Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-α in macrophage cell line and rat Kupffer cells

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Abstract

Recent studies have demonstrated that administration of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis. However, the mechanism responsible for the beneficial effect of AM/AMBP-1 remains unknown. The aim of this study therefore was to determine whether AM/AMBP-1 directly reduces lipopolysaccharide (LPS)-induced secretion of TNF-α from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from normal rats. TNF-α release and gene expression were determined by ELISA and RT-PCR, respectively. The results indicated that LPS increased TNF-α production from RAW cells by 38–63-fold in a dose- and time-dependent manner. Although incubation with AM or AMBP-1 alone inhibited LPS-induced TNF-α release by 14–22% and 13–22%, respectively, AM and AMBP-1 in combination significantly suppressed TNF-α production (by 24–35%). Moreover, the upregulated TNF-α mRNA by LPS stimulation was significantly reduced by AM/AMBP-1, but not by AM or AMBP-1 alone. In the Kupffer cells primary culture, AM or AMBP-1 alone inhibited LPS-induced TNF-α production by 52% and 44%, respectively. Co-culture with AM/AMBP-1 markedly reduced TNF-α production (by 90%). Moreover, AM or AMBP-1 alone decreased TNF-α mRNA expression by 41% and 36%, respectively, whereas the combination of AM/AMBP-1 decreased its expression by 63%. These results indicate that AM and AMBP-1 in combination effectively suppress LPS-induced TNF-α expression and release especially from primary cultured Kupffer cells, suggesting that the downregulatory effect of AM/AMBP-1 on proinflammatory cytokine TNF-α may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.

Introduction

Sepsis and septic shock are considered to be the major causes of morbidity and mortality in patients with severe trauma, burns, or blood loss [1]. Uncontrolled sepsis plays a central role in the development of multiple organ failure [2]. Studies have shown that circulating macrophages and tissue-fixed macrophages such as Kupffer cells are involved in immunologic and metabolic responses to sepsis [3], [4]. They produce proinflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6] and other inflammatory mediators that lead to tissue injury [5], [6]. In addition to its signaling effects among cell populations, TNF-α also enhances macrophage activation and causes cellular dysfunction [7].

Adrenomedullin (AM) is a potent vasodilator peptide which was originally isolated from a human pheochromocytoma and reported by Kitamura et al. [8]. AM acts as a circulating hormone which elicits various biological activities in a paracrine or autocrine manner. Our recent studies have shown that upregulation of AM plays a major role in initiating the hyperdynamic response during the early stage of sepsis and the reduced vascular responsiveness to AM appears to be responsible for the transition from the hyperdynamic phase to the hypodynamic phase during the progression of polymicrobial sepsis [9], [10], [11].

A specific AM binding protein (i.e., AMBP-1) in human plasma was recently identified [12] and the purified protein was reported to be identified to human complement factor H [13]. AMBP-1 enhances AM-mediated induction of cAMP in fibroblast; augments the AM-mediated growth of a cancer cell line; and suppresses the bactericidal capability of AM on Escherichia coli [13]. Conversely, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I [13]. Recent findings of the interaction between AM and AMBP-1 have opened a new avenue for further understanding of the AM pathobiology in sepsis [14]. Our recent studies have demonstrated that administration of AM and AMBP-1 in combination maintains cardiovascular stability and reduces mortality in sepsis [15]. It remains unknown, however, whether AM/AMBP-1 play any role in downregulating proinflammatory cytokine, TNF-α production during sepsis. Therefore, we hypothesized that AM/AMBP-1 directly reduce lipopolysaccharide (LPS)-stimulated secretion of TNF-α from murine macrophage-like cell line RAW 264.7 cells and Kupffer cells isolated from the normal rat. The present study was conducted to test this hypothesis.

Section snippets

Cell culture

Murine macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO Life Technologies, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. RAW 264.7 cells were plated at a density of 5×105/well in a 24-well plate. For all experiments, cells were grown to

Effects of AM and AMBP-1 on TNF-α production from RAW 264.7 cells stimulated by LPS

As shown in Table 1A, incubation of RAW 264.7 cells with LPS at concentrations from 2.5 to 100 ng/ml for a period of 4 h increased supernatant TNF-α levels by 38–63-fold (dose-dependent). Despite 13–22% decreases in TNF-α production following the addition of AM (100 nM) or AMBP-1 (50 nM), these differences were not statistically significant. However, incubation with AM and AMBP-1 in combination significantly decreased LPS-induced TNF-α secretion from RAW 264.7 cells compared with LPS alone

Discussion

Adrenomedullin is a potent vasodilatory peptide first isolated from extracts of human pheochromocytoma by monitoring the elevating activity of platelet cAMP [8]. It belongs to the calcitonin gene peptide superfamily based on its homology with calcitonin gene-related peptide (CGRP), amylin and calcitonin [21]. The expression of AM has been demonstrated in various tissues and biological fluids such as plasma, cerebrospinal fluid, sweat, amniotic fluid, urine, and milk [22], [23]. It is now

Acknowledgements

This study was supported by National Institutes of Health grant R01 GM 57468.

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