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UPRES Lipids & Nutrition, Faculty of Life Sciences, Université de Bourgogne, Dijon 21000, France
2To whom correspondence should be addressed. E-mail: naim.khan{at}u-bourgogne.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • (n-3) fatty acids • hypertension • calcium • immunomodulation • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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2
3)
. Greenland Eskimos
and, to a lesser extent, some Japanese, with high dietary intake of
(n-3) PUFA from seafood, have a low incidence of cardiovascular
diseases compared with Western populations (4
,5)
. In
fact, unlike saturated fatty acids and cholesterol, which have been
implicated in atherogenesis, the (n-3) PUFA such as eicosapentaenoic
acid [EPA, 20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)] are
considered to be vasodilators and antihypertensive agents
(6)
. In an experimental model of hypertension, i.e.,
spontaneously hypertensive rats (SHR), a diet containing EPA-rich
fish oils can attenuate the development of hypertension in young and
old animals (7
,8)
. In another experimental study of
humans, an EPA-rich diet lowered blood pressure, platelet
aggregation and improved plasma lipid concentration (9)
.
In addition to the antihypertensive effects, (n-3) PUFA possess
immunosuppressive properties (10
,11)
. Epidemiologic
studies have demonstrated a decreased incidence of inflammatory
diseases in Greenland Eskimos and Japanese people (12)
. In
a clinical study, diet supplementation with fish oils exerted a
therapeutic anti-inflammatory effect in patients suffering from
rheumatoid arthritis (13)
. The intravenous administration
of triacylglycerols containing EPA into healthy volunteers suppressed
natural killer (NK) cell activity in these subjects (14)
.
Feeding (n-3) PUFA–containing oils decreased the production of
interleukin (IL)-1, IL-6 and tumor necrosis factor-
(TNF-) by
murine peritoneal macrophages (15)
. Consistent with these
animal experiments, Endres et al. (16)
and Meydani et al.
(17)
observed that fish oil supplementation of humans
diminished the ability of peripheral blood monocytes to produce
TNF-, IL-1 and IL-1ß. Mitogen-stimulated lymphocytes of
fish oil–fed mice secreted less IL-4 and IL-10 compared with those of
safflower oil–fed mice (15)
. Manhart et al.
(18)
compared the effects of different treatments and
observed that after feeding rats a diet containing (n-3) fatty acids
for 10 d, the production of immunoglobulin (Ig) A was diminished
in the Peyer’s patches. DHA inhibited not only the infiltration of
CD4+ cells but also the expression of mRNA for interferon-
, IL-6,
IL-1ß and IL-2 in mice fed a DHA-rich diet (19)
. DHA
has also been found to diminish significantly the expression of CD4 and
CD8 on circulating T lymphocytes (20)
. EPA seems to be a
more potent immunosuppressive agent than DHA (21)
. It has
been shown that the immunosuppressive effects of (n-3) PUFA in
different animal models are mediated via intervention with T-cell
signaling (11
,22)
.
Evidence has been put forth in favor of the hypothesis that
hypertension may be associated with malfunctioning of the immune system
(23)
. Circulating immunoglobulins have been found to be
higher in subjects with essential hypertension compared with normal
subjects (24
,25)
, and the presence of autoantibodies
against nuclear structures has been reported in malignant hypertension
(25)
. The sera of SHR have also been found to contain
autoantibodies against the ß-adrenoceptor (26)
.
Atherosclerosis may be an autoimmune disease, caused by the molecular
mimicry between microbial and human 60-kDa heat shock proteins
(27)
. Abnormal activation of the immune system of SHR,
along with the loss of suppressor T cells, has been reported
(28)
.
Although an early study showed that administration of exogenous IL-2
prevents the increases in blood pressure (BP) in SHR (29)
,
several later studies reported that not only did exogenous IL-2 fail to
lower the increases in BP in animals (30)
and humans
(31)
, but IL-2 therapy also favored the development of
renal dysfunction in almost all of the 72 patients (32)
.
In fact, the idea that high levels of circulating cytokines including
IL-2 are associated with the development of hypertension is receiving
strong support (33
34
35)
. Peters et al. (36)
recently demonstrated that proinflammatory cytokines are significantly
higher in patients with essential hypertension. Whether altered immune
function is a primary factor in the pathogenesis of hypertension or
secondary to tissue damage of vascular beds induced by hypertension is
unknown.
Several plausible mechanisms of action of (n-3) PUFA, implicated in the
decrease of BP, have been proposed, i.e., substitution of (n-6) PUFA in
plasma membrane phospholipids, reduced production of eicosanoids of
(n-6) family and inhibition of cyclooxygenase and lipoxygenase
activities (12)
. Some studies have tried to demonstrate
that (n-3) PUFA can modulate the expression of membrane markers on T
lymphocytes (10
,37)
. However, little is known about the
modulation of the second-messenger cascade, which is implicated in
(n-3) PUFA-induced immunosuppression during hypertension.
Therefore, the present study was conducted to elucidate the role of a
(n-3) PUFA-rich diet in T-cell calcium signaling in
hypertensive rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The culture medium RPM1640 and L-glutamine were obtained from Biowhitaker (Liège, Belgium). The fluorescent probe, Fura-2/AM, was procured from Molecular Probes (Eugene, OR). [3H]-Thymidine was purchased from Amersham Radiochemicals (Saclay, France). All other chemicals including thapsigargin, ionomycin and caffeine were obtained from Sigma Chemical (St. Louis, MO). The PUFA/EPAX diet was prepared by substituting control diet with EPAX-7010, which was generously provided by Polaris, Quimper, France.
Diets and animals.
Male normotensive 3-mo-old Wistar-Kyoto (WKY) rats
(n = 20) and SHR (n = 20),
weighing from 250 to 430 g, were obtained from IFA-CREDO
(L’arbresle, France). After 1 wk of acclimation, rats were fed control
or PUFA/EPAX diet for 2 mo. The chemical composition of the control
diet was as follows (g/kg dry diet): starch, 587; casein, 200;
cellulose, 50; sucrose, 50; mineral mix, 40; vitamin mix, 20;
DL-methionine, 3; vegetable oil-Isio-4 (Lesieur,
Neuilly-sur-Seine, France), 50. In the PUFA/EPAX diet, half of the
vegetable oil-Isio-4 was replaced by EPAX-7010. The compositions of the
mineral and the vitamin mixtures were identical to that described by
Frenoux et al. (7)
. Isio-4 oil contained the following
(mg/g): 18:2 (n-6), 47.2; total (n-3), 1.7; and monounsaturated fatty
acids, 40.2 (largely 18:1). EPAX-7010, in the form of ethyl ester,
contained 
85% (n-3) PUFA, i.e., EPA, 70%; DHA, 12%; and
-tocopherol, 2.1–3.2%. EPAX-7010 was tightly sealed under a stream
of nitrogen to avoid lipid oxidation and kept at 4°C.
Diets were prepared every day and the rats consumed them ad libitum. Uneaten food was discarded; food cups and water bottles were washed frequently. Rats were housed individually in stainless steel cages in a room maintained at 22°C with a 12-h light:dark cycle. Rats were weighed each week and systolic BP was determined four times by the tail-cuff method. The rats were completely anesthetized by intraperitoneal administration of sodium pentobarbital (0.2 mL/100 g body). The abdominal cavity was opened and whole blood was drawn from the abdominal aorta and the spleens were removed. The study was conducted in accordance with the university guidelines for the care and use of laboratory animals.
Isolation and preparation of splenic T cells.
The removed spleens were immediately transferred to the petri dishes, containing RPMI 1640 complete medium, supplemented with the following: 25 mmol/L HEPES; 2 mmol/L L-glutamine; 1 x 105 U/L penicillin; 100 mL/L streptomycin; and 100 mL/L fetal calf serum (FCS). The spleens were teased apart using a wire gauge. The number of viable cells was determined using the trypan blue exclusion test. After centrifugation (200 x g, 5 min), the cells were resuspended in PBS, pH 7.4, and placed in a sterile petri dish for 1 h at 37°C to remove the macrophages by adherence. T lymphocytes were isolated by panning. In brief, the unadhered cells were decanted and centrifuged (200 x g, 5 min) once with PBS-containing bovine serum albumin (2 g/L BSA), and were transferred to the petri dishes that were previously coated with anti-rat IgG (37.5 mg/15 mL) overnight at 4°C. Hence, selective depletion of B lymphocytes was accomplished because they adhered to the substratum of the petri dishes. After an incubation of 1 h at 4°C, the T-lymphocyte–rich supernatant was decanted and centrifuged (200 x g, 5 min) twice with PBS-BSA and resuspended in the same buffer. This technique provided us with an enriched (99%) T-cell population as verified by flow cyotofluorimetry (not shown). The cell viability was again checked by employing the trypan blue test.
Measurement of Ca2+ signaling.
The T cells (2 x 109/L), isolated by the panning technique, were washed with PBS, pH 7.4, and then loaded with Fura-2/AM (1 µmol/L) for 60 min at 37°C in loading buffer which contained the following (in mmol/L): NaCl, 110; KCI, 5.4; NaHCO3, 25; MgCl2, 0.8; KH2PO4, 0.4; HEPES-Na, 20; NaHPO4, 0.33; and CaCl2, 1.2; the pH was adjusted to 7.4.
After loading, the cells were washed three times (2000 x g, 10 min) and remained suspended in the identical
buffer. Intracellular free calcium ([Ca2+]i)
was measured according to Grynkiewicz et al. (38)
. The
fluorescence intensities were measured in the ratio mode in a PTI
spectrofluorometer at 340 nm and 380 nm (excitation filters) and 510 nm
(emission filters). The cells were stirred continuously throughout the
experiment. The test molecules were added into the cuvettes in small
volumes with no interruptions in recordings. The
[Ca2+]i were calculated using the following
equation: [Ca2+]i = Kd x (R - Rmin)/(Fmax
- F)(Sf2/Sb2).
A value of 224 for Kd was added into the
calculations. Rmax and
Rmin values were obtained by addition of
ionomycin (5 µmol/L) and MnCl2 (2 mmol/L),
respectively. All of the experiments were performed at 35°C instead
of 37°C to minimize the leakage of Fura-2. For each rat,
[Ca2+]i measurements were done at least in
quadruplicate.
T-cell blastogenesis.
For T-cell blastogenesis, all splenocytes, without eliminating accessory and B cells, were used. The cells (2 x 105/well) were cultured in 96-well flat-bottomed tissue culture plates (Nunc, Paris, France) in the presence or absence of concanavalin A (Con-A) at 1 µg/well. This concentrations of Con-A was found optimal to activate T cells (results not shown). Cells were distributed in six replicates as follows: 180 µL cell suspension, 20 µL mitogen or medium (RPMI1640 containing 10 mL/L FCS). Plates were incubated for 72 h at 37°C in a 5% CO2/air atmosphere. After 60 h, 20 µL of [3H]-thymidine (740 GBq/mmol, 29.6 kBq/well) was added. At the end of the incubation, cells were collected using a cell harvester (Dynatech, Vienna, Austria), trapping their DNA onto glass filtermats. When the filter circles were dried, we placed them in plastic minivials (Packard, Paris, France) with 4 mL Optifluor-O (Packard), and the radioactivity was recorded in a scintillation counter (Packard). The index of stimulation was determined as follows: radiolabel of cells with mitogen/radiolabel of unstimulated cells.
Interleukin-2 quantification.
Splenic T lymphocytes (2 x 105 cells/well) were isolated and cultured in the presence or absence of Con-A (1 µg/well) as described above. After 72 h, the supernatants were removed by centrifugation (200 x g, 5 min) and stored at -80°C. Culture supernatants were thawed, gently vortexed and centrifuged (100 x g, 1 min) and an aliquot (50 µL) was used for IL-2 quantification using a commercially available kit (R & D System, Oxford, UK).
Analysis of phospholipids.
The lipids from T lymphocytes were extracted according to the method of
Bligh and Dyer (39)
. Phospholipids were separated on
silica gel by TLC, using the solvent chloroform/methanol/acetic acid at
35:14:2.7 (v/v/v). The phospholipids were scraped and extracted from
silica by using chloroform/methanol/2 mol/L NaCl at 5:5:1 (v/v/v). The
plasma membrane phospholipids, after methylation at 80°C for 20 min
by BF3/methanol, were analyzed on TLC plates. The spots
from the TLC plates were scraped off and fatty acids were extracted
with 2 mL isooctane and separated by gas-liquid chromatography in a
Packard Model 417 gas-liquid chromatograph, equipped with a flame
ionization detector and a 30-m capillary gas column coated with
carbowax 20M. The analysis conditions were as follows: oven, 194°C
and injector and ionizing detector, 240°C. Helium was used as carrier
gas, with a flow rate of 0.4 mL/min. Analysis of fatty acid peaks was
achieved with reference to the internal standards (Nu-Chek-Prep,
Elysian, MN) by using DELSI ENICA 31 (Delsi Nermag, Rungis, France).
The fatty acid levels were expressed as g/100 g total fatty acids.
Statistical analysis.
Results are shown as means ± SEM. Statistical analyses of data were carried out using STATISTICA (version 4.1, Statsoft, Paris, France). The significance of the differences between mean values was determined by two-way ANOVA followed by the Least Significant Difference (LSD) test. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Feeding the PUFA/EPAX diet did not completely normalize the BP of SHR, but it did decrease the BP in these rats compared with SHR fed the control diet (SHR control, 251 ± 16 mm Hg vs. SHR PUFA/EPAX, 209 ± 2 mm Hg; P < 0.001). The PUFA/EPAX diet did not affect BP in WKY rats (WKY control, 161 ± 16 mm Hg vs. WKY PUFA/EPAX, 178 ± 7 mm Hg). The PUFA/EPAX diet did not affect the body weight of either group of rats (data not shown).
PUFA/EPAX dietary treatment exerts immunosuppressive effects.
Con-A–stimulated T-cell proliferation was not significantly
different between SHR and WKY rats (Fig. 1
). However, feeding the PUFA/EPAX diet suppressed Con-A–stimulated
T-cell blastogenesis in both strains (Fig. 1)
.
|
). PUFA/EPAX feeding diminished IL-2 secretion in T cells of SHR, but
not in those of WKY rats.
|
The basal intracellular free calcium concentrations,
[Ca2+]i, in T cells of
SHR were lower than those of WKY rats fed the control diet. Feeding
PUFA/EPAX to WKY rats did not alter basal
[Ca2+]i in T cells but it
significantly increased basal
[Ca2+]i in SHR
(Fig. 3
).
|
.
TG-induced increases in
[Ca2+]i were greater in T
cells of SHR than in those of WKY rats fed the control diet. Feeding
the PUFA/EPAX diet increased in WKY rats and decreased in SHR the
increases in [Ca2+]i in T
cells (Fig. 4
). The capacity of ionomycin as a channel opener was higher in T cells
of SHR compared with T lymphocytes of WKY rats fed the control diet
(Fig. 5
). Furthermore, the PUFA/EPAX diet did not influence the
ionomycin-induced increases in
[Ca2+]i in T cells of
either strain of rats (Fig. 5)
.
|
|
,
in the lymphocytes of either strain of rats (increases in
[Ca2+]i, WKY rats fed the
control diet, 12.9 ± 0.6 nmol/L vs. WKY rats fed the
PUFA/EPAX diet, 13.7 ± 0.8 nmol/L; and SHR fed the control diet,
14.1 ± 0.5 nmol/L vs. SHR fed the PUFA/EPAX diet, 15.9 ± 0.2 nmol/L). (n-3) PUFA replace (n-6) PUFA in T-cell phospholipids.
EPA and DHA, which were absent from the phospholipids of T cells of
rats fed the control diet, were present in both strains of rats fed the
PUFA/EPAX diet. The concentrations of EPA and DHA were significantly
higher in SHR than in WKY rats fed the PUFA/EPAX diet (Table 1
). The concentrations of arachidonic acid were lower in the
phospholipids of rats fed the PUFA/EPAX diet compared with those fed
the control diet . The concentrations of arachidonic acid were higher
in T cells from SHR than in those from WKY rats (Table 1)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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8
9)
. It is interesting to note that Con-A–stimulated T-cell proliferation was not significantly different in the strains of rats and the PUFA/EPAX diet significantly curtailed T-cell blastogenesis. Nonetheless, the secretion of IL-2 by mitogen-stimulated T cells was higher in SHR than that in WKY rats. These observations suggest that T cells from SHR secrete more IL-2 than those from WKY rats. It is possible that in vivo activation of the immune system during hypertension may be responsible for this difference in ex vivo secretion.
Our hypothesis is supported by several studies that have demonstrated
the abnormal activation of the immune system during hypertension
(24
,25
,28
,36)
, and high secretion of cytokines is
correlated with the severity of hypertension in humans
(33
,35)
. Because the T cells from SHR secrete high
concentrations of IL-2, the immunosuppressive action of (n-3) PUFA
should be more pronounced in these cells. As anticipated, the PUFA/EPAX
diet decreased IL-2 secretion only in mitogen-stimulated T cells of
SHR. Our observations agree well with the findings of several authors
who have also observed the decreased production of IL-2
(16)
and suppressed mitogen-induced T-cell
proliferation in fish oil–fed rats (41)
and mice
(11
,22)
.
Substantial evidence has been gained from studies of platelets from
patients with primary hypertension (42)
and from SHR
(43)
in support of the notion that the concentration of
[Ca2+]i is modified in
clinical and experimental hypertension. Investigators have not reached
a consensus because some studies have shown an increase in
[Ca2+]i in peripheral
(43)
and spleen T cells (44)
, whereas others
have shown no significant differences in
[Ca2+]i in lymphocytes
from hypertensive subjects compared with normotensive individuals
(45)
. This discrepancy is due largely to heterogeneity of
lymphocyte populations and differences in the protocols and techniques
used to determine increases in
[Ca2+]i. In our study, we
employed the double excitation technique, i.e., excitation at two
wavelengths to determine increases in
[Ca2+]i. This technique
corrects the error that is usually caused by the shift in the spectrum
from one wavelength to another during the increases in
[Ca2+]i if the cells are
excited at one wavelength. We found that basal
[Ca2+]i in T cells from
SHR were lower than those from WKY rats, and feeding the PUFA/EPAX
diet, without affecting the basal
[Ca2+]i in lymphocytes
from WKY rats, increased basal
[Ca2+]i in lymphocytes
from SHR. These observations demonstrate that T cells from SHR have
lower basal calcium concentrations compared with those from WKY rats,
and the EPA-rich diet can correct the reduced basal
[Ca2+]i in the former
without significantly influencing it in the latter.
We employed different agents to probe whether the mechanisms of calcium mobilization in T cells were modified during hypertension. Thapsigargin, which increases [Ca2+]i of the endoplasmic reticulum (ER) pool, evoked greater increases in T cells from SHR compared with those from WKY rats. Feeding the PUFA/EPAX diet exerted opposite effects in T cells, i.e., TG-induced increases were lower in SHR and potentiated in the WKY rats compared with the respective control diet–fed rats. These observations support the hypothesis that the PUFA/EPAX diet is affecting calcium homeostasis in these rats. The mechanisms of action of EPA (largely present in the experimental diet) and DHA (present in small amounts in the diet) in the modulation of intracellular calcium homeostasis are not well understood.
However, the PUFA/EPAX diet–induced decreases in TG response in SHR
can be substantiated in part by the findings of a recent study in which
a diet enriched with EPA and DHA exerted not only antineoplastic
effects but also decreased TG-induced increases in
[Ca2+]i in cancer cells
(46)
. Similarly, Gamberuchi et al. (47)
showed that in vitro addition of EPA and DHA can significantly diminish
TG-induced increases in
[Ca2+]i. We have also
observed that DHA can empty the ER pool in human Jurkat T cells
(48)
. Nonetheless, the stimulatory effects of PUFA/EPAX
diet on TG-induced increases in
[Ca2+]i in T cells of WKY
rats remain unexplained.
The mobilization of
[Ca2+]i by caffeine, an
agent that recruits calcium from the CICR pool, did not differ whether
the rats were fed the control or experimental diet. Ionomycin, a
calcium channel opener, induced a greater increase in
[Ca2+]i in T cells from
SHR compared with those from WKY rats, regardless of diet treatment.
Hence, we hypothesize that the plasma membranes of T cells from SHR may
be different in their composition from those of WKY rats; thus, the
former may be more sensitive than the latter to the action of
ionomycin. This is supported by the findings of Ebata et al.
(49)
who reported an increased density of
L-type calcium channels in heart and brain of SHR. The
density of calcium channels in T cells in SHR has not been examined. It
is possible that plasma membrane integrity may be altered in T cells
from SHR as evidenced by the difference in the composition of
phospholipids of T cells, particularly the contents of arachidonic
acid, in SHR and WKY rats. How this overall modification in plasma
membrane phospholipids is involved in the modulation of calcium
channels in T cells from SHR and WKY rats remains an unanswered
question. However, a number of anomalies have been described in
lymphocytes from hypertensive rats and humans including increased
Na+ influx and K+ efflux,
enhanced Na+-H+ and
Na+/Ca2+ exchanger
activities (43
,50)
.
Although further studies are required to establish a relationship between the mobilization of [Ca2+]i in T cells and its effect on therapeutic interventions, particularly on the use of known immunosuppressors, during the progression of cardiovascular pathology in hypertensive subjects, our study clearly demonstrates that EPA- and, in part, DHA-rich diets exert antihypertensive effects by inhibiting the secretion of IL-2 and modulating calcium homeostasis in T cells of these rats.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BP, blood pressure; BSA,
bovine serum albumin; [Ca2+]i, intracellular
free calcium concentrations; CICR, Ca2+-induced
Ca2+-release; Con-A, concavalin-A; DHA, docosahexaenoic
acid; EPA, eicosapentaenoic acid; ER, endoplasmic reticulum; FCS, fetal
calf serum; Ig, immunoglobulin; IL, interleukin; NK, natural killer;
PUFA/EPAX, polyunsaturated fatty acid diet containing EPAX-7010; SHR,
spontaneously hypertensive rats; TG, thapsigargin; TNF-, tumor
necrosis factor-; WKY, Wistar-Kyoto.
Manuscript received April 18, 2001. Initial review completed May 14, 2001. Revision accepted June 11, 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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