The Journal of Nutrition
Dietary Fish Oil Increases the Number of Splenic
Macrophages Secreting TNF-a and IL-10 But
Decreases the Secretion of These Cytokines by
Splenic T Cells from Mice1,2
Dagbjort H. Petursdottir and Ingibjorg Hardardottir*
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Iceland, 101 Reykjavik, Iceland
Dietary fish oil has immunomodulatory effects that are partly mediated by its effects on cytokine secretion. In this paper,
we examine whether dietary fish oil has different effects on cytokine secretion by T cells and macrophages. Female BalbC
mice were fed diets supplemented with 18% fish oil 1 2% corn oil or 20% corn oil. Concanavalin A (ConA)- and LPS-
induced TNF-a and IL-10 secretion by splenocytes was examined using ELISA. Dietary fish oil decreased ConA induced-,
butincreasedLPS-induced, TNF-aand IL-10secretionbytotalmurinesplenocytes.Dietaryfishoilincreasedthenumberof
splenocytes secreting TNF-a and IL-10, following stimulation with LPS, by 123 and 38%, respectively, but did not affect
cytokine secretion by each cell, as determined using enzyme-linked immunospot. Spleens from mice fed the fish oil diet
had over 2-fold higher proportion of macrophages with high expression of CD11b than spleens from mice fed the corn
oil diet. In addition, fish oil increased the proportion of total and CD11b1splenocytes that expressed the LPS receptor
complex molecules, CD14 and toll-like receptor (TLR)4/myeloid differentiation factor-2 (MD-2), by 85 and 28%, respec-
tively. The increased proportion of macrophages expressing the LPS receptor complex molecules, CD14 and TLR4/MD-2,
in spleens from mice fed the fish oil diet may explain the increased number of cells that secreted the cytokines after LPS
stimulation.J. Nutr. 137: 665–670, 2007.
The immunomodulatory effects of dietary fish oil are generally
thought to be antiinflammatory (1). This hypothesis is based on
results from human studies indicating that dietary fish oil or
(n-3) PUFA arebeneficial inseveralinflammatory disorders (2–4).
The antiinflammatory effects of dietary fish oil are thought to be
mediated in part by decreased proinflammatory cytokine pro-
duction, shown, for example, by decreased circulating IL-1b
levels in patients with rheumatoid arthritis (5) and decreased
LPS-induced TNF-a and IL-1b secretion by human peripheral
blood mononuclear cells (6–8). Decreased IL-2 secretion by
murine T lymphocytes stimulated with concanavalin A (ConA)3
or anti-CD3/anti-CD28 (9,10) has also been shown following
feeding with low- (5 g/100 g) or high- (18 g/100 g) fat diets
containing (n-3) PUFA or fish oil (9–11). In addition, a number
of studies have shown that dietary fish oil, or (n-3) PUFA, de-
crease mitogen-induced proliferation by lymphocytes in rodents
and men fed low- or high-fat diets (7,10,12–16). The decreased
lymphoproliferation may be dependent on dietary fish oil mod-
ification of lipid rafts and displacement of signaling proteins and
cytokine receptors from lipid rafts (17,18). Displacement of
signaling proteins and cytokine receptors from lipid rafts could
lead to decreased intracellular signaling and dietary fish oil has
indeed been shown to decrease events linked to intracellular
signaling, such as formation of diacylglycerol and ceramide (10),
tyrosine phosphorylation of protein kinase C-g (19), and recruit-
ment of protein kinase C-u to lipid rafts (9).
Although dietary fish oil has antiinflammatory effects on
cytokine secretion by circulating monocytes and T cells, as de-
scribed above, results from several studies show that dietary fish
oil has proinflammatory effects on cytokine secretion by macro-
phages. We and others have shown that feeding mice fish oil
increases LPS-induced TNF-a and IL-1b secretion by resident
peritoneal macrophages (20–27) but decreases secretion of the
antiinflammatory cytokine, IL-10 (25). In addition, dietary fish
oil increases TNF-a secretion by murine splenocytes after stimu-
lation with LPS (28,29). These proinflammatory effects were
seen in mice fed diets containing 5 (29), 10 (22–24,26), or 20
g/100 g fat (25), with (n-3) PUFA being as low as 1.5 g/100 g in
one of these studies (22). The increased TNF-a secretion by
resident peritoneal macrophages from mice fed fish oil is partly
1Supported by a grant from the Icelandic Research Council’s Research Fund and
Graduate Education Fund (DHP) and The Research Fund of the University of
2Supplemental Tables 1 and 2 and Supplemental Figure 1 are available with the
online posting of this paper at jn.nutrition.org.
3Abbreviations used: ConA, concanavalin A; ELISpot, enzyme-linked immuno-
spot; MD-2, myeloid differentiation factor-2; TLR, toll-like receptor.
* To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
0022-3166/07 $8.00 ª 2007 American Society for Nutrition.
Manuscript received 22 September 2006. Initial review completed 6 November 2006. Revision accepted 10 December 2006.
by guest on June 6, 2013
Supplemental Material can be found at:
explained by decreased prostaglandin production (22,25,27),
but other mechanisms at work remain to be elucidated.
As discussed above, dietary fish oil seems to have different
effects on cytokine secretion by T cells and circulating mono-
cytes than on tissue macrophages. Thus, the purpose of this
study was to determine whether dietary fish oil has different
effects on cytokine secretion by splenic T cells and macrophages
obtained from the same animals and using similar experimental
setup. We chose to monitor the secretion of 1 proinflammatory
cytokine, TNF-a, and 1 antiinflammatory cytokine, IL-10, be-
cause these cytokines are often monitored following induction
of macrophages with LPS (30,31). We also explored a possible
mechanism by which dietary fish oil affects cytokine secretion by
Materials and Methods
Animals and diets. All experimental procedures using laboratory an-
imals complied with the National Research Council’s Guide for the Care
and Use of Laboratory Animals. Female BalbC mice weighing 18–20 g
(Bomholtgaard) were randomly divided into 2 groups of 10 mice each.
Mice were housed 5 per cage at 25?C with a 12-h light and dark cycle.
We designed experimental diets according to AIN-93 guidelines (32) with
modification in fat content. The experimental diets were based on a
nutritionally complete diet made for the addition of 200 g/kg of fat (ICN
Pharmaceuticals), as previously described (25). The fish oil diet contained
180 g/kg menhaden fish oil and 20 g/kg corn oil (ICN Pharmaceuticals).
The corn oil diet contained 200 g/kg corn oil. The antioxidant tert-
butylhydroquinone (ICN Biomedicals) (1.2 mmol/L) was added to the oils
to prevent their deterioration (33). Diets were prepared in bulk and daily
portions packed in zip-lock bags, flushed with nitrogen, sealed, and stored
at 220?C. Mice consumed water and food ad libitum. The mice were fed
the experimental diets for 5 to 6 wk. The length of feeding did not influ-
ence the effect of dietary fish oil on the parameters measured. An equal
numberof animalsfromeachdietarygroupwaskilled ateach timepoint.
Fatty acid analysis. Fatty acid composition of the diets was analyzed,
as described previously (25, Supplemental Table 1). Fatty acid compo-
sition of hepatic phospholipids from mice fed the fish oil and corn oil
diets was analyzed, as described previously (25, Supplemental Table 2).
A higher proportion of (n-3) PUFA and a lower proportionof (n-6) PUFA
in hepatic phospholipids from mice fed the fish oil diet, compared with
that in hepatic phospholipids from mice fed the corn oil diet, demon-
strate the effectiveness of the diets in changing tissue fatty acid compo-
Isolation and activation of splenocytes. Mice were anesthetized with
isoflurane (Abbot Scandinavia) and blood was collected by axillary
bleeding. Serum from 2–3 mice in the same dietary group was pooled,
heat inactivated at 56?C for 40 min, and used as homologous serum in
cell cultures. The mice were killed by cervical dislocation. Spleens were
removed aseptically postmortem, cut, and passed through a wire mesh to
obtain a single cell suspension. The cells were treated with lysing buffer
(0.15 mol/L NH4Cl, 1 mmol/L KHCO3, 0.1 mmol/L Na2EDTA) to lyse
red blood cells. For cytokine measurements, 5 3 109cells/L were
cultured in 0.2 mL/well on a 96-well plate with 5% homologous serum
at 37?C in an atmosphere of 5% CO2. The cells were stimulated for 24 h
with 2 mg/L LPS, (E. coli 055:195, Fluka Chemie) or for 48 h with
8 mg/L ConA (Sigma-Aldrich).
TNF-a and IL-10 quantitation. After incubation of cells, the culture
plateswerecentrifuged,supernatants collected, andstoredat270?C. We
measured TNF-a and IL-10 with Duo Set ELISA kits (R&D Systems).
Determination of the numbers of TNF-a and IL-10 secreting cells.
Numbers of TNF-a and IL-10 secreting cells were determined by
enzyme-linked immunospot (ELISpot; R&D Systems). For TNF-a,
1.25 3 107cells/L and for IL-10, 2 3 108cells/L were incubated with
or without LPS (2 mg/L) for 24 h on polyvinilidine difluoridemicroplates
(Millipore) coated with anti-mouse TNF-a or IL-10. Spots were devel-
oped using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium
(R&D Systems) and automatically evaluated with a computer-assisted
video image analyzer (KS ELISpot, version 4.8, Zeiss).
Flow cytometric analysis of splenocytes producing TNF-a. Marker
expression on TNF-a producing cells from mice receiving standard chow
was examined usingflowcytometry. Cells(5 3 109cells/L) were cultured
with LPS (2 mg/L) and brefeldin A (1 mL/L) (BD Pharmingen), a protein
transport inhibitor, for 2 h. We then incubated the cells with anti-mouse
CD3-FITC, CD19-PE, CD11b-PE, CD49b-PE (BD Pharmingen), CD169-
FITC, and F4/80-PE (Serotec). The cells were fixed and permeabilized
using Leukoperm Permeabilization kit (Serotec) and stained with anti-
bodies against the intracellular markers CD68-PE (FA-11) or MOMA-
2-PE (Serotec) and anti-mouse TNF-a-APC (eBioscience). Cells were
incubated with anti-mouse CD16/32 (2 mg/106cells) (Mouse Fc Block,
BD Pharmingen) prior to incubation with immunoglobulin G antibodies
to block unspecific binding. Appropriate isotypic control for each anti-
body was used to set the quadrants and evaluate background staining.
We performed flow cytometry with a Becton Dickinson FACScalibur
flow cytometer equipped with an argon ion laser. A total of 100,000
events were collected for each analysis. Data were analyzed using FCS
express V3 (de Novo Software).
Flow cytometric analysis of splenocyte subpopulations. Cells were
incubated with mouse Fc Block (2 mg/106cells) and stained with 1 or
more of the following monoclonal antibodies: CD3-FITC (17A2), CD4-
PE (RM4–5), CD8-PerCP (53–6.7), CD19-PE (1D3), CD45-FITC (30-
F11), CD49b-PE (DX5), CD11c-PE (HL3) (Pharmingen), F4/80-PE
(A3–1), CD169-FITC (3d6.112) (Serotec), CD11b-PE, or PE-Cy5 (M1/
70) (eBioscience). Cells were also stained intracellularly with CD68-PE,
MOMA-2-PE, CD14-FITC (Sa2–8), and toll-like receptor (TLR)-4/
myeloid differentiation factor-2 (MD-2)-PE (MTS510) (eBioscience)
using Cytofix/Cytoperm kit (Becton Dickinson) or Leukoperm. Cells
were then washed and fixed in 1% paraformaldehyde. Appropriate
isotypic control for each antibody was used to set the quadrants and
evaluate background staining. We collected a total of 25,000 events.
Isolation and activation of splenic CD11b positive macrophages.
CD11b positive macrophages were isolated using magnetic activated cell
sorting (Miltenyi Biotech). After isolation, 1 3 109cells/L were cultured,
0.2 mL/well, on a 96-well plate with 5% homologous serum, and stim-
ulated with LPS (2 mg/L) for 24 h. Approximately 90% of the isolated
cells had high expression of CD11b (CD11bhigh), as determined by flow
cytometry in both dietary groups.
Statistical analysis. We analyzed differences between dietary groups by
an unpaired Student’s t test using StatsDirect statistical program, version
2.3.3 (StatsDirect) and differences were significant if P , 0.05 (two-
Mouse growth, spleen weights, and cell counts. Body
weights and relative weight gains did not differ between the
groups. Mice fed the fish oil diet had heavierspleens (1716 6 mg)
the fish oil diet also had a greater spleen cell count (98 6 4 3106
cells/spleen) than those fed the corn oil diet (71 6 4 3106cells/
spleen, P , 0.0001).
LPS and Con A induced TNF-a and IL-10 secretion by total
splenocytes. After stimulation with LPS, total splenocytes from
mice fed the fish oil diet secreted significantly more TNF-a and
IL-10 than splenocytes from mice fed the corn oil diet (Fig.
1A,B). However, when stimulated with ConA, total splenocytes
from mice fed the fish oil diet secreted significantly less TNF-a
666Petursdottir and Hardardottir
by guest on June 6, 2013
and IL-10 than splenocytes from mice fed the corn oil diet (Fig.
The number of splenocytes that secreted TNF-a and IL-10
following stimulation with LPS. The number of splenocytes
that secreted TNF-a following stimulation with LPS was higher
in mice fed the fish oil diet (1698 6 226 spots/105cells) than in
micefed the corn oil diet (7606 109 spots/105cells, P ¼ 0.0025).
The number of splenocytes secreting IL-10 was higher in mice
fed the fish oil diet (149 6 6 spots/105cells) than in mice fed the
corn oil diet (108 6 15 spots/105cells, P ¼ 0.03). The mean
TNF-a and IL-10 secretion per cell was similar in splenocyte
cultures from mice fed the fish oil diet and the corn oil diet
(results not shown).
Marker expression on splenocytes that produced TNF-a. A
large proportion (;90%) of the cells that produced TNF-a were
macrophages expressing CD11b, MOMA-2, and CD68 and
;70% expressed F4/80. Hardly any cells producing TNF-a
expressed the macrophage marker CD169, the NK cell marker
CD49b, or the T and B cell markers CD3 and CD19 (Fig. 2).
Marker expression on splenocytes. Spleens from mice fed
the fish oil diet had a higher proportion of macrophages with
from mice fed the corn oil diet (Table 1). The proportion of
MOMA-2 positive macrophages, which are mainly macrophages
from the periarteriolar lymphoid sheet and marginal zone (34),
was also increased in spleens from mice fed the fish oil diet com-
pared with that in spleens from mice fed the corn oil diet. On the
other hand, the proportion of red pulp macrophages, expressing
F4/80, was lower in spleens from mice fed the fish oil diet than in
spleens from mice fed the corn oil diet. Dietary fish oil did not
affect the proportion of macrophages expressing macrosialin
(CD68) or the proportion of metallophilic macrophages express-
ing CD169 (Table 1).
Examination of other surface markers demonstrated that the
from mice fed the fish oil diet than in spleens from mice fed the
corn oil diet, but the proportion of splenic total leukocytes
(CD45), B cells (CD19), T cells (CD3, CD4, CD8), and dendritic
cells (CD11c) was similar in spleens from mice fed the corn oil
diet and spleens from mice fed the fish oil diet (Table 1).
LPS induced TNF-a and IL-10 secretion by CD11b1
splenocytes. Cell yield after isolation of CD11b1cells was
70% higher in the fish oil group (6.0 6 0.4 3 106cells) com-
pared with that in the corn oil group (3.6 6 0.4 3 106cells, P ¼
0.0014). When the same number of CD11b1cells was cultured
and stimulated with LPS, isolated CD11b1splenocytes from
mice fed the fish oil diet secreted more TNF-a and IL-10 than
TNF-a-producing splenocytes of BALB/c mice receiving standard chow. Cells
were stimulated with LPS (2 mg/L), stained with appropriate markers, and
analyzed by flow cytometry. (A) Representative dot plots of LPS-induced TNF-a
staining of cells and marker expression. Numbers represent the proportion of
gated cells in each quadrant. (B) The proportion of TNF-a positive splenocytes
expressing the indicated markers. Values are mean 6 SEM, n ¼ 3.
Flow cytometric analysis for identification and characterization of
and IL-10 (B) secretion by total splenocytes from mice fed the corn oil (open
bars) or the fish oil diet (closed bars). Cells were stimulated with LPS (2 mg/L)
or ConA (8 mg/L) for 24 h and 48 h, respectively. Values are means 6 SEM,
n ¼ 10. * Different from corn oil, P , 0.05.
The effects of dietary fish oil on LPS- and ConA-induced TNF-a (A)
Marker expression on splenocytes from mice fed
diets containing corn oil or fish oil1
MarkerCorn oilFish oil
Other cell types
2.8 6 0.2
6.3 6 0.3
10.3 6 0.8
4.2 6 0.2
2.7 6 0.3
7.3 6 0.7*
10.7 6 0.9*
7.3 6 0.5*
4.0 6 0.3
3.2 6 0.4
10.7 6 0.6
98.8 6 1.7
53.7 6 1.7
24.2 6 0.7
11.1 6 0.3
3.8 6 0.6
7.1 6 0.4*
97.8 6 1.1
57.0 6 1.1
24.2 6 0.7
10.9 6 0.9
4.0 6 1.1
1Values are means 6 SEM, n ¼ 5–8. * Different from corn oil, P , 0.05.
Fish oil affects TNF-a and IL-10 secretion667
by guest on June 6, 2013
CD11b1splenocytes from mice fed the corn oil diet (Fig. 3).
TNF-a and IL-10 secretion by total splenocytes from mice fed
the corn oil and the fish oil diet in the same experiment is shown
Relative expression of the LPS receptor complex. Spleens
from mice fed the fish oil diet had a higher proportion (32%) of
isolated CD11b1cells expressing CD14 and TLR4/MD-2 than
spleens from mice fed the corn oil diet (Fig. 4A). In comparison,
the proportion of total splenocytes expressing CD14 and TLR4/
MD-2 was 85% higher in mice fed the fish oil diet than in mice
fed the corn oil diet (Fig. 4B). Dietary fish oil did not affect the
mean fluorescence intensity for CD14 and TLR4/MD-2 on
splenocytes from mice fed the different diets (data not shown).
The results from this study demonstrate that dietary fish oil has
different effects on TNF-a and IL-10 secretion by splenocytes
stimulated with ConA or LPS (Fig. 1). The TNF-a and IL-10
measured, following stimulation with ConA, was most likely
secreted by T cells, as splenocytes depleted of CD90.11T cells
secreted no detectable TNF-a and IL-10 following stimulation
with ConA (results not shown). ConA is a plant lectin and a
well-characterized mitogen that stimulates T cells by binding to
the CD3 molecule and perhaps also via a pathway dependent on
the T cell surface protein, CD2 (35). On the other hand, TNF-a
and IL-10 measured following stimulation with LPS was most
likely secreted by macrophages, as LPS binds to CD14 (36)
expressed on monocytes and macrophages (37,38) and signals
into the cell through the TLR4/MD-2 complex (39). LPS also
stimulates B cells through a complex containing TLR4 and the
TLR protein, RP105 (40), leading to enhanced antigen-presenting
capacity, enhanced proliferation, and secretion of LPS-neutralizing
antibodies (41,42), but TNF-a and IL-10 secretion by B cells has
been shown at later time points and using higher concentration
of LPS than in this study (43,44). In addition, characterization of
the cells secreting TNF-a after LPS stimulation in our study
revealed that few cells expressing the B cell marker, CD19,
produced TNF-a (Fig. 2). Thus, our results indicate that dietary
fish oil differently affects cytokine secretion by T cells and mac-
rophages, decreasing TNF-a and IL-10 secretion by T cells and
increasing the secretion of these cytokines by splenic macro-
Decreased ConA-induced TNF-a secretion by splenocytes
from mice fed (n-3) PUFA has been shown previously (45) and
we have also seen decreased TNF-a secretion by total splenocytes
and isolated CD90 positive T cells, following stimulation with
in agreement with results by Ly et al. (46) who showed decreased
anti-CD3/anti-CD28 induced IL-10 secretion by CD41T cells
from mice fed DHA. Yaqoob et al. (47) showed no effect of
dietary fish oil on IL-10 secretion by splenic lymphocytes stim-
ulated with ConA, which can possibly be explained by the shorter
stimulation time (24 h) used in that study, compared with the
stimulation time (48 h) in this study and the one by Ly et al. (46).
A number of studies also showed decreased ConA and anti-CD3/
anti-CD28 induced IL-2 secretion and proliferation of splenic
results indicate that dietary (n-3) PUFA have antiinflammatory
effects on cytokine secretion and proliferation of T cells.
Results from this study showing increased TNF-a secretion
by splenic macrophages from mice fed fish oil are in agreement
with results by others (28,29), but, to our knowledge, the effects
of dietary fish oil on LPS-induced IL-10 secretion by splenocytes
has not been investigated previously. The increase in LPS-
induced TNF-a and IL-10 secretion by splenocytes in this study
proved to be due to an increase in the number of cells secreting
these cytokines, but not to increased TNF-a and IL-10 secretion
by each cell (demonstrated byELISpot). Thus, the mechanism by
which dietary fish oil affects cytokine secretion by splenic mac-
rophages may be very different from the mechanism by which
they affect cytokine secretion by T cells, which has been attrib-
uted, e.g., to the ability of (n-3) PUFA to affect the production of
lipid messengers, to modify action of nuclear receptors, and to
affect lipid rafts in cell membranes [reviewed in (49)]. Whether
increased LPS-induced TNF-a secretion by splenocytes (28,29)
or resident peritoneal macrophages (20–25) from mice fed fish
oil seen in previous studies is due to an increase in the number of
cells secreting the cytokine is not known. Results from our
laboratory show that dietary fish oil does not affect the number
of resident peritoneal macrophages secreting TNF-a, but, rather,
TLR4/MD-2 in isolated CD11b1splenocytes (A) and total splenocytes (B).
Values are means 6 SEM, n ¼ 4 for CD11b1macrophages, n ¼ 5 for total
splenocytes. * Different from corn oil, P , 0.05.
The effects of dietary fish oil on intracellular expression of CD14 and
secretion by isolated CD11b1splenic macrophages and total splenocytes from
mice fed the corn oil diet (open bars) or the fish oil diet (closed bars).
Macrophages expressing CD11b (1 3 109cells/L) and total splenocytes (5 3 109
cells/L) were stimulated with LPS (2 mg/L) for 24 h. Values are means 6 SEM,
n ¼ 10. * Different from corn oil, P , 0.05.
The effects of dietary fish oil on LPS-induced TNF-a (A) and IL-10 (B)
668Petursdottir and Hardardottir
by guest on June 6, 2013
increases TNF-a secretion per cell (I. Skuladottir, D. Petursdottir,
I. Hardardottir, unpublished results). Thus, the mechanism by
which dietary fish oil increases TNF-a secretion by resident
peritoneal macrophages may be different from the mechanism
by which dietary fish oil increases cytokine secretion by splenic
macrophages. In fact, the increased TNF-a secretion by resident
peritoneal macrophages from mice fed the fish oil diet has been
partly explained by decreased prostaglandin (PG)-E2production
(22,25,27), but decreased PGE2production does not explain the
increase in TNF-a secretion by splenic macrophages in this study,
because blocking prostaglandin production with indomethacine
had no effect on LPS-induced TNF-a and IL-10 secretion (results
not shown). Further underlining that dietary fish oil has different
effects on cytokine secretion by macrophages from different body
compartments, our previous results showed that dietary fish oil
decreased IL-10 secretion by resident peritoneal macrophages
(25), which is in contrast to the increase in IL-10 secretion by
splenic macrophages from mice fed the fish oil diet in this study.
Most of the cells producing TNF-a, following stimulation
with LPS, were macrophages expressing CD11bhigh, MOMA-2,
and CD68 (Fig. 2). Spleens from mice fed the fish oil diet had a
higher proportion of cells expressing CD11bhighand MOMA-2
than spleens from mice fed the corn oil diet (Table 1), with these
markers being predominantly expressed on the same cells (results
not shown). Thus, the increase in the number of cells secreting
TNF-a and IL-10, demonstrated by ELISpot, could be explained
by the increase in the proportion of these cells in spleens from
mice fed the fish oil diet. However, when CD11b1cells were
isolated (;90% were CD11bhigh) and the same number of
CD11b1cells from mice fed the fish oil diet and the corn oil diet
plated and stimulated with LPS, isolated CD11b1splenocytes
from mice fed the fish oil diet secreted significantly more TNF-a
and IL-10 than isolated CD11b1splenocytes from mice fed the
corn oil diet (Fig. 3), indicating that that the increase in the
proportion of CD11bhighcells in spleens from mice fed the fish
oil diet is not sufficient to explain the increase in TNF-a and
IL-10 secretion. Because there was not an increase in cytokine
secretion per cell, there must be a higher proportion of cells
producing TNF-a and IL-10 within the CD11bhighpopulation in
spleens from mice fed the fish oil diet than in spleens from mice
fed the corn oil diet. That this could be the case was demon-
strated by showing that a higher proportion of isolated CD11b1
cells from mice fed the fish oil diet expressed the LPS receptor
complex molecules, CD14 and TLR4/MD-2, compared with
that in CD11b1cells from mice fed the corn oil diet (Fig. 4).
Furthermore, the increase in the proportion of cells expressing
CD14 and TLR4/MD-2 among isolated CD11b1cells and total
splenocytes from mice fed the fish oil diet correlated with the
increase in cytokine secretion by isolated CD11b1and total
splenocytes, respectively (Figs. 4 and 3). Thus, we conclude that
the increase in the proportion of cells expressing CD14 and
TLR4/MD-2 from mice fed the fish oil diet was probably respon-
sible for the increase in TNF-a and IL-10 secretion by splenocytes
from mice fed the fish oil diet.
The increased proportion of CD11bhighmacrophages in
spleens from mice fed fish oil in this study may be caused by
infiltration of monocytes from blood, because there was an
increase in the proportion of circulating monocytes expressing
CD11bhighin mice fed the fish oil diet compared with that in
mice fed the corn oil diet (results not shown). An increase in the
proportion of macrophages in the spleen and an increase in the
proportion of circulating monocytes in mice fed fish oil has been
shown previously (50). In that study, there was an increase in
anti-F4/80 staining in the red pulp area in spleens from mice fed
fish oil, whereas the results from this study show an increase
in cells expressing other macrophage markers, with a modest
decrease in the proportion of red pulp macrophages. The reason
for the discrepancy in the results from the 2 studies may be due
to the different methods used.
The decreased proportion of F4/80 red pulp macrophages
and NK-cells expressing CD49bhighin spleens from mice fed the
fish oil diet probably reflects no change in absolute number of
these cells, as there is an increase in total cell count in spleens
from mice fed the fish oil diet. Whether this proportional
decrease in F4/80 macrophages and NK-cells is likely to affect
immune function is difficult to predict.
In summary, results from this study demonstrated that dietary
fish oil had different effects on cytokine secretion by splenocytes
stimulated with ConA and LPS, probably reflecting different
effects of dietary fish oil on cytokine secretion by T cells and
macrophages. Furthermore, the results demonstrated that die-
tary fish oil increased the number of cells secreting TNF-a and
IL-10 after LPS stimulation and this increase is probably due to
an increase in the number of splenocytes from mice fed the fish
oil diet expressing the LPS receptor complex proteins, CD14 and
TLR4/MD-2. Increased proportion of macrophages expressing
the LPS receptor molecules may be important for primary de-
fenses and immunosurveillance.
1.Calder PC. Immunoregulatory and anti-inflammatory effects of n-3
polyunsaturated fatty acids. Braz J Med Biol Res. 1998;31:467–90.
Belluzzi A, Brignola C, Campieri M, Pera A, Boschi S, Miglioli M.
Effect of an enteric-coated fish-oil preparation on relapses in Crohn’s
disease. N Engl J Med. 1996;334:1557–60.
Donadio JV Jr, Larson TS, Bergstralh EJ, Grande JP. A randomized trial
of high-dose compared with low-dose omega-3 fatty acids in severe IgA
nephropathy. J Am Soc Nephrol. 2001;12:791–9.
Kremer JM, Bigauoette J, Michalek AV, Timchalk MA, Lininger L,
Rynes RI, Huyck C, Zieminski J, Bartholomew LE. Effects of manip-
ulation of dietary fatty acids on clinical manifestations of rheumatoid
arthritis. Lancet. 1985;1:184–7.
Kremer JM, Lawrence DA, Petrillo GF, Litts LL, Mullaly PM, Rynes RI,
Stocker RP, Parhami N, Greenstein NS, et al. Effects of high-dose fish
oil on rheumatoid arthritis after stopping nonsteroidal antiinflamma-
tory drugs. Clinical and immune correlates. Arthritis Rheum. 1995;38:
Endres S, Ghorbani R, Kelley VE, Georgilis K, Lonnemann G, van der
Meer JW, Cannon JG, Rogers TS, Klempner MS, et al. The effect of
dietary supplementation with n-3 polyunsaturated fatty acids on the
synthesis of interleukin-1 and tumor necrosis factor by mononuclear
cells. N Engl J Med. 1989;320:265–71.
Meydani SN, Endres S, Woods MM, Goldin BR, Soo C, Morrill-
Labrode A, Dinarello CA, Gorbach SL. Oral (n-3) fatty acid supple-
mentation suppresses cytokine production and lymphocyte proliferation:
comparison between young and older women. J Nutr. 1991;121:
Trebble T, Arden NK, Stroud MA, Wootton SA, Burdge GC, Miles EA,
Ballinger AB, Thompson RL, Calder PC. Inhibition of tumour necrosis
factor-alpha and interleukin 6 production by mononuclear cells fol-
lowing dietary fish-oil supplementation in healthy men and response to
antioxidant co-supplementation. Br J Nutr. 2003;90:405–12.
Fan YY, Ly LH, Barhoumi R, McMurray DN, Chapkin RS. Dietary
docosahexaenoic acid suppresses T cell protein kinase C theta lipid raft
recruitment and IL-2 production. J Immunol. 2004;173:6151–60.
10. Jolly CA, Jiang YH, Chapkin RS, McMurray DN. Dietary (n-3) polyun-
saturated fatty acids suppress murine lymphoproliferation, interleukin-2
secretion, and the formation of diacylglycerol and ceramide. J Nutr.
11. Pompos LJ, Fritsche KL. Antigen-driven murine CD41 T lymphocyte
proliferation and interleukin-2 production are diminished by dietary
(n-3) polyunsaturated fatty acids. J Nutr. 2002;132:3293–300.
Fish oil affects TNF-a and IL-10 secretion669
by guest on June 6, 2013
12. Endres S, Meydani SN, Ghorbani R, Schindler R, Dinarello CA. Dietary
supplementation with n-3 fatty acids suppresses interleukin-2 produc-
tion and mononuclear cell proliferation. J Leukoc Biol. 1993;54:599–
13. Fowler KH, Chapkin RS, McMurray DN. Effects of purified dietary n-3
ethyl esters on murine T lymphocyte function. J Immunol. 1993;151:
14. Oarada M, Furukawa H, Majima T, Miyazawa T. Fish oil diet affects on
oxidative senescence of red blood cells linked to degeneration of spleen
cells in mice. Biochim Biophys Acta. 2000;1487:1–14.
15. Ly LH, Smith R, Switzer KC, Chapkin RS, McMurray DN. Dietary
eicosapentaenoic acid modulates CTLA-4 expression in murine CD41
T-cells. Prostaglandins Leukot Essent Fatty Acids. 2006;74:29–37.
16. Triboulot C, Hichami A, Denys A, Khan NA. Dietary (n-3) polyunsat-
urated fatty acids exert antihypertensive effects by modulating calcium
signaling in T cells of rats. J Nutr. 2001;131:2364–9.
17. Li Q, Wang M, Tan L, Wang C, Ma J, Li N, Li Y, Xu G, Li J.
Docosahexaenoic acid changes lipid composition and interleukin-2
receptor signaling in membrane rafts. J Lipid Res. 2005;46:1904–13.
18. Stulnig TM, Berger M, Sigmund T, Raederstorff D, Stockinger H,
Waldhausl W. Polyunsaturated fatty acids inhibit T cell signal trans-
duction by modification of detergent-insoluble membrane domains. J Cell
19. Sanderson P, Calder PC. Dietary fish oil appears to prevent the
activation of phospholipase C-gamma in lymphocytes. Biochim Biophys
20. Blok WL, Vogels MT, Curfs JH, Eling WM, Buurman WA, van der
Meer JW. Dietary fish-oil supplementation in experimental gram-
negative infection and in cerebral malaria in mice. J Infect Dis. 1992;
21. Chang HR, Arsenijevic D, Pechere JC, Piguet PF, Mensi N, Girardier L,
Dulloo AG. Dietary supplementation with fish oil enhances in vivo
synthesis of tumor necrosis factor. Immunol Lett. 1992;34:13–7.
22. Hardardottir I, Kinsella JE. Tumor necrosis factor production by murine
resident peritoneal macrophages is enhanced by dietary n-3 polyunsat-
urated fatty acids. Biochim Biophys Acta. 1991;1095:187–95.
23. Hardardottir I, Kinsella JE. Increasing the dietary (n-3) to (n-6)
polyunsaturated fatty acid ratio increases tumor necrosis factor
production by murine resident peritoneal macrophages without an
effect on elicited peritoneal macrophages. J Nutr. 1992;122:1942–51.
24. Lokesh BR, Sayers TJ, Kinsella JE. IL-1 and TNF synthesis by mouse
peritoneal macrophages is enhanced by dietary n-3 polyunsaturated
fatty acids. Immunol Lett. 1990;23:281–6.
25. Petursdottir DH, Olafsdottir I, Hardardottir I. Dietary fish oil increases
tumor necrosis factor secretion but decreases interleukin-10 secretion by
murine peritoneal macrophages. J Nutr. 2002;132:3740–3.
26. Watanabe S, Hayashi H, Onozaki K, Okuyama H. Effect of dietary
alpha-linolenate/linoleate balance on lipopolysaccharide-induced tumor
necrosis factor production in mouse macrophages. Life Sci. 1991;48:
27. Ertel W, Morrison MH, Ayala A, Chaudry IH. Modulation of
macrophage membrane phospholipids by n-3 polyunsaturated fatty
acids increases interleukin 1 release and prevents suppression of cellular
immunity following hemorrhagic shock. Arch Surg. 1993;128:15–20.
28. Albers R, Bol M, Bleumink R, Willems A, Blonk C, Pieters R. Effects of
dietary lipids on immune function in a murine sensitisation model. Br J
29. Barber MD, Fearon KC, Ross JA. Eicosapentaenoic acid modulates the
immune response but has no effect on a mimic of antigen-specific
responses. Nutrition. 2005;21:588–93.
30. Gratchev A, Kzhyshkowska J, Kothe K, Muller-Molinet I, Kannookadan
S, Utikal J, Goerdt S. Mphi1 and Mphi2 can be re-polarized by Th2 or
Th1 cytokines, respectively, and respond to exogenous danger signals.
31. Meng F, Lowell CA. Lipopolysaccharide (LPS)-induced macrophage
activation and signal transduction in the absence of Src-family kinases
Hck, Fgr, and Lyn. J Exp Med. 1997;185:1661–70.
32. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for
laboratory rodents: final report of the American Institute of Nutrition
ad hoc writing committee on the reformulation of the AIN-76A rodent
diet. J Nutr. 1993;123:1939–51.
33. Fritsche KL, Johnston PV. Rapid autoxidation of fish oil in diets without
added antioxidants. J Nutr. 1988;118:425–6.
34. Lang T, Ave P, Huerre M, Milon G, Antoine JC. Macrophage subsets
harbouring Leishmania donovani in spleens of infected BALB/c mice:
localization and characterization. Cell Microbiol. 2000;2:415–30.
35. Wimer BM, Mann PL. Mitogen information summaries. Cancer Biother
36. Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a
receptor for complexes of lipopolysaccharide (LPS) and LPS binding
protein. Science. 1990;249:1431–3.
37. Griffin JD, Ritz J, Nadler LM, Schlossman SF. Expression of myeloid
differentiation antigens on normal and malignant myeloid cells. J Clin
38. Todd RF III, Nadler LM, Schlossman SF. Antigens on human monocytes
identified by monoclonal antibodies. J Immunol. 1981;126:1435–42.
39. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF. A family of
human receptors structurally related to Drosophila Toll. Proc Natl Acad
Sci USA. 1998;95:588–93.
40. Ogata H, Su I, Miyake K, Nagai Y, Akashi S, Mecklenbrauker I,
Rajewsky K, Kimoto M, Tarakhovsky A. The toll-like receptor protein
RP105 regulates lipopolysaccharide signaling in B cells. J Exp Med.
41. Kearney JF, Lawton AR. B lymphocyte differentiation induced by
lipopolysaccharide. I. Generation of cells synthesizing four major
immunoglobulin classes. J Immunol. 1975;115:671–6.
42. Oliver AM, Martin F, Kearney JF. IgMhighCD21high lymphocytes
enriched in the splenic marginal zone generate effector cells more rap-
idly than the bulk of follicular B cells. J Immunol. 1999;162:7198–207.
43. Gieni RS, Umetsu DT, DeKruyff RH. Ly1- (CD5-) B cells produce
interleukin (IL)-10. Cell Immunol. 1997;175:164–70.
44. O’Garra A, Stapleton G, Dhar V, Pearce M, Schumacher J, Rugo H,
Barbis D, Stall A, Cupp J, et al. Production of cytokines by mouse B
cells: B lymphomas and normal B cells produce interleukin 10. Int
45. Sun D, Krishnan A, Zaman K, Lawrence R, Bhattacharya A, Fernandes
G. Dietary n-3 fatty acids decrease osteoclastogenesis and loss of bone
mass in ovariectomized mice. J Bone Miner Res. 2003;18:1206–16.
46. Ly LH, Smith R III, Chapkin RS, McMurray DN. Dietary n-3
polyunsaturated fatty acids suppress splenic CD4(1) T cell function
in interleukin (IL)-10(2/2) mice. Clin Exp Immunol. 2005;139:202–9.
47. Yaqoob P, Calder PC. The effects of dietary lipid manipulation on
the production of murine T cell-derived cytokines. Cytokine. 1995;7:
48. Arrington JL, Chapkin RS, Switzer KC, Morris JS, McMurray DN.
Dietary n-3 polyunsaturated fatty acids modulate purified murine T-cell
subset activation. Clin Exp Immunol. 2001;125:499–507.
49. Stulnig TM. Immunomodulation by polyunsaturated fatty acids: mech-
anisms and effects. Int Arch Allergy Immunol. 2003;132:310–21.
50. Blok WL, de Bruijn MF, Leenen PJ, Eling WM, van Rooijen N, Stanley
ER, Buurman WA, van der Meer JW. Dietary n-3 fatty acids increase
spleen size and postendotoxin circulating TNF in mice; role of mac-
rophages, macrophage precursors, and colony-stimulating factor-1.
J Immunol. 1996;157:5569–73.
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