Adrenomedullin and adrenomedullin binding protein-1 downregulate TNF-α in macrophage cell line and rat Kupffer cells☆
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.
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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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This work was performed while the authors were at the University of Alabama at Birmingham, Birmingham, AL 35294.