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High-magnesium concentration and cytokine production in human whole blood model

Magnesium Research. Volume 22, Numéro 2, 93-6, June 2009, Short communication

DOI : 10.1684/mrh.2009.0168


Auteur(s) : Wojciech Nowacki, Corinne Malpuech-Brugère, Edmond Rock, Yves Rayssiguier , Department of Veterinary Prevention and Immunology, Faculty of Veterinary Medicine, University of Environmental and Life Sciences, Wroclaw, Poland, INRA, UMR 1019 Nutrition Humaine, Saint-Genès Champanelle, France, Clermont Université, UFR Médecine, UMR 1019 Nutrition Humaine, Clermont-Ferrand, France, CRNH Auvergne, Clermont-Ferrand, France.



Auteur(s) : Wojciech Nowacki1, Corinne Malpuech-Brugère2,3,4, Edmond Rock2,3,4, Yves Rayssiguier2,3,4

1Department of Veterinary Prevention and Immunology, Faculty of Veterinary Medicine, University of Environmental and Life Sciences, Wroclaw, Poland
2INRA, UMR 1019 Nutrition Humaine, Saint-Genès Champanelle, France
3Clermont Université, UFR Médecine, UMR 1019 Nutrition Humaine, Clermont-Ferrand, France
4CRNH Auvergne, Clermont-Ferrand, France

There are several papers showing epidemiological, clinical and experimental evidence on the relationship between magnesium status and the inflammatory response [1-3]. It is well established that Mg deficiency in experimental animals leads to the increased leukocyte and macrophage activation, release of inflammatory cytokines and acute phase proteins and excessive production of free radicals [4]. In vitro studies have shown that a reduction in extracellular Mg resulted in phagocyte and vascular cell activation [4-8]. On the other hand, an increase in extracellular Mg in these models reduced the inflammatory response [7, 9]. As shown by Bussière et al. [7], increasing extracellular Mg concentration in the incubation medium decreased the superoxide anion production by polymorphonuclear leukocytes following activation by opsonized zymozan. An anti-inflammatory effect of Mg has also been shown in endothelial cells [10-13]. This relationship between extracellular Mg and the inflammatory response was mainly ascribed to its calcium antagonist properties [5]. However, despite large available data from ex vivo studies on cells from experimental animals there are no studies performed on human circulating cells.

In the present work we examined the effect of increasing Mg2+ concentration on the inflammatory response to septic shock using an ex vivo human whole blood model.

Material and methods

The protocol of the whole blood stimulation with lipopolysaccharide (LPS) was similar to that previously described [14, 15]. Blood from 13 healthy volunteers (5 men and 8 women) was collected into heparinised tubes (Vacutainer®, lithium heparin, Becton Dickinson, Le Pont-De-Claix, France). The study was approved by the local ethics committee. 20 μL of whole blood was diluted in 170 μL of Hank’s Buffered Salt Solution (HBSS) supplemented with penicillin and streptomycin (100 U/100 μg per mL) containing various concentrations of magnesium, as MgSO4. Ten μL of LPS (E. coli O111B4) in HBSS or 10 μL of HBSS alone were then added to the blood samples to give a final concentration of 500 ng of LPS/mL. The resulting final dilution of blood was ten fold and final concentrations of magnesium were 1, 3 and 10 mM. All samples were prepared in triplicate in 96-well cell culture plates. Plates were incubated at 37°C in a 5% CO2 atmosphere. Cell viability (white blood cells) was assessed using the trypan blue exclusion method and gave viability results of more than 90%. After 18 hrs incubation, whole blood samples were centrifuged (250 g) and supernatants were stored at -80°C until cytokine measurements. TNF-α, IL-6 and IL 8 were the cytokines measured, using commercial kits following manufacturer’s instructions (Genzyme, Cambridge, MA, USA). Measurements were made in duplicate. Results are expressed as means ± SEM. The results were analyzed by one-way ANOVA with Student-Newman-Keuls post hoc test. Differences with p-values of < 0.05 were considered significant.

Results and discussion

The results show that under basal (without LPS) conditions, only the high (10 mM) MgSO4 concentration inhibited the spontaneous production of the proinflammatory cytokines studied by incubated whole blood (figure 1). In the LPS-stimulated conditions there was only a tendency for an inhibitory effect of 10 mM MgSO4 for IL-6 and IL-8 production observed (figure 1).

The whole blood model has been extensively used and is considered as a useful tool for investigating immunomodulating effects on a mixed white blood cell population [16]. This test was also proposed as a prognostic indicator for patients at high risk for developing a sepsis syndrome [17]. Several laboratories have used this experimental approach but it is difficult to compare the results between these studies because of a lack of standardization with regard to LPS concentration, incubation time, blood dilution, cytokine studied and anticoagulant used. For this reason in the present work we extrapolated previously published approaches to select our own experimental conditions. Within the conditions studied, our results show that, in the conditions studied, the cytoprotective and anti-inflammatory action of MgSO4 appears relatively weak and is only observed with the highest Mg2+ concentration. Other more extended studies with more appropriate experimental conditions are needed to precise this action with regard to the intensity of septic shock and inflammatory response. However, the results of the present experiment are supported by previously performed in vivo studies, consisting of assessing the influence of plasma Mg concentration on cytokine production in rats after endotoxin challenge [18]. A significant increase in TNF-α plasma levels was observed in Mg-deficient rats compared to rats fed the control diet. Mg-deficient rats that received Mg replacement therapy before endotoxin challenge had significantly lower TNF-α plasma values than those receiving saline before endotoxin. These data clearly indicate that increasing plasma Mg of Mg-deficient rats by Mg supplementation prior to endotoxin challenge results in lower TNF-α production. This in vivo performed experiment also indicates that there were no significant differences in plasma TNF-α values in control animals that received Mg or saline before endotoxin challenge, despite a marked increase in plasma Mg levels. These results suggest that the efficiency of Mg supplementation on cytokine production depends on the Mg status and might explain the weak efficiency of increasing Mg concentration in human blood from healthy non Mg-deficient volunteers.


1 Weglicki WB, Mak IT, Kramer JH, Dickens BF, Cassidy MM, Stafford RE, et al. Role of free radicals and substance P in magnesium deficiency. Cardiovasc Res 1996; 31: 677-82.

2 Mazur A, Maier JA, Rock E, Gueux E, Nowacki W, Rayssiguier Y. Magnesium and the inflammatory response: potential physiopathological implications. Arch Biochem Biophys 2007; 458: 48-56.

3 King DE. Inflammation and elevation of C-reactive protein: Does magnesium play a key role? Magnes Res 2009; 22: 57-8.

4 Malpuech-Brugère C, Nowacki W, Daveau M, Gueux E, Linard C, Rock E, et al. Inflammatory response following acute magnesium deficiency in the rat. Biochim Biophys Acta 2000; 501: 91-8.

5 Malpuech-Brugère C, Rock E, Astier C, Nowacki W, Mazur A, Rayssiguier Y. Exacerbated immune stress response during experimental magnesium deficiency results from abnormal cell calcium homeostasis. Life Sci 1998; 63: 1815-22.

6 Bussière FI, Zimowska W, Gueux E, Rayssiguier Y, Mazur A. Stress protein expression cDNA array study supports activation of neutrophils during acute magnesium deficiency in rats. Magnes Res 2002; 15: 37-42.

7 Bussière FI, Gueux E, Rock E, Girardeau JP, Tridon A, Mazur A, et al. Increased phagocytosis and production of reactive oxygen species by neutrophils during magnesium deficiency in rats and inhibition by high magnesium concentration. Br J Nutr 2002; 87: 107-13.

8 Rayssiguier Y, Gueux E, Nowacki W, Rock E, Mazur A. High fructose consumption combined with low dietary magnesium intake may increase the incidence of the metabolic syndrome by inducing inflammation. Magnes Res 2006; 19: 237-43.

9 Bussière FI, Mazur A, Fauquert JL, Labbe A, Rayssiguier Y, Tridon A. High magnesium concentration in vitro decreases human leukocyte activation. Magnes Res 2002; 15: 43-8.

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12 Almoznino-Sarafian D, Berman S, Mor A, Shteinshnaider M, Gorelik O, Tzur I, et al. Magnesium and C-reactive protein in heart failure: an anti-inflammatory effect of magnesium administration? Eur J Nutr 2007; 46: 230-7.

13 Rochelson B, Dowling O, Schwartz N, Metz CN. Magnesium sulfate suppresses inflammatory responses by human umbilical vein endothelial cells (HuVECs) through the NFkappaB pathway. J Reprod Immunol 2007; 73: 101-7.

14 Setrakian JC, Yee J, Christou NV. Reduced tumor necrosis factor alpha production in lipopolysaccharide-treated whole blood from patients in the intensive care unit. Arch Surg 1994; 129: 187-92.

15 Monneret G, Pachot A, Laroche B, Picollet J, Bienvenu J. Procalcitonin and calcitonin gene-related peptide decrease LPS-induced TNF production by human circulating blood cells. Cytokine 2000; 12: 762-4.

16 Wang JE, Solberg R, Okkenhaug C, Jorgensen PF, Krohn CD, Aasen AO. Cytokine modulation in experimental endotoxemia: characterization of an ex vivo whole blood model. Eur Surg Res 2000; 32: 65-73.

17 Flach R, Majetschak M, Heukamp T, Jennissen V, Flohé S, Börgermann J, et al. Relation of ex vivo stimulated blood cytokine synthesis to post-traumatic sepsis. Cytokine 1999; 11: 173-8.

18 Malpuech-Brugère C, Nowacki W, Rock E, Gueux E, Mazur A, Rayssiguier Y. Enhanced tumor necrosis factor-alpha production following endotoxin challenge in rats is an early event during magnesium deficiency. Biochim Biophys Acta 1999; 1453: 35-40.


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