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.код для вставкиСкачать
ARTHRITIS & RHEUMATISM 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 TNFRs. 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. 1 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: [email protected] edu.au. Submitted for publication July 25, 2005; accepted in revised form November 28, 2006. 799 800 MOGHADDAMI ET AL 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. MATERIALS AND METHODS 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 Sigma-Aldrich. 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 ⫻ 7 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 NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 801 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. RESULTS 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 expression. 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 802 MOGHADDAMI ET AL 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. NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 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 shown). 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 803 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) (http://www.serva.de/products/knowledge/061311. shtml). 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. 804 MOGHADDAMI ET AL 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. DISCUSSION 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 surface. We also tested the effects of the above-described inhibitors when added individually at concentrations equivalent to those found in the cocktail (http:// www.sigmaaldrich.com/sigma/bulletin/inhib1bul.pdf), 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 NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 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 (28,32). 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 shedding. 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 805 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⫹ signaling. 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 (http://www.serva.de/products/sheets/proteases. 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 806 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 MOGHADDAMI ET AL 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. ACKNOWLEDGMENTS 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. AUTHOR CONTRIBUTIONS 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. REFERENCES 1. Ferrante A, Kowanko IC, Bates EJ. Mechanisms of host tissue damage by neutrophils activated by cytokines. In: Coffey RG, editor. Granulocyte responses to cytokines: basic and clinical research. New York: Marcel Dekker; 1992. p. 499–521. 2. Schimmer RC, Schrier DJ, Flory CM, Dykens J, Tung DK, Jacobson PB, et al. Streptococcal cell wall-induced arthritis: requirements for neutrophils, P-selectin, intercellular adhesion molecule-1, macrophage-inflammatory protein-2. J Immunol 1997;159:4103–8. NOVEL ACTION OF n-3 POLYUNSATURATED FATTY ACIDS 3. Wipke BT, Allen PM. Essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis. J Immunol 2001;167:1601–8. 4. Emery P. Adalimumab therapy: clinical findings and implications for integration into clinical guidelines for rheumatoid arthritis. Drugs Today (Barc) 2005;41:155–63. 5. Kowanko IC, Ferrante A. Adhesion and TNF priming in neutrophil-mediated cartilage damage. Clin Immunol Immunopathol 1996;79:36–42. 6. Kowanko IC, Ferrante A, Clemente G, Youssef PP, Smith M. Tumor necrosis factor priming of peripheral blood neutrophils from rheumatoid arthritis patients. J Clin Immunol 1996;16: 216–21. 7. Nozaki N, Yamaguchi S, Yamaoka M, Okuyama M, Nakamura H, Tomoike H. Enhanced expression and shedding of tumor necrosis factor receptors from mononuclear leukocytes in human heart failure. J Mol Cell Cardiol 1998;30:2003–12. 8. Tai DI, Tsai SL, Chen TC, Lo SK, Chang YH, Liaw YF. Modulation of tumor necrosis factor receptors 1 and 2 in chronic hepatitis B and C: the differences and implications in pathogenesis. J Biomed Sci 2001;8:321–7. 9. Moghaddami N, Costabile M, Grover PK, Jersmann HP, Huang ZH, Hii CS, et al. Unique effect of arachidonic acid on human neutrophil TNF receptor expression: up-regulation involving protein kinase C, extracellular signal-regulated kinase, and phospholipase A2. J Immunol 2003;171:2616–24. 10. Calder PC. Dietary modification of inflammation with lipids. Proc Nutr Soc 2002;61:345–58. 11. Endres S, De Caterina R, Schmidt EB, Kristensen SD. n-3 polyunsaturated fatty acids: update 1995. Eur J Clin Invest 1995; 25:629–38. 12. Meydani SN. Effect of n-3 polyunsaturated fatty acids on cytokine production and their biologic function. Nutrition 1996;12:S8–14. 13. Deckelbaum RJ, Akabas SR. n-3 Fatty acids and cardiovascular diseases: navigating towards recommendations [editorial]. Am J Clin Nutr 2006;84:1–2. 14. Ferrante A, Thong YH. A rapid one-step procedure for purification of mononuclear and polymorphonuclear leukocytes from human blood using a modification of the Hypaque-Ficoll technique. J Immunol Methods 1978;24:389–93. 15. Hii CS, Ferrante A, Edwards YS, Huang ZH, Hartfield PJ, Rathjen DA, et al. Activation of mitogen-activated protein kinase by arachidonic acid in rat liver epithelial WB cells by a protein kinase C-dependent mechanism. J Biol Chem 1995;270: 4201–4. 16. Hii CS, Huang ZH, Bilney A, Costabile M, Murray AW, Rathjen DA, et al. Stimulation of p38 phosphorylation and activity by arachidonic acid in HeLa cells, HL60 promyelocytic leukemic cells, and human neutrophils: evidence for cell type-specific activation of mitogen-activated protein kinases. J Biol Chem 1998; 273:19277–82. 17. Hardy SJ, Robinson BS, Ferrante A, Hii CS, Johnson DW, Poulos A, et al. Polyenoic very-long-chain fatty acids mobilize intracellular calcium from a thapsigargin-insensitive pool in human neutrophils: the relationship between Ca⫹ mobilization and superoxide production induced by long- and very-long-chain fatty acids. Biochem J 1995;311(Pt 2):689–97. 18. Badwey JA, Curnutte JT, Karnovsky ML. Cis-polyunsaturated fatty acids induce high levels of superoxide production in human neutrophils. J Biol Chem 1981;256:12640–3. 19. Steinbeck MJ, Robinson JM, Karnovsky MJ. Activation of the neutrophil NADPH-oxidase by free fatty acids requires the ionized carboxyl groups and partitioning into membrane lipids. J Leukoc Biol 1991;49:360–8. 20. Poulos A, Robinson BS, Ferrante A, Harvey DP, Hardy SJ, Murray AW. Effect of 22-32 carbon n-3 polyunsaturated fatty acids on superoxide production in human neutrophils: synergism 807 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. of docosahexaenoic acid with f-met-leu-phe and phorbol ester. Immunology 1991;73:102–8. Bates EJ, Ferrante A, Harvey D, Nandoskar M, Poulos A. Docosahexaenoic acid (22:6 n-3) but not eicosapentaenoic acid (20:5 n-3) can induce neutrophil-mediated injury of cultured endothelial cells: involvement of neutrophil elastase. J Leukoc Biol 1993;54:590–8. Ferrante A, Hii CS, Costabile M. Regulation of neutrophil functions by fatty acids. In: Gabrilovich DI, editor. The neutrophils: new outlook for old cells. 2nd ed. London: Imperial College Press; 2005. p. 169–228. Parker PJ, Murray-Rust J. PKC at a glance. J Cell Sci 2004;117(Pt 2):131–2. Lew PD, Monod A, Waldvogel FA, Dewald B, Baggiolini M, Pozzan T. Quantitative analysis of the cystolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils. J Cell Biol 1986;102:2197–204. Porteu F, Nathan CF. Mobilizable intracellular pool of p55 (type 1) tumor necrosis factor receptors in human neutrophils. J Leukoc Biol 1992;52:122–4. Dri P, Gasparini C, Menegazzi R, Cramer R, Alberi L, Presani G, et al. TNF-induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase. J Immunol 2000;165:2165–72. Gasparini C, Menegazzi R, Patriarca P, Dri P. Evidence that elastase is the TNF-R75 shedding enzyme in resting human polymorphonuclear leukocytes. FEBS Lett 2003;553:360–4. Bannenberg GL, Chiang N, Ariel A, Arita M, Tjonahen E, Gotlinger KH, et al. Molecular circuits of resolution: formation and actions of resolvins and protectins. J Immunol 2005;174: 4345–55. Winkler JD, Sung CM, Hubbard WC, Chilton FH. Influence of arachidonic acid on indices of phospholipase A2 activity in the human neutrophil. Biochem J 1993;291:825–31. Robinson BS, Hii CS, Poulos A, Ferrante A. Effect of tumor necrosis factor-␣ on the metabolism of arachidonic acid in human neutrophils. J Lipid Res 1996;37:1234–45. Nichols RC, Vanderhoek JY. 5-Hydroxyeicosanoids selectively stimulate the human neutrophil 15-lipoxygenase to use endogenous substrate. J Exp Med 1990;171:367–75. Serhan CN, Hong S, Gronert K, Colgan SP, Devchand PR, Mirick G, et al. Resolvins: a family of bioactive products of omega-3 fatty acid transformation circuits initiated by aspirin treatment that counter proinflammation signals. J Exp Med 2002;196:1025–37. Arita M, Bianchini F, Aliberti J, Sher A, Hong S, Yang R, et al. Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1. J Exp Med 2005;201:713–22. Corey SJ, Rosoff PM. Unsaturated fatty acids and lipoxygenase products regulate phagocyte NADPH oxidase activity by nondetergent mechanism. J Lab Clin Med 1991;118:343–51. Hii CS, Moghadammi N, Dunbar A, Ferrante A. Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signalling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family. J Biol Chem 2001;276:27246–55. Dulin NO, Sorokin A, Douglas JG. Arachidonate-induced tyrosine phosphorylation of epidermal growth factor receptor and ShcGrb2-Sos association. Hypertension 1998;32:1089–93. McPhail LC, Clayton CC, Snyderman R. A potential second messenger role for unsaturated fatty acids: activation of Ca2⫹dependent protein kinase. Science 1984;224:622–-5. Porteu F, Brockhaus M, Wallach D, Engelmann H, Nathan CF. Human neutrophil elastase releases a ligand-binding fragment from the 75-kDa tumor necrosis factor (TNF) receptor: compari- 808 son with the proteolytic activity responsible for shedding of TNF receptors from stimulated neutrophils. J Biol Chem 1991;266: 18846–53. 39. Ohmann HB, Campos M, McDougall L, Lawman MJ, Babiuk LA. Expression of tumor necrosis factor-␣ receptors on bovine macrophages, lymphocytes and polymorphonuclear leukocytes, internalization of receptor-bound ligand, and some functional effects. Lymphokine Res 1990;9:43–58. 40. Schleiffenbaum B, Fehr J. The tumor necrosis factor receptor and human neutrophil function: deactivation and cross-deactivation of tumor necrosis factor-induced neutrophil responses by receptor down-regulation. J Clin Invest 1990;86:184–95. MOGHADDAMI ET AL 41. Porteu F, Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med 1990;172:599–607. 42. Bates EJ, Ferrante A, Harvey DP, Poulos A. Polyunsaturated fatty acids increase neutrophil adherence and integrin receptor expression. J Leukoc Biol 1993;53:420–6. 43. Simopoulos AP. Essential fatty acids in health and chronic disease. Am J Clin Nutr 1999;70:560–9. 44. Sperling RI. Dietary omega-3 fatty acids: effects on lipid mediators of inflammation and rheumatoid arthritis. Rheum Dis Clin North Am 1991;17:373–89. 45. Arend WP, Dayer JM. Cytokine and cytokine inhibitors or antagonists in rheumatoid arthritis. Arthritis Rheum 1990;33:305–15.