Original Article
Selective 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

https://doi.org/10.1016/j.yjmcc.2004.01.001Get rights and content

Abstract

An increase in circulating brain natriuretic peptide (BNP) but not atrial natriuretic factor (ANF) is observed coincident with cardiac allograft rejection that is reversed upon treatment with anti-lymphocyte therapy suggesting that pro-inflammatory cytokines may uniquely modulate BNP gene expression and secretion. This study tested pro-inflammatory cytokines or conditioned medium (CM) derived from mixed– lymphocyte reaction (MLR) cultures in their ability to modulate ANF or BNP mRNA expression, secretion, as well as BNP promoter activity in cultured neonatal rat cardiocytes. IL-1β and TNF-α elicited a significant dose- and time-dependent increase in BNP mRNA, and secretion, whereas, ANF mRNA levels and secretion did not change. IL-1β and TNF-α rapidly increased phosphorylated p38 MAP kinase abundance and activity. Inhibition of p38 MAP kinase with SB203580 abolished IL-1β- and TNF-α-stimulated increase in BNP mRNA, promoter activity and secretion. MLR-CM in 20%, 50% and 100% proportions increased BNP but not ANF secretion. The MLR-induced increases in BNP secretion were completely abolished by SB203580 pre-treatment. These investigations show that exposure of cultured rat cardiocytes to specific pro-inflammatory cytokines as well as MLR-CM results in the only known instance of upregulation of cardiac BNP at the transcriptional and translational levels without a corresponding increase in ANF gene expression. Furthermore, these effects are dependent on signaling by p38 MAP kinase. In all, the findings reveal a unique dis-coordinated expression of BNP and ANF to inflammatory cytokines and offers an opportunity to better understand the differential regulation of these two cardiac-derived endocrine hormones that share receptors as well as biological properties.

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.

References (21)

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