involved in clonal expansion and activation of CD4+ and CD8+ T-lymphocytes (IL-2 and IFN-γ), B-lymphocytes (IL-2 and IL-4), the upregulation of MHC I and II antigens (IFN-γ and TNF-α) and they also increase the expression of adhesion molecules (IL-1β, IFN-γ and TNF-α) [5], [6].
In the present study, we investigated whether pro-inflammatory and immunoregulatory cytokines or conditioned medium (CM) harvested from allo-activated, mixed T-lymphocyte reactions (MLR) (an in vitro model of transplantation immunity) were capable of selectively upregulating BNP gene expression and secretion in vitro. We also determined the involvement of specific signaling pathways in mediating cytokine-stimulated changes in BNP gene expression, as well as the effect of BNP-stimulating cytokines on BNP promoter activity in ventricular cardiocytes. We report that specific cytokines and CM from MLR cultures upregulate BNP but not ANF at the transcriptional and translational levels through a specific signaling mechanism.
Section snippets
Neonatal rat ventricular cardiocyte culture
The experimental protocol was approved by the University of Ottawa Animal Care Committee in accordance with the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. The cardiocyte isolation is a modified procedure of Argentin et al. [7]. Apical 1/3 of the ventricles from 2- to 4-d-old Sprague–Dawley rats were isolated, washed in Joklik modified MEM (Gibco), minced into 1 mm or less fragments and subjected to four sequential 15-min digestions at 37 °C in 0.1%
IL-1β and TNF-α selectively increase BNP secretion
Fig. 1A shows BNP secretion from neonatal rat ventricular cardiocytes cultured in SF medium after exposure to IL-1β or TNF-α for 48 h. BNP secretion was significantly increased with respect to vehicle using 0.5 ng/ml of IL-1β for 48 h. Additional increases of IL-1β to 1 and 10 ng/ml further increased BNP levels. Similarly, treatment with TNF-α at concentrations of 1 and 20 ng/ml also increased BNP secretion in the medium.
Fig. 1B shows ANF secretion into the medium after exposure to IL-1β or
Discussion
ANF and BNP are potent modulators of vascular tone, fluid balance, and electrolyte balance and cardiovascular growth. As a rule, under pathophysiological conditions such as chronic hypertension or chronic congestive heart failure, the expression and secretion of both hormones are significantly augmented. An exception to this rule is seen in acute cardiac allograft rejection episodes when BNP and not ANF plasma levels are increased [2]. Furthermore, successful treatment of rejection with
Acknowledgments
This work was supported by grants from the Ontario Heart and Stroke Foundation and the Canadian Institute for Health Research (CIHR). We would like to acknowledge the expert technical assistance of Amalia Ponce.
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