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Novel action of n-3 polyunsaturated fatty acidsInhibition of arachidonic acidinduced increase in tumor necrosis factor receptor expression on neutrophils and a role for proteases.

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Vol. 56, No. 3, March 2007, pp 799–808
DOI 10.1002/art.22432
© 2007, American College of Rheumatology
Novel Action of n-3 Polyunsaturated Fatty Acids
Inhibition of Arachidonic Acid–Induced Increase in Tumor Necrosis Factor
Receptor Expression on Neutrophils and a Role for Proteases
Nahid Moghaddami,1 James Irvine,1 Xiuhui Gao,2 Phulwinder K. Grover,2 Maurizio Costabile,3
Charles S. Hii,1 and Antonio Ferrante4
docosahexaenoic acid led to a marked inhibition of the
AA-induced up-regulation of TNFRs I and II. Such
pretreatment, however, did not prevent AA from stimulating the activities of PKC and ERK-1/2, which is
required for the actions of AA or its ability to mobilize
Ca2ⴙ. Nevertheless, treatment with n-3 PUFAs caused
the stimulation of serine proteases that could cleave the
Conclusion. These findings suggest a mechanism
by which the n-3 PUFAs inhibit the inflammatory
response in RA, by regulating the ability of AA to
increase TNFR expression. These results help fill the
gaps in our knowledge regarding the mechanisms of
action of n-3 PUFAs, thus allowing us to make specific
recommendations for the use of n-3 PUFAs in the
regulation of inflammatory diseases.
Objective. Neutrophils and tumor necrosis factor
(TNF) play important roles in the pathogenesis of
rheumatoid arthritis (RA). Modulation of TNF receptors (TNFRs) may contribute to the regulation of tissue
damage, and n-6 polyunsaturated fatty acids (PUFAs)
such as arachidonic acid (AA) can increase the expression of TNFRI and TNFRII on neutrophils. Because the
n-3 PUFAs are antiinflammatory in RA, we examined
whether, as a novel mechanism of action, n-3 PUFAs can
antagonize the AA-induced increase in TNFR expression.
Methods. Human neutrophils were treated with
PUFAs and examined for changes in surface expression
of TNFRs by flow cytometry. Translocation of protein
kinase C (PKC) and activation of ERK-1/2 MAPK were
determined by Western blotting. Intracellular calcium
mobilization was measured in Fura 2–loaded cells by
luminescence spectrometry.
Results. Pretreatment of neutrophils with nanomolar levels of n-3 PUFAs, eicosapentaenoic acid, or
Extensive neutrophil accumulation in the synovial fluid and synovial tissue of patients with rheumatoid
arthritis (RA) has been observed during both the early
and the exacerbation phases of this inflammatory disease. When activated, these cells release tissuedamaging substances, including oxygen-derived reactive
species, hypochlorous acid, and elastase, all of which
have been implicated in neutrophil-mediated damage of
cartilage tissue (1). Furthermore, conclusive evidence of
a role for neutrophils comes from studies involving
experimental animal models of arthritis, the results of
which have shown that neutrophil depletion protects
against this inflammatory disease (2,3). A role for tumor
necrosis factor (TNF) in the pathogenesis of RA has
been demonstrated on the basis of the protective effects
of anti-TNF antibody therapy in humans (4). Among the
many-recognized mechanisms by which TNF promotes
pathogenesis is its ability to prime neutrophils for in-
Supported by the National Health and Medical Research
Council of Australia.
Nahid Moghaddami, PhD, James Irvine, BSc, Charles S. Hii,
PhD: Children, Youth and Women’s Health Services, and University
of Adelaide, Adelaide, South Australia, Australia; 2Xiuhui Gao, PhD,
Phulwinder K. Grover, PhD: Children, Youth and Women’s Health
Services, Adelaide, South Australia, Australia; 3Maurizio Costabile,
PhD: Children, Youth and Women’s Health Services, and University
of South Australia, Adelaide, South Australia, Australia; 4Antonio
Ferrante, FRCPath, PhD: Children, Youth and Women’s Health
Services, University of Adelaide, and University of South Australia,
Adelaide, South Australia, Australia.
Address correspondence and reprint requests to Antonio
Ferrante, FRCPath, PhD, Department of Immunopathology, Women’s and Children’s Hospital, 72 King William Road, North Adelaide,
South Australia 5006, Australia. E-mail: antonio.ferrante@adelaide.
Submitted for publication July 25, 2005; accepted in revised
form November 28, 2006.
creased cartilage damage (5,6). Thus, modulation of the
expression of the TNF receptors (TNFRs) on neutrophils may alter the tissue-damaging potential of these
cells. It has been demonstrated that the levels of
TNFR in the tissue are modulated under a variety of
conditions (7,8).
Recently, we demonstrated that n-6 polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA)
up-regulate the expression of TNFRs on neutrophils and
increase the response of the cells to TNF (9). The
beneficial and protective effects of the n-3 PUFAs on
inflammatory diseases are attributable to their ability to
produce metabolites, which are less biologically active
than AA metabolites (10). Supplementation with n-3–
enriched foods also reduces the production of proinflammatory cytokines such as interleukin-1␤ (IL-1␤),
IL-6, and TNF (11,12). Particularly interesting is our
finding that the n-3 PUFAs, eicosapentaenoic acid
(EPA), docosahexaenoic acid (DHA), and linolenic acid
failed to increase TNFR expression on neutrophils (9).
It remains of interest therefore to determine if cells
preexposed to n-3 PUFAs display altered responses to
AA, in relation to the previously observed up-regulation
induced by AA; confirmation of this would be consistent
with the antiinflammatory properties of n-3 PUFAs. The
prevailing view is that there are several gaps in our
knowledge of the mechanisms of action and properties
of n-3 PUFAs that limit our ability to make specific
recommendations on their use in inflammatory conditions (13). Identification of their effects on TNFR
expression on inflammatory cells would be an important
step to help fill such gaps, particularly since TNF is a key
cytokine in the pathogenesis of RA.
Thus, in the present study we found that low
amounts of DHA or EPA can indeed prevent the
AA-induced up-regulation of TNFR expression on neutrophils. The possibility that the n-3 PUFAs could cause
TNFR shedding was supported by our findings that the
inability of DHA to increase the surface expression of
TNFR could be totally reversed by inhibitors of serine
proteases. These data are evidence of a novel property
of n-3 PUFAs that may contribute to their antiinflammatory characteristics.
Fatty acids. PUFAs were purchased from SigmaAldrich (St. Louis, MO). Stocks of the fatty acids were made
up in chloroform and stored at ⫺70oC. On the day of use, the
chloroform was evaporated under nitrogen, and the fatty acids
were prepared in ethanol. The purity and quality of these fatty
acids were ensured by analysis via thin layer chromatography
and gas chromatography mass spectrometry.
Protease inhibitors. Inhibition studies were carried
out with a protease inhibitor cocktail containing 4-(2aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A,
E-64, bestatin, leupeptin, and aprotinin, as well as individual
protease inhibitors. All protease inhibitors were obtained from
Neutrophils. Neutrophils were isolated from the blood
of healthy volunteers by the rapid single-step method, involving centrifugation of blood on Hypaque-Ficoll (14). The
neutrophils, harvested from the lower leukocyte band, were
96–99% pure and ⬎99% viable, as judged by their ability to
exclude trypan blue.
Surface expression of TNFR. Surface expression of
TNFRI and TNFRII was measured by flow cytometry, essentially as described previously (9). Neutrophils (106 cells/ml)
were treated for various times with the PUFA or an equivalent
amount of vehicle (ethanol) as control. The cells were washed
in ISOTON II (Beckman-Coulter, Gladesville, Australia) supplemented with 0.1% (weight/volume) bovine serum albumin
and then incubated for 30 minutes on ice with anti-human
TNFRI and TNFRII monoclonal antibodies (mAb) (or ␥1/␥2
isotype–matched mAb) (R&D Systems, Minneapolis, MN).
The cells were then incubated with fluorescein isothiocyanate–
conjugated goat anti-mouse IgG antibodies (AMRAD Operations, Melbourne, Australia) for 30 minutes, fixed with paraformaldehyde (1% volume/volume), and analyzed on a BD
Biosciences fluorescence-activated cell sorter (FACS) (FACScan; Becton Dickinson, Mountain View, CA). Results were
expressed as the mean fluorescence intensity, corrected for the
values of the isotype-matched negative controls.
Measurement of protein kinase C (PKC) and ERK-1/2
activation. The translocation of PKC was determined as described previously (15). Briefly, neutrophils were pretreated
with various concentrations of DHA (for 30 minutes) or an
equivalent amount of ethanol, and then stimulated with AA
(30 ␮M for 5 minutes). After harvesting, the cells were
sonicated and centrifuged (at 100,000g for 30 minutes), and
particulate fractions were extracted with 2% Triton X-100.
Proteins were separated by 12% sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and transferred to nitrocellulose (Schleicher & Schuell, Dassel, Germany). PKC isozymes
were detected using anti-PKC isozyme-specific antibodies
(Santa Cruz Biotechnology, Santa Cruz, CA) and detected by
enhanced chemiluminescence (15).
Dual phosphorylation of ERK-1/2 was assayed as
described previously (16). Briefly, neutrophils were pretreated
with DHA, and then stimulated with AA and lysed. The
samples were subjected to Western blot analysis using an
anti–active ERK antibody (New England Biolabs, Beverly,
MA). The blots were stripped and reprobed with anti–ERK-2
antibody (Santa Cruz Biotechnology).
Measurement of intracellular Ca2ⴙ. Neutrophils (1 ⫻
10 cells/ml) in Hank’s balanced salt solution (HBSS) were
loaded in the dark with Fura 2 AM (1 ␮M) in a shaking water
bath (for 30 minutes at 37°C). The cells were washed twice and
resuspended in cold HBSS (6 ⫻ 106 cells/ml). Calcium mobilization was determined using a PerkinElmer LS50B luminescence spectrometer and Fluorescence Data Manager software
as described previously (17). Briefly, the neutrophils were
placed in the reaction cuvette and incubated for 5 minutes in
the dark at 37°C. Baseline fluorescence (excitation at 340 nm,
emission at 510 nm, slit width 5.0 nm for both) was measured.
Ethanol or DHA was then added and fluorescence was monitored for 30 minutes, after which AA (30 ␮M) was added.
After 5 minutes, Triton X-100, at a final concentration of
0.15% (v/v), was added and maximal fluorescence was determined over 3 minutes. The minimum fluorescence over 3
minutes was then determined, following the addition of Tris
base (pH 10.0, 40 mM final concentration) and EGTA (12.5
mM final concentration).
Statistical analysis. Statistical comparisons were carried out using Student’s 2-tailed t-test for paired or unpaired
data, with analysis using Graphpad software (Cricket Software,
Philadelphia, PA). Where appropriate, multiple comparisons
with a single control were performed using analysis of variance
with Dunnett’s modification.
Effects of DHA and EPA on the AA-induced
up-regulation of TNFR expression. We previously demonstrated that DHA and EPA reduce, whereas AA
increases, TNFR expression on neutrophils (9). In those
studies (9), we demonstrated that AA dose-dependently
(5–30 ␮M) increased expression of TNFRs I and II on
neutrophils. Kinetics studies demonstrated that this
effect reached a maximum at 30–40 minutes of AA
treatment. We postulated that although both n-6 and
n-3 PUFAs could stimulate neutrophil responses, such
as respiratory burst, degranulation, and the adherence
and surface expression of ␤2 integrins (18–22), the n-3
PUFA is most likely responsible for promoting the
cleavage of the TNFRs. We thus postulated that EPA or
DHA would prevent AA from up-regulating TNFR
Neutrophils were treated with EPA or DHA (20
␮M each) for 30 minutes and then challenged with AA
(30 ␮M). After 30 minutes of pretreatment, the expression of TNFRs I and II was examined by flow cytometry.
As expected, AA caused an increase in TNFR expression (Figure 1). Pretreatment of the cells with either
EPA or DHA blocked the AA-induced increase in
TNFR expression (Figure 1), and this was observed in
the majority of the cells. The n-3 fatty acids alone caused
a small reduction in fluorescence intensity compared
with ethanol-treated control cells (Figure 1), as observed
previously (9). Under our experimental conditions, the
fatty acids did not affect the viability of the neutrophils,
as judged by their ability to exclude trypan blue (results
not shown).
Further studies were conducted to identify the
concentration of EPA and DHA required for the inhi-
Figure 1. Suppression of the arachidonic acid (AA)–induced increase
in tumor necrosis factor receptor I (TNFRI) and TNFRII expression
on neutrophils by eicosapentaenoic acid (EPA) and docosahexaenoic
acid (DHA). Neutrophils were pretreated with EPA (A) or DHA (B)
(20 ␮M each) for 30 minutes, and then stimulated with AA (30 ␮M).
After 30 minutes, the levels of TNFR on the cell surface were
measured by flow cytometry. Results, expressed as a percent of control
(C) (diluent) TNFR expression, are the mean and SEM of 3 experiments. ⴱ ⫽ P ⬍ 0.001 versus control neutrophils, by Student’s t-test.
bition of an AA-induced increase in TNFR expression.
Neutrophils were pretreated with various concentrations of EPA (500, 2,500, or 5,000 nM), DHA (0.3, 3, 50,
500, or 2,500 nM), or an equivalent amount of diluent
for 30 minutes, followed by treatment with AA or
diluent for 30 minutes. The cells were then examined for
TNFR expression. The results revealed that both EPA
and DHA caused a significant inhibition of the AAinduced up-regulation of TNFR (Figure 2). It was
evident that the maximum inhibitory effects of EPA
were achieved at 500 nM (Figure 2). The inhibitory
effects of DHA on TNFRI expression were evident at
concentrations of ⱖ3 nM, whereas the expression of
TNFRII was suppressed by DHA at concentrations of
ⱖ50 nM (Figure 2).
We also investigated the consequence of DHA
pretreatment on the ability of TNF to stimulate superoxide production. Neutrophils were pretreated with either DHA (0.2 ␮M for 30 minutes) or vehicle, and
then incubated with AA (10 ␮M for 30 minutes). TNF
(100 units/ml) was added and superoxide production was
determined by a lucigenin-based assay (9). Pretreatment
with DHA reduced the amount of superoxide produced
to 61 ⫾ 14% (mean ⫾ SEM; n ⫽ 5) of the response seen
in vehicle-pretreated cells (P ⬍ 0.05), reflecting the
Figure 2. Effects of varying EPA and DHA concentrations on the
AA-induced up-regulation of TNFRI and TNFRII expression in
neutrophils. Neutrophils were pretreated with varying concentrations
of EPA, DHA, or diluent (ethanol) at 37°C for 30 minutes. The cells
were then incubated with AA (30 ␮M) or ethanol for 30 minutes at
37°C. The expression of the TNFRs was examined by flow cytometry.
Results showing the effect of EPA or DHA on the AA-induced change
in receptor expression are the mean ⫾ SEM of 3 experiments. ⴱ ⫽ P
⬍ 0.01 versus ethanol pretreatment, by analysis of variance followed by
Dunnett’s modification. See Figure 1 for definitions.
suppression of AA-induced TNFR expression by DHA,
such that the response to a TNF challenge is decreased.
Effect of DHA on the AA-induced activation of
PKC and ERK-1/2 and the mobilization of Ca2ⴙ. AA
stimulates the activities of several intracellular signaling
molecules, including PKC and ERK-1/2 (22), which we
have previously demonstrated to be required for AA to
cause the up-regulation of TNFR expression (9). We
therefore examined whether DHA inhibited AAinduced activation of these signaling molecules.
To investigate the effects of DHA on AAstimulated PKC activation, neutrophils were pretreated
with 0.2 ␮M of DHA for 30 minutes, and then stimulated
with AA. After 5 minutes, the cells were sonicated and
particulate fractions were prepared and subjected to
Western blot analysis using anti-PKC␣, anti-PKC␤1,
anti-PKC␤2, and/or anti-PKC␦ antibodies. An association of classical and novel PKC isozymes with a particulate (membrane) compartment is essential to the activation and function of these isozymes (23), and this step
is subjected to regulation by PUFAs (15,16). As observed in previous studies (15,16), AA caused an increase in particulate fraction–associated PKC (Figures
3a and b). Surprisingly, pretreatment with DHA (0.2
Figure 3. Stimulation of the activation of protein kinase C (PKC) and
ERK-1/2 and the mobilization of intracellular Ca2⫹ by AA, without
prevention by DHA. Neutrophils were pretreated with DHA at 0.2 ␮M
(a) or 5 or 20 ␮M (b) for 30 minutes, and then incubated with AA (30
␮M) for 5 minutes. The cells were then processed for the determination of PKC isozyme translocation and ERK-1/2 activation (a and b) by
Western blotting using anti-PKC isozyme-specific antisera and an
anti–active ERK antibody, respectively. The amount of ERK-2 that
was present in the lanes was determined by stripping and reprobing the
blots with an anti–ERK-2 antibody. The blots show a typical experimental run, representative of 3 experiments. DHA also did not prevent
AA from mobilizing intracellular Ca2⫹ (c). In these studies, the cells
were preloaded with Fura 2 and the levels of intracellular Ca2⫹ were
estimated as described in Materials and Methods. Either ethanol or
DHA (0.2 ␮M) was added at 15 minutes, followed by AA (30 ␮M) at
45 minutes. Inset shows the mean and SEM percent of AA-induced
increase (n ⫽ 4 experiments) in intracellular Ca2⫹ levels in the
neutrophils that had been preexposed to either ethanol or DHA (P ⬎
0.05 versus ethanol controls). See Figure 1 for other definitions.
␮M) did not prevent AA from activating PKC␣, PKC␤I,
and PKC␤II (Figure 3a).
We next examined the effects of DHA at 5 ␮M
and 20 ␮M. The results showed that at these higher
concentrations, DHA alone caused a variable increase in
the amount of particulate fraction–associated PKC, including PKC␦, that was still detectable at 35 minutes
after the addition of the PUFA (Figure 3b). The ability
of AA to promote PKC␣ and PKC␦ translocation was
not affected by the higher concentrations of DHA.
Interestingly, pretreatment with 5 ␮M DHA enhanced
the effect of AA on PKC␤ translocation, whereas 20 ␮M
DHA tended to reduce the AA-mediated association of
PKC␤I with the particulate fraction (Figure 3b).
To assess the level of ERK-1/2 activation, the
cells were treated in the same manner as described
above and lysed. The lysates were then subjected to
Western blot analysis using an anti–active ERK antibody. Consistent with previous findings (15,16), AA was
able to stimulate ERK-1/2 activity, but this was not
prevented by pretreatment with DHA (0.2 ␮M) (Figure
3a). Higher doses of DHA (5 ␮M and 20 ␮M) also did
not inhibit the ability of AA to stimulate the activation
of ERK-1/2 and, in fact, enhanced the activation of
ERK-1/2 by AA (Figure 3b). Taken together, these
results suggest that the inhibitory actions of the n-3 fatty
acids, especially at lower concentrations, were unlikely
to have been exerted at the level of PKC or ERK-1/2.
Since Ca2⫹ plays an important role in neutrophil
activation, including degranulation (18), we also investigated whether pretreatment with DHA would prevent
AA from mobilizing intracellular Ca2⫹ (17). The results
(shown in Figure 3c) demonstrated that DHA (0.2 ␮M)
alone had no effect on either the concentration of
intracellular Ca2⫹ or the ability of AA to mobilize
intracellular Ca2⫹. Higher doses of DHA (5 ␮M and 20
␮M), although causing a small degree of Ca2⫹ mobilization, also did not prevent AA from stimulating Ca2⫹
mobilization but enhanced the effect of AA (results not
Increased TNFR expression by DHA in the presence of protease inhibitors. The inhibition of surface
TNFR expression by DHA and EPA is intriguing,
because both n-3 fatty acids stimulate degranulation
from the specific and azurophilic granule compartments
(22,24) that should have increased the expression of
TNFR (25). Furthermore, higher doses of DHA also
caused the activation of PKC and Ca2⫹ mobilization,
and this should have resulted in increased receptor
expression. To understand how the actions of the n-3
fatty acids on TNFR expression were achieved, we
Figure 4. Stimulation of TNFR expression on neutrophils by DHA in
the presence of protease inhibitors (PIs). The ability of a cocktail of
PIs to alter the effects of DHA on TNFRI and TNFRII expression on
neutrophils was examined by flow cytometry. The results (a and b) are
the mean and SEM fluorescence intensity, expressed as a percent of
the control (vehicle-treated) values in 3 experiments. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01 versus control cells, by analysis of variance followed by
Dunnett’s modification. The responses to AA (30 ␮M) under these
conditions were 295 ⫾ 42% (n ⫽ 3) and 445 ⫾ 157% (n ⫽ 5) of
controls for TNFRI and TNFRII, respectively. Representative histograms (c and d) show the flow cytometry profiles of the effects of PIs
on the ability of DHA to increase TNFR expression on neutrophils.
See Figure 1 for other definitions.
tested the hypothesis that these fatty acids reduced the
surface expression of the receptors by causing receptor
cleavage. In neutrophils, shedding of TNFR caused by
TNF or n-formyl-methionyl-L-leucyl-L-phenylalanine
(fMLP) has been reported to be due to proteases such as a
metalloproteinase (TNFRI and TNFRII) and a serine
protease such as elastase (TNFRII) (25–27), although
the possible involvement of other proteases cannot be
excluded. We therefore used a protease inhibitor cocktail containing AEBSF (serine protease inhibitor), pepstatin A (aspartic protease inhibitor), E-64 (cysteine
protease inhibitor), bestatin (aminopeptidase and metalloprotease inhibitor), leupeptin (serine and cysteine protease inhibitor), and aprotinin (serine protease inhibitor)
To 1 ⫻ 106 neutrophils in 0.5 ml HBSS, we added
10 ␮l of protease inhibitor or an equivalent volume of
HBSS. The cells were incubated at 37°C for 10 minutes.
of both TNFRI and TNFRII (Figure 5). These results
imply that a serine protease whose activity was sensitive
to AEBSF was involved in mediating the effect of DHA
on the expression of TNFRs I and II on neutrophils.
Figure 5. Increased expression of TNFRI and TNFRII by 4-(2aminoethyl)benzenesulfonyl fluoride (AEBSF) and protease inhibitor
(PI) cocktail in the presence of DHA. Neutrophils were incubated with
DHA or vehicle in the presence or absence of AEBSF (2 ␮M) or PI for
30 minutes. After washing, the expression of the TNFRs was assessed
by flow cytometry. Results are the mean and SEM of 3 experiments,
presented as the percent of TNFR expression on control cells. Neither
AEBSF nor PI alone affected TNFR expression. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽
P ⬍ 0.01 versus DHA alone. See Figure 1 for other definitions.
DHA (20 ␮M) was then added and the cells were
incubated for a further 30 minutes. As observed previously, DHA alone reduced the basal expression of
TNFRs I and II (Figures 4a and b). Interestingly,
whereas the addition of the protease inhibitor cocktail
alone did not affect the basal expression of the TNFRs,
the addition of the protease inhibitor cocktail together
with DHA produced a marked increase in the surface
expression of both TNFRI and TNFRII (Figures 4a and
b). Results of FACS analysis (histograms in Figures 4c
and d) showed that addition of the protease inhibitor
cocktail and DHA caused a higher level of fluorescence
intensity compared with that in control cells. These
results imply that DHA activated a protease that was
inhibited by the protease inhibitor cocktail, while it
simultaneously recruited additional TNFRs to the cell
We also tested the effects of the above-described
inhibitors when added individually at concentrations
equivalent to those found in the cocktail (http://, to
investigate the class of protease that was involved in the
action of DHA. Whereas aprotinin, bestatin, E-64, leupeptin, and pepstatin A did not affect the surface
expression of TNFR, either in the presence or absence
of DHA (results not shown), coincubation of the neutrophils with AEBSF and DHA increased the expression
An intriguing finding of the present study is that
preexposure of neutrophils to EPA or DHA can suppress the AA-induced up-regulation of TNFRI and
TNFRII. This novel finding demonstrates a further
dissociation between the actions of the n-6 and n-3
PUFAs, despite the observations that PUFAs from both
series are very effective at stimulating the neutrophil
respiratory burst, degranulation, and adherence (22).
This action highlights an important property of n-3 fatty
acids that is likely to also constitute a novel mechanism
by which the perceived antiinflammatory actions of n-3
fatty acids are mediated.
The suppressive effects of the n-3 fatty acids were
observed at nanomolar levels, whereas their stimulatory
effects on neutrophil respiratory burst activity and degranulation occur at micromolar levels (20–22). Studies
in mice have demonstrated that the levels of nonesterified DHA and EPA can increase rapidly during the
inflammatory response (28). Thus, levels of DHA and
EPA have been reported to reach 141 nM and 172 nM,
respectively, within 2 hours after the initiation of an
inflammatory response in the peritoneal cavity. These
findings suggest that the suppressive effects of the n-3
PUFAs on TNFR expression are likely to be observed at
sites of inflammation.
Furthermore, neutrophils are likely to be exposed
to micromolar levels of nonesterified AA during cellular
activation. For example, it has been reported that although nonesterified AA is undetectable in unstimulated neutrophils, activated neutrophils have been found
to contain more than 800 pmoles of AA/107 cells (29),
estimated in excess of 110 ␮M. However, 85% of this AA
is released into the extracellular space (29). Our own
studies have demonstrated that whereas unstimulated
neutrophils contained 15 pmoles of AA/107 neutrophils
(⬃2 ␮M), this level increased to 125 pmoles and 1,800
pmoles/107 neutrophils (⬃17 ␮M and ⬃250 ␮M, respectively) when the cells were incubated with TNF and
A23187, respectively (30). Others have reported higher
levels of nonesterified AA in neutrophils (31). Following
dietary supplementation with fish oil at 1 gm/day for 3
months, our previous studies demonstrated that the ratio
of n-3 PUFA:n-6 PUFA in the red blood cells of subjects
reached 1:2.2 (Mukaro V, et al: unpublished observations). Thus, it is likely that the levels of n-3 PUFAs
tested in this study can be achieved, at least under
inflammatory conditions. However, it is not known
whether the suppressive effects of the n-3 PUFAs were
caused by the PUFAs per se or could be attributed to
their metabolites, including resolvins, docosatrienes,
and neutroprotectins that, via their counterregulatory
effects on neutrophil recruitment and inhibition of cytokine and chemokine production, have been proposed to
participate in the resolution of inflammation (32,33).
Neutrophils can generate some of these metabolites
Owing to its ability to stimulate degranulation
from both the specific and the azurophilic granules (22),
it is most likely that AA causes the mobilization of
TNFR from storage granules. However, to date, the only
evidence presented has been for the storage of TNFRI
in specific granules (25). TNFRII was found to be
associated with only the plasma membrane fraction (25).
Nevertheless, our previous findings suggest that TNFRII
is likely to be mobilized from intracellular locations,
because its expression on the plasma membrane was
increased by AA with kinetics similar to those of the
AA-induced increase in TNFRI expression (9).
Our findings with regard to the n-3 PUFAs are
interesting. Although EPA and DHA are potent stimulators of degranulation, our results suggest that these
PUFAs also have other unidentified concurrent actions
that cause a net reduction in the surface expression of
TNFR. This is also evident in the majority of neutrophil ligands. For example, A23187 and fMLP in the
presence of cytochalasin B are strong stimulators of
degranulation (22), but instead of increasing the surface
expression of TNFR, these agents cause a loss of surface
expression. This has been attributed to their ability to
also stimulate proteolytic cleavage of the receptors
(26,27). Thus, the net expression of a receptor class on
the cell surface is likely to reflect the balance between
mobilization from intracellular stores and loss caused by
Because of the existence of multiple cellular
targets for AA, EPA, and DHA, it is difficult to readily
decipher the biochemical mechanisms involved in TNFR
regulation by the PUFAs. Badwey et al (18) pointed out
that AA readily partitions into membranes, which results
in a change to the physical properties of the membranes
(19). However, Corey and Rosoff (34) excluded the
possibility of such a nonspecific action, and our recent
studies (35) and those of other investigators (36) have
demonstrated that cell activation by AA can occur via
members of the ErbB receptor family. In addition, AA
can stimulate the activities of signaling molecules such as
PKC, ERK-1/2, p38, phosphatidylinositol 3-kinase, and
the cytosolic phospholipase A2 (cPLA2) (15,16,35–37).
We have previously demonstrated that AA acted via
PKC, ERK-1/2, and cPLA2 to elicit the increase in
TNFR expression on neutrophils (9). Surprisingly, low
concentrations (0.2 ␮M) of DHA did not prevent the
activation of PKC and ERK-1/2 by AA nor did this low
concentration of DHA prevent the ability of AA to
mobilize intracellular Ca2⫹. At higher concentrations (5
␮M and 20 ␮M), DHA caused some activation of the
kinases and the mobilization of Ca2⫹, and amplified the
effect of AA on these parameters. These results suggest
that the inhibitory effects of DHA were unlikely to have
been caused by inhibition of PKC, ERK-1/2, and Ca2⫹
We were interested in identifying the mechanism
of action of this antagonistic effect of the n-3 PUFAs on
AA. Our results suggest that DHA caused the proteolytic cleavage of TNFRs I and II, since the addition of a
protease inhibitor cocktail totally altered the action of
DHA. Thus, whereas DHA per se reduced the surface
expression of TNFRs I and II, the fatty acid uncharacteristically caused a substantial increase in TNFR expression in the presence of broad-spectrum inhibitors
of serine, cysteine, and aspartic proteases and aminopeptidases. Although these findings support the involvement of one or more proteases, it is not known whether
the enzyme is membrane-bound or soluble. A previous
attempt, by Dri et al (26), to demonstrate the release of
proteases in the presence of fMLP failed to demonstrate whether such release occurred, since their results
could be interpreted in a number of ways. Nevertheless,
those authors did show that a membrane-bound, non–
matrix metalloproteinase, possibly ADAM-17, may be
involved in cleaving TNFRI in response to either TNF or
fMLP (26). In contrast, shedding of TNFRII has been
reported to require a metalloproteinase and the serine
protease elastase (26,27,38).
Surprisingly, our results show that DHA was able
to increase the surface expression of TNFRI independent of metalloproteinases, since bestatin, which can
also inhibit metalloproteinases, had no effect on TNFR
expression. Only AEBSF, an irreversible serine protease
inhibitor (
pdf), increased the level of TNFR expression in the
presence of DHA. Although aprotinin and leupeptin
also inhibit serine proteases, these did not affect TNFR
expression. The reason for this is not clear but may be
related to the fact that these are reversible inhibitors of
serine proteases. Consistent with this idea, disiopropylfluorophosphate, used in previous studies to demonstrate the role of elastase (27), is also an irreversible
inhibitor of serine proteases. These results imply that
DHA utilizes a combination of proteases that differed
from that used by TNF or fMLP (26,27). It is unlikely
that DHA contributes to tissue damage by activating
these proteases, since the low level of protease activation
that occurs at low DHA concentrations contrasts greatly
with the high level of protease activation that occurs
when neutrophils are exposed to micromolar concentrations of AA.
Whereas inflammatory mediators such as
fMLP, lipopolysaccharide, the complement fragment
C5a, leukotriene B4, granulocyte–macrophage colonystimulating factor, TNF, and platelet-activating factor
are able to reduce the level of TNFR expression (26,
39–41), AA not only failed to down-regulate these receptors but caused a further increase in these receptors
in the presence of fMLP (9). The stimulatory effect of
AA on TNFR expression has great significance in the
regulation of inflammation, in which this PUFA is likely
to play a role in ensuring, maintaining, and increasing
the ability of TNF to stimulate/prime neutrophils migrating to the inflammatory site and thereby promote
their antimicrobial function. Indeed, we have previously
demonstrated that pretreatment of neutrophils with
AA resulted in a significant increase in superoxide
release when the neutrophils were subsequently challenged with TNF, as compared with that in cells that
had not been preexposed to AA (9). Clearly, AA contributes to amplification of the components of the
inflammatory response by increasing the expression of
not only CR3 (42) but also TNFR on neutrophils (9).
Interestingly, pretreatment with DHA can suppress
superoxide production in response to TNF in AAchallenged neutrophils.
Manipulation of dietary fatty acid uptake can
alter the metabolism of AA within inflammatory cells.
Furthermore, epidemiologic and clinical evidence consistently demonstrates the beneficial effects of n-3 fatty
acids in the treatment of RA (43,44). These effects of
n-3 PUFAs are attributed to their ability to competitively inhibit the enzymes that metabolize AA and to
produce metabolites that are less biologically active than
AA metabolites. Supplementation with n-3–enriched
foods also reduces the production of the proinflammatory cytokines, such as IL-1␤, IL-6, and TNF (11,43,44).
Particularly interesting is our finding that not only did
n-3 PUFAs, EPA, DHA, and linolenic acid fail to
increase TNFR expression on neutrophils (9), but also
EPA and DHA significantly depressed TNFR expression and inhibited the ability of AA to up-regulate these
receptors. Thus, herein we have described another functional aspect of n-3 PUFAs in that they may lead to the
inhibition of inflammation.
Esterification of increased amounts of n-3 PUFAs
in membrane phospholipids is likely not only to compromise the generation of highly inflammatory eicosanoids
and cytokines, but also to prevent the increased expression of TNFR on neutrophils and indeed promote the
generation of soluble TNFRs, thus protecting against
pathogenesis (45). As discussed above, the levels of n-3
PUFAs tested appear to be achievable. Since the concentrations of n-3 PUFAs required to inhibit TNFR
expression are much lower than those required to inhibit
eicosanoid and cytokine production, the results suggest
that this may be a more important mechanism by which
n-3 fatty acids and fish oil suppress the inflammatory
processes and immune responses. Our findings provide
new insights into the mechanisms by which AA amplifies
the inflammatory response, and demonstrate the mechanisms by which DHA and EPA can depress this response.
We are grateful to Matthew Leach and Hisham Yassin
for technical assistance, and to Yong Qin Li, Tricia Harvey,
and the technical staff of the Department of Immunopathology
for assistance with blood preparation and separation.
Dr. Ferrante had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Moghaddami, Irvine, Gao, Grover, Hii, Ferrante.
Acquisition of data. Moghaddami, Irvine, Gao, Grover, Costabile.
Analysis and interpretation of data. Moghaddami, Irvine, Gao,
Grover, Costabile, Hii, Ferrante.
Manuscript preparation. Moghaddami, Hii, Ferrante.
Statistical analysis. Moghaddami, Irvine, Hii.
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