ARTHRITIS & RHEUMATISMVol. 56, No. 3, March 2007, pp 799–808DOI 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 FactorReceptor 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

Objective. Neutrophils and tumor necrosis factor(TNF) play important roles in the pathogenesis ofrheumatoid arthritis (RA). Modulation of TNF recep-tors (TNFRs) may contribute to the regulation of tissuedamage, and n-6 polyunsaturated fatty acids (PUFAs)such as arachidonic acid (AA) can increase the expres-sion of TNFRI and TNFRII on neutrophils. Because then-3 PUFAs are antiinflammatory in RA, we examinedwhether, as a novel mechanism of action, n-3 PUFAs canantagonize the AA-induced increase in TNFR expres-sion.

Methods. Human neutrophils were treated withPUFAs and examined for changes in surface expressionof TNFRs by flow cytometry. Translocation of proteinkinase C (PKC) and activation of ERK-1/2 MAPK weredetermined by Western blotting. Intracellular calciummobilization was measured in Fura 2–loaded cells byluminescence spectrometry.

Results. Pretreatment of neutrophils with nano-molar levels of n-3 PUFAs, eicosapentaenoic acid, or

docosahexaenoic acid led to a marked inhibition of theAA-induced up-regulation of TNFRs I and II. Suchpretreatment, however, did not prevent AA from stimu-lating the activities of PKC and ERK-1/2, which isrequired for the actions of AA or its ability to mobilizeCa2�. Nevertheless, treatment with n-3 PUFAs causedthe stimulation of serine proteases that could cleave theTNFRs.

Conclusion. These findings suggest a mechanismby which the n-3 PUFAs inhibit the inflammatoryresponse in RA, by regulating the ability of AA toincrease TNFR expression. These results help fill thegaps in our knowledge regarding the mechanisms ofaction of n-3 PUFAs, thus allowing us to make specificrecommendations for the use of n-3 PUFAs in theregulation of inflammatory diseases.

Extensive neutrophil accumulation in the syno-vial fluid and synovial tissue of patients with rheumatoidarthritis (RA) has been observed during both the earlyand the exacerbation phases of this inflammatory dis-ease. When activated, these cells release tissue-damaging substances, including oxygen-derived reactivespecies, hypochlorous acid, and elastase, all of whichhave been implicated in neutrophil-mediated damage ofcartilage tissue (1). Furthermore, conclusive evidence ofa role for neutrophils comes from studies involvingexperimental animal models of arthritis, the results ofwhich have shown that neutrophil depletion protectsagainst this inflammatory disease (2,3). A role for tumornecrosis factor (TNF) in the pathogenesis of RA hasbeen demonstrated on the basis of the protective effectsof anti-TNF antibody therapy in humans (4). Among themany-recognized mechanisms by which TNF promotespathogenesis is its ability to prime neutrophils for in-

Supported by the National Health and Medical ResearchCouncil of Australia.

1Nahid Moghaddami, PhD, James Irvine, BSc, Charles S. Hii,PhD: Children, Youth and Women’s Health Services, and Universityof Adelaide, Adelaide, South Australia, Australia; 2Xiuhui Gao, PhD,Phulwinder K. Grover, PhD: Children, Youth and Women’s HealthServices, Adelaide, South Australia, Australia; 3Maurizio Costabile,PhD: Children, Youth and Women’s Health Services, and Universityof South Australia, Adelaide, South Australia, Australia; 4AntonioFerrante, FRCPath, PhD: Children, Youth and Women’s HealthServices, University of Adelaide, and University of South Australia,Adelaide, South Australia, Australia.

Address correspondence and reprint requests to AntonioFerrante, FRCPath, PhD, Department of Immunopathology, Wom-en’s and Children’s Hospital, 72 King William Road, North Adelaide,South Australia 5006, Australia. E-mail: antonio.ferrante@adelaide.edu.au.

Submitted for publication July 25, 2005; accepted in revisedform November 28, 2006.

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creased cartilage damage (5,6). Thus, modulation of theexpression of the TNF receptors (TNFRs) on neutro-phils may alter the tissue-damaging potential of thesecells. It has been demonstrated that the levels ofTNFR in the tissue are modulated under a variety ofconditions (7,8).

Recently, we demonstrated that n-6 polyunsatu-rated fatty acids (PUFAs) such as arachidonic acid (AA)up-regulate the expression of TNFRs on neutrophils andincrease the response of the cells to TNF (9). Thebeneficial and protective effects of the n-3 PUFAs oninflammatory diseases are attributable to their ability toproduce metabolites, which are less biologically activethan AA metabolites (10). Supplementation with n-3–enriched foods also reduces the production of proin-flammatory cytokines such as interleukin-1� (IL-1�),IL-6, and TNF (11,12). Particularly interesting is ourfinding that the n-3 PUFAs, eicosapentaenoic acid(EPA), docosahexaenoic acid (DHA), and linolenic acidfailed to increase TNFR expression on neutrophils (9).It remains of interest therefore to determine if cellspreexposed to n-3 PUFAs display altered responses toAA, in relation to the previously observed up-regulationinduced by AA; confirmation of this would be consistentwith the antiinflammatory properties of n-3 PUFAs. Theprevailing view is that there are several gaps in ourknowledge of the mechanisms of action and propertiesof n-3 PUFAs that limit our ability to make specificrecommendations on their use in inflammatory condi-tions (13). Identification of their effects on TNFRexpression on inflammatory cells would be an importantstep to help fill such gaps, particularly since TNF is a keycytokine in the pathogenesis of RA.

Thus, in the present study we found that lowamounts of DHA or EPA can indeed prevent theAA-induced up-regulation of TNFR expression on neu-trophils. The possibility that the n-3 PUFAs could causeTNFR shedding was supported by our findings that theinability of DHA to increase the surface expression ofTNFR could be totally reversed by inhibitors of serineproteases. These data are evidence of a novel propertyof n-3 PUFAs that may contribute to their antiinflam-matory characteristics.

MATERIALS AND METHODS

Fatty acids. PUFAs were purchased from Sigma-Aldrich (St. Louis, MO). Stocks of the fatty acids were madeup in chloroform and stored at �70oC. On the day of use, thechloroform was evaporated under nitrogen, and the fatty acidswere prepared in ethanol. The purity and quality of these fatty

acids were ensured by analysis via thin layer chromatographyand gas chromatography mass spectrometry.

Protease inhibitors. Inhibition studies were carriedout with a protease inhibitor cocktail containing 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), pepstatin A,E-64, bestatin, leupeptin, and aprotinin, as well as individualprotease inhibitors. All protease inhibitors were obtained fromSigma-Aldrich.

Neutrophils. Neutrophils were isolated from the bloodof healthy volunteers by the rapid single-step method, involv-ing centrifugation of blood on Hypaque-Ficoll (14). Theneutrophils, harvested from the lower leukocyte band, were96–99% pure and �99% viable, as judged by their ability toexclude trypan blue.

Surface expression of TNFR. Surface expression ofTNFRI and TNFRII was measured by flow cytometry, essen-tially as described previously (9). Neutrophils (106 cells/ml)were treated for various times with the PUFA or an equivalentamount of vehicle (ethanol) as control. The cells were washedin ISOTON II (Beckman-Coulter, Gladesville, Australia) sup-plemented with 0.1% (weight/volume) bovine serum albuminand then incubated for 30 minutes on ice with anti-humanTNFRI and TNFRII monoclonal antibodies (mAb) (or �1/�2isotype–matched mAb) (R&D Systems, Minneapolis, MN).The cells were then incubated with fluorescein isothiocyanate–conjugated goat anti-mouse IgG antibodies (AMRAD Opera-tions, Melbourne, Australia) for 30 minutes, fixed with para-formaldehyde (1% volume/volume), and analyzed on a BDBiosciences fluorescence-activated cell sorter (FACS) (FACS-can; Becton Dickinson, Mountain View, CA). Results wereexpressed as the mean fluorescence intensity, corrected for thevalues of the isotype-matched negative controls.

Measurement of protein kinase C (PKC) and ERK-1/2activation. The translocation of PKC was determined as de-scribed previously (15). Briefly, neutrophils were pretreatedwith various concentrations of DHA (for 30 minutes) or anequivalent amount of ethanol, and then stimulated with AA(30 �M for 5 minutes). After harvesting, the cells weresonicated and centrifuged (at 100,000g for 30 minutes), andparticulate fractions were extracted with 2% Triton X-100.Proteins were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocel-lulose (Schleicher & Schuell, Dassel, Germany). PKC isozymeswere detected using anti-PKC isozyme-specific antibodies(Santa Cruz Biotechnology, Santa Cruz, CA) and detected byenhanced chemiluminescence (15).

Dual phosphorylation of ERK-1/2 was assayed asdescribed previously (16). Briefly, neutrophils were pretreatedwith DHA, and then stimulated with AA and lysed. Thesamples were subjected to Western blot analysis using ananti–active ERK antibody (New England Biolabs, Beverly,MA). The blots were stripped and reprobed with anti–ERK-2antibody (Santa Cruz Biotechnology).

Measurement of intracellular Ca2�. Neutrophils (1 �107 cells/ml) in Hank’s balanced salt solution (HBSS) wereloaded in the dark with Fura 2 AM (1 �M) in a shaking waterbath (for 30 minutes at 37°C). The cells were washed twice andresuspended in cold HBSS (6 � 106 cells/ml). Calcium mobi-lization was determined using a PerkinElmer LS50B lumines-cence spectrometer and Fluorescence Data Manager softwareas described previously (17). Briefly, the neutrophils were

800 MOGHADDAMI ET AL

placed in the reaction cuvette and incubated for 5 minutes inthe 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 mon-itored for 30 minutes, after which AA (30 �M) was added.After 5 minutes, Triton X-100, at a final concentration of0.15% (v/v), was added and maximal fluorescence was deter-mined over 3 minutes. The minimum fluorescence over 3minutes was then determined, following the addition of Trisbase (pH 10.0, 40 mM final concentration) and EGTA (12.5mM final concentration).

Statistical analysis. Statistical comparisons were car-ried out using Student’s 2-tailed t-test for paired or unpaireddata, with analysis using Graphpad software (Cricket Software,Philadelphia, PA). Where appropriate, multiple comparisonswith a single control were performed using analysis of variancewith Dunnett’s modification.

RESULTS

Effects of DHA and EPA on the AA-inducedup-regulation of TNFR expression. We previously dem-onstrated that DHA and EPA reduce, whereas AAincreases, TNFR expression on neutrophils (9). In thosestudies (9), we demonstrated that AA dose-dependently(5–30 �M) increased expression of TNFRs I and II onneutrophils. Kinetics studies demonstrated that thiseffect reached a maximum at 30–40 minutes of AAtreatment. We postulated that although both n-6 andn-3 PUFAs could stimulate neutrophil responses, suchas respiratory burst, degranulation, and the adherenceand surface expression of �2 integrins (18–22), the n-3PUFA is most likely responsible for promoting thecleavage of the TNFRs. We thus postulated that EPA orDHA would prevent AA from up-regulating TNFRexpression.

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 expres-sion of TNFRs I and II was examined by flow cytometry.As expected, AA caused an increase in TNFR expres-sion (Figure 1). Pretreatment of the cells with eitherEPA or DHA blocked the AA-induced increase inTNFR expression (Figure 1), and this was observed inthe majority of the cells. The n-3 fatty acids alone causeda small reduction in fluorescence intensity comparedwith ethanol-treated control cells (Figure 1), as observedpreviously (9). Under our experimental conditions, thefatty acids did not affect the viability of the neutrophils,as judged by their ability to exclude trypan blue (resultsnot shown).

Further studies were conducted to identify theconcentration of EPA and DHA required for the inhi-

bition of an AA-induced increase in TNFR expression.Neutrophils were pretreated with various concentra-tions of EPA (500, 2,500, or 5,000 nM), DHA (0.3, 3, 50,500, or 2,500 nM), or an equivalent amount of diluentfor 30 minutes, followed by treatment with AA ordiluent for 30 minutes. The cells were then examined forTNFR expression. The results revealed that both EPAand DHA caused a significant inhibition of the AA-induced up-regulation of TNFR (Figure 2). It wasevident that the maximum inhibitory effects of EPAwere achieved at 500 nM (Figure 2). The inhibitoryeffects of DHA on TNFRI expression were evident atconcentrations of �3 nM, whereas the expression ofTNFRII was suppressed by DHA at concentrations of�50 nM (Figure 2).

We also investigated the consequence of DHApretreatment on the ability of TNF to stimulate super-oxide production. Neutrophils were pretreated with ei-ther DHA (0.2 �M for 30 minutes) or vehicle, andthen incubated with AA (10 �M for 30 minutes). TNF(100 units/ml) was added and superoxide production wasdetermined by a lucigenin-based assay (9). Pretreatmentwith DHA reduced the amount of superoxide producedto 61 � 14% (mean � SEM; n � 5) of the response seenin vehicle-pretreated cells (P � 0.05), reflecting the

Figure 1. Suppression of the arachidonic acid (AA)–induced increasein tumor necrosis factor receptor I (TNFRI) and TNFRII expressionon neutrophils by eicosapentaenoic acid (EPA) and docosahexaenoicacid (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 weremeasured by flow cytometry. Results, expressed as a percent of control(C) (diluent) TNFR expression, are the mean and SEM of 3 experi-ments. � � P � 0.001 versus control neutrophils, by Student’s t-test.

NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 801

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 ofPKC and ERK-1/2 and the mobilization of Ca2�. AAstimulates the activities of several intracellular signalingmolecules, including PKC and ERK-1/2 (22), which wehave previously demonstrated to be required for AA tocause the up-regulation of TNFR expression (9). Wetherefore examined whether DHA inhibited AA-induced activation of these signaling molecules.

To investigate the effects of DHA on AA-stimulated PKC activation, neutrophils were pretreatedwith 0.2 �M of DHA for 30 minutes, and then stimulatedwith AA. After 5 minutes, the cells were sonicated andparticulate fractions were prepared and subjected toWestern blot analysis using anti-PKC�, anti-PKC�1,anti-PKC�2, and/or anti-PKC� antibodies. An associa-tion of classical and novel PKC isozymes with a partic-ulate (membrane) compartment is essential to the acti-vation and function of these isozymes (23), and this stepis subjected to regulation by PUFAs (15,16). As ob-served in previous studies (15,16), AA caused an in-crease in particulate fraction–associated PKC (Figures3a and b). Surprisingly, pretreatment with DHA (0.2

Figure 3. Stimulation of the activation of protein kinase C (PKC) andERK-1/2 and the mobilization of intracellular Ca2� by AA, withoutprevention 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 determina-tion of PKC isozyme translocation and ERK-1/2 activation (a and b) byWestern blotting using anti-PKC isozyme-specific antisera and ananti–active ERK antibody, respectively. The amount of ERK-2 thatwas present in the lanes was determined by stripping and reprobing theblots with an anti–ERK-2 antibody. The blots show a typical experi-mental run, representative of 3 experiments. DHA also did not preventAA from mobilizing intracellular Ca2� (c). In these studies, the cellswere preloaded with Fura 2 and the levels of intracellular Ca2� wereestimated as described in Materials and Methods. Either ethanol orDHA (0.2 �M) was added at 15 minutes, followed by AA (30 �M) at45 minutes. Inset shows the mean and SEM percent of AA-inducedincrease (n � 4 experiments) in intracellular Ca2� levels in theneutrophils that had been preexposed to either ethanol or DHA (P �0.05 versus ethanol controls). See Figure 1 for other definitions.

Figure 2. Effects of varying EPA and DHA concentrations on theAA-induced up-regulation of TNFRI and TNFRII expression inneutrophils. Neutrophils were pretreated with varying concentrationsof EPA, DHA, or diluent (ethanol) at 37°C for 30 minutes. The cellswere then incubated with AA (30 �M) or ethanol for 30 minutes at37°C. The expression of the TNFRs was examined by flow cytometry.Results showing the effect of EPA or DHA on the AA-induced changein receptor expression are the mean � SEM of 3 experiments. � � P� 0.01 versus ethanol pretreatment, by analysis of variance followed byDunnett’s modification. See Figure 1 for definitions.

802 MOGHADDAMI ET AL

�M) did not prevent AA from activating PKC�, PKC�I,and PKC�II (Figure 3a).

We next examined the effects of DHA at 5 �Mand 20 �M. The results showed that at these higherconcentrations, DHA alone caused a variable increase inthe amount of particulate fraction–associated PKC, in-cluding PKC�, that was still detectable at 35 minutesafter the addition of the PUFA (Figure 3b). The abilityof AA to promote PKC� and PKC� translocation wasnot affected by the higher concentrations of DHA.Interestingly, pretreatment with 5 �M DHA enhancedthe effect of AA on PKC� translocation, whereas 20 �MDHA tended to reduce the AA-mediated association ofPKC�I with the particulate fraction (Figure 3b).

To assess the level of ERK-1/2 activation, thecells were treated in the same manner as describedabove and lysed. The lysates were then subjected toWestern blot analysis using an anti–active ERK anti-body. Consistent with previous findings (15,16), AA wasable to stimulate ERK-1/2 activity, but this was notprevented by pretreatment with DHA (0.2 �M) (Figure3a). Higher doses of DHA (5 �M and 20 �M) also didnot inhibit the ability of AA to stimulate the activationof ERK-1/2 and, in fact, enhanced the activation ofERK-1/2 by AA (Figure 3b). Taken together, theseresults suggest that the inhibitory actions of the n-3 fattyacids, especially at lower concentrations, were unlikelyto have been exerted at the level of PKC or ERK-1/2.

Since Ca2� plays an important role in neutrophilactivation, including degranulation (18), we also investi-gated whether pretreatment with DHA would preventAA 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 ofintracellular Ca2� or the ability of AA to mobilizeintracellular Ca2�. Higher doses of DHA (5 �M and 20�M), although causing a small degree of Ca2� mobili-zation, also did not prevent AA from stimulating Ca2�

mobilization but enhanced the effect of AA (results notshown).

Increased TNFR expression by DHA in the pres-ence of protease inhibitors. The inhibition of surfaceTNFR expression by DHA and EPA is intriguing,because both n-3 fatty acids stimulate degranulationfrom the specific and azurophilic granule compartments(22,24) that should have increased the expression ofTNFR (25). Furthermore, higher doses of DHA alsocaused the activation of PKC and Ca2� mobilization,and this should have resulted in increased receptorexpression. To understand how the actions of the n-3fatty acids on TNFR expression were achieved, we

tested the hypothesis that these fatty acids reduced thesurface expression of the receptors by causing receptorcleavage. In neutrophils, shedding of TNFR caused byTNF or n-formyl-methionyl-L-leucyl-L-phenylalanine(fMLP) has been reported to be due to proteases such as ametalloproteinase (TNFRI and TNFRII) and a serineprotease such as elastase (TNFRII) (25–27), althoughthe possible involvement of other proteases cannot beexcluded. We therefore used a protease inhibitor cock-tail containing AEBSF (serine protease inhibitor), pep-statin A (aspartic protease inhibitor), E-64 (cysteineprotease inhibitor), bestatin (aminopeptidase and met-alloprotease inhibitor), leupeptin (serine and cysteine pro-tease inhibitor), and aprotinin (serine protease inhibitor)(http://www.serva.de/products/knowledge/061311.shtml).

To 1 � 106 neutrophils in 0.5 ml HBSS, we added10 �l of protease inhibitor or an equivalent volume ofHBSS. The cells were incubated at 37°C for 10 minutes.

Figure 4. Stimulation of TNFR expression on neutrophils by DHA inthe presence of protease inhibitors (PIs). The ability of a cocktail ofPIs to alter the effects of DHA on TNFRI and TNFRII expression onneutrophils was examined by flow cytometry. The results (a and b) arethe mean and SEM fluorescence intensity, expressed as a percent ofthe control (vehicle-treated) values in 3 experiments. � � P � 0.05;�� � P � 0.01 versus control cells, by analysis of variance followed byDunnett’s modification. The responses to AA (30 �M) under theseconditions were 295 � 42% (n � 3) and 445 � 157% (n � 5) ofcontrols for TNFRI and TNFRII, respectively. Representative histo-grams (c and d) show the flow cytometry profiles of the effects of PIson the ability of DHA to increase TNFR expression on neutrophils.See Figure 1 for other definitions.

NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 803

DHA (20 �M) was then added and the cells wereincubated for a further 30 minutes. As observed previ-ously, DHA alone reduced the basal expression ofTNFRs I and II (Figures 4a and b). Interestingly,whereas the addition of the protease inhibitor cocktailalone did not affect the basal expression of the TNFRs,the addition of the protease inhibitor cocktail togetherwith DHA produced a marked increase in the surfaceexpression of both TNFRI and TNFRII (Figures 4a andb). Results of FACS analysis (histograms in Figures 4cand d) showed that addition of the protease inhibitorcocktail and DHA caused a higher level of fluorescenceintensity compared with that in control cells. Theseresults imply that DHA activated a protease that wasinhibited by the protease inhibitor cocktail, while itsimultaneously recruited additional TNFRs to the cellsurface.

We also tested the effects of the above-describedinhibitors when added individually at concentrationsequivalent to those found in the cocktail (http://www.sigmaaldrich.com/sigma/bulletin/inhib1bul.pdf), toinvestigate the class of protease that was involved in theaction of DHA. Whereas aprotinin, bestatin, E-64, leu-peptin, and pepstatin A did not affect the surfaceexpression of TNFR, either in the presence or absenceof DHA (results not shown), coincubation of the neu-trophils with AEBSF and DHA increased the expression

of both TNFRI and TNFRII (Figure 5). These resultsimply that a serine protease whose activity was sensitiveto AEBSF was involved in mediating the effect of DHAon the expression of TNFRs I and II on neutrophils.

DISCUSSION

An intriguing finding of the present study is thatpreexposure of neutrophils to EPA or DHA can sup-press the AA-induced up-regulation of TNFRI andTNFRII. This novel finding demonstrates a furtherdissociation between the actions of the n-6 and n-3PUFAs, despite the observations that PUFAs from bothseries are very effective at stimulating the neutrophilrespiratory burst, degranulation, and adherence (22).This action highlights an important property of n-3 fattyacids that is likely to also constitute a novel mechanismby which the perceived antiinflammatory actions of n-3fatty acids are mediated.

The suppressive effects of the n-3 fatty acids wereobserved at nanomolar levels, whereas their stimulatoryeffects on neutrophil respiratory burst activity and de-granulation occur at micromolar levels (20–22). Studiesin mice have demonstrated that the levels of nonesteri-fied DHA and EPA can increase rapidly during theinflammatory response (28). Thus, levels of DHA andEPA have been reported to reach 141 nM and 172 nM,respectively, within 2 hours after the initiation of aninflammatory response in the peritoneal cavity. Thesefindings suggest that the suppressive effects of the n-3PUFAs on TNFR expression are likely to be observed atsites of inflammation.

Furthermore, neutrophils are likely to be exposedto micromolar levels of nonesterified AA during cellularactivation. For example, it has been reported that al-though nonesterified AA is undetectable in unstimu-lated neutrophils, activated neutrophils have been foundto contain more than 800 pmoles of AA/107 cells (29),estimated in excess of 110 �M. However, 85% of this AAis released into the extracellular space (29). Our ownstudies have demonstrated that whereas unstimulatedneutrophils contained 15 pmoles of AA/107 neutrophils(�2 �M), this level increased to 125 pmoles and 1,800pmoles/107 neutrophils (�17 �M and �250 �M, respec-tively) when the cells were incubated with TNF andA23187, respectively (30). Others have reported higherlevels of nonesterified AA in neutrophils (31). Followingdietary supplementation with fish oil at 1 gm/day for 3months, our previous studies demonstrated that the ratioof n-3 PUFA:n-6 PUFA in the red blood cells of subjects

Figure 5. Increased expression of TNFRI and TNFRII by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) and protease inhibitor(PI) cocktail in the presence of DHA. Neutrophils were incubated withDHA or vehicle in the presence or absence of AEBSF (2 �M) or PI for30 minutes. After washing, the expression of the TNFRs was assessedby flow cytometry. Results are the mean and SEM of 3 experiments,presented as the percent of TNFR expression on control cells. NeitherAEBSF nor PI alone affected TNFR expression. � � P � 0.05; �� �P � 0.01 versus DHA alone. See Figure 1 for other definitions.

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reached 1:2.2 (Mukaro V, et al: unpublished observa-tions). Thus, it is likely that the levels of n-3 PUFAstested in this study can be achieved, at least underinflammatory conditions. However, it is not knownwhether the suppressive effects of the n-3 PUFAs werecaused by the PUFAs per se or could be attributed totheir metabolites, including resolvins, docosatrienes,and neutroprotectins that, via their counterregulatoryeffects on neutrophil recruitment and inhibition of cyto-kine and chemokine production, have been proposed toparticipate in the resolution of inflammation (32,33).Neutrophils can generate some of these metabolites(28,32).

Owing to its ability to stimulate degranulationfrom both the specific and the azurophilic granules (22),it is most likely that AA causes the mobilization ofTNFR from storage granules. However, to date, the onlyevidence presented has been for the storage of TNFRIin specific granules (25). TNFRII was found to beassociated with only the plasma membrane fraction (25).Nevertheless, our previous findings suggest that TNFRIIis likely to be mobilized from intracellular locations,because its expression on the plasma membrane wasincreased by AA with kinetics similar to those of theAA-induced increase in TNFRI expression (9).

Our findings with regard to the n-3 PUFAs areinteresting. Although EPA and DHA are potent stimu-lators of degranulation, our results suggest that thesePUFAs also have other unidentified concurrent actionsthat cause a net reduction in the surface expression ofTNFR. This is also evident in the majority of neutro-phil ligands. For example, A23187 and fMLP in thepresence of cytochalasin B are strong stimulators ofdegranulation (22), but instead of increasing the surfaceexpression of TNFR, these agents cause a loss of surfaceexpression. This has been attributed to their ability toalso stimulate proteolytic cleavage of the receptors(26,27). Thus, the net expression of a receptor class onthe cell surface is likely to reflect the balance betweenmobilization from intracellular stores and loss caused byshedding.

Because of the existence of multiple cellulartargets for AA, EPA, and DHA, it is difficult to readilydecipher the biochemical mechanisms involved in TNFRregulation by the PUFAs. Badwey et al (18) pointed outthat AA readily partitions into membranes, which resultsin a change to the physical properties of the membranes(19). However, Corey and Rosoff (34) excluded thepossibility of such a nonspecific action, and our recentstudies (35) and those of other investigators (36) have

demonstrated that cell activation by AA can occur viamembers of the ErbB receptor family. In addition, AAcan stimulate the activities of signaling molecules such asPKC, ERK-1/2, p38, phosphatidylinositol 3-kinase, andthe cytosolic phospholipase A2 (cPLA2) (15,16,35–37).We have previously demonstrated that AA acted viaPKC, ERK-1/2, and cPLA2 to elicit the increase inTNFR expression on neutrophils (9). Surprisingly, lowconcentrations (0.2 �M) of DHA did not prevent theactivation of PKC and ERK-1/2 by AA nor did this lowconcentration of DHA prevent the ability of AA tomobilize intracellular Ca2�. At higher concentrations (5�M and 20 �M), DHA caused some activation of thekinases and the mobilization of Ca2�, and amplified theeffect of AA on these parameters. These results suggestthat the inhibitory effects of DHA were unlikely to havebeen caused by inhibition of PKC, ERK-1/2, and Ca2�

signaling.We were interested in identifying the mechanism

of action of this antagonistic effect of the n-3 PUFAs onAA. Our results suggest that DHA caused the proteo-lytic cleavage of TNFRs I and II, since the addition of aprotease inhibitor cocktail totally altered the action ofDHA. Thus, whereas DHA per se reduced the surfaceexpression of TNFRs I and II, the fatty acid uncharac-teristically caused a substantial increase in TNFR ex-pression in the presence of broad-spectrum inhibitorsof serine, cysteine, and aspartic proteases and amino-peptidases. Although these findings support the involve-ment of one or more proteases, it is not known whetherthe enzyme is membrane-bound or soluble. A previousattempt, by Dri et al (26), to demonstrate the release ofproteases in the presence of fMLP failed to demon-strate whether such release occurred, since their resultscould be interpreted in a number of ways. Nevertheless,those authors did show that a membrane-bound, non–matrix metalloproteinase, possibly ADAM-17, may beinvolved in cleaving TNFRI in response to either TNF orfMLP (26). In contrast, shedding of TNFRII has beenreported to require a metalloproteinase and the serineprotease elastase (26,27,38).

Surprisingly, our results show that DHA was ableto increase the surface expression of TNFRI indepen-dent of metalloproteinases, since bestatin, which canalso inhibit metalloproteinases, had no effect on TNFRexpression. Only AEBSF, an irreversible serine proteaseinhibitor (http://www.serva.de/products/sheets/proteases.pdf), increased the level of TNFR expression in thepresence of DHA. Although aprotinin and leupeptinalso inhibit serine proteases, these did not affect TNFR

NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 805

expression. The reason for this is not clear but may berelated to the fact that these are reversible inhibitors ofserine proteases. Consistent with this idea, disiopro-pylfluorophosphate, used in previous studies to demon-strate the role of elastase (27), is also an irreversibleinhibitor of serine proteases. These results imply thatDHA utilizes a combination of proteases that differedfrom that used by TNF or fMLP (26,27). It is unlikelythat DHA contributes to tissue damage by activatingthese proteases, since the low level of protease activationthat occurs at low DHA concentrations contrasts greatlywith the high level of protease activation that occurswhen neutrophils are exposed to micromolar concentra-tions of AA.

Whereas inflammatory mediators such asfMLP, lipopolysaccharide, the complement fragmentC5a, leukotriene B4, granulocyte–macrophage colony-stimulating factor, TNF, and platelet-activating factorare able to reduce the level of TNFR expression (26,39–41), AA not only failed to down-regulate these re-ceptors but caused a further increase in these receptorsin the presence of fMLP (9). The stimulatory effect ofAA on TNFR expression has great significance in theregulation of inflammation, in which this PUFA is likelyto play a role in ensuring, maintaining, and increasingthe ability of TNF to stimulate/prime neutrophils mi-grating to the inflammatory site and thereby promotetheir antimicrobial function. Indeed, we have previouslydemonstrated that pretreatment of neutrophils withAA resulted in a significant increase in superoxiderelease when the neutrophils were subsequently chal-lenged with TNF, as compared with that in cells thathad not been preexposed to AA (9). Clearly, AA con-tributes to amplification of the components of theinflammatory response by increasing the expression ofnot only CR3 (42) but also TNFR on neutrophils (9).Interestingly, pretreatment with DHA can suppresssuperoxide production in response to TNF in AA-challenged neutrophils.

Manipulation of dietary fatty acid uptake canalter the metabolism of AA within inflammatory cells.Furthermore, epidemiologic and clinical evidence con-sistently demonstrates the beneficial effects of n-3 fattyacids in the treatment of RA (43,44). These effects ofn-3 PUFAs are attributed to their ability to competi-tively inhibit the enzymes that metabolize AA and toproduce metabolites that are less biologically active thanAA metabolites. Supplementation with n-3–enrichedfoods also reduces the production of the proinflamma-tory 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 toincrease TNFR expression on neutrophils (9), but alsoEPA and DHA significantly depressed TNFR expres-sion and inhibited the ability of AA to up-regulate thesereceptors. Thus, herein we have described another func-tional aspect of n-3 PUFAs in that they may lead to theinhibition of inflammation.

Esterification of increased amounts of n-3 PUFAsin membrane phospholipids is likely not only to compro-mise the generation of highly inflammatory eicosanoidsand cytokines, but also to prevent the increased expres-sion of TNFR on neutrophils and indeed promote thegeneration of soluble TNFRs, thus protecting againstpathogenesis (45). As discussed above, the levels of n-3PUFAs tested appear to be achievable. Since the con-centrations of n-3 PUFAs required to inhibit TNFRexpression are much lower than those required to inhibiteicosanoid and cytokine production, the results suggestthat this may be a more important mechanism by whichn-3 fatty acids and fish oil suppress the inflammatoryprocesses and immune responses. Our findings providenew insights into the mechanisms by which AA amplifiesthe inflammatory response, and demonstrate the mech-anisms by which DHA and EPA can depress this re-sponse.

ACKNOWLEDGMENTS

We are grateful to Matthew Leach and Hisham Yassinfor technical assistance, and to Yong Qin Li, Tricia Harvey,and the technical staff of the Department of Immunopathologyfor assistance with blood preparation and separation.

AUTHOR CONTRIBUTIONS

Dr. Ferrante had full access to all of the data in the study andtakes responsibility for the integrity of the data and the accuracy of thedata 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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