ARTICLE
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.
References
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