HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, Y. Articles by Chen, L. H. Search for Related Content PubMed PubMed Citation Articles by Zhao, Y. Articles by Chen, L. H.

Eicosapentaenoic Acid Prevents LPS-Induced TNF- Expression by Preventing NF-B Activation

Graduate Center for Nutritional Sciences (Y.Z., L.H.C.), Internal Medicine (S.J.-B., S.B.), University of Kentucky, Lexington, KY

Address reprint requests to: Dr. Linda Chen, Graduate Center for Nutritional Sciences, 204 Funkhouser Building, University of Kentucky, Lexington, KY 40506-0054. Phone: (859) 257-3288. E-mail: lchen{at}uky.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
Background: Many studies have shown that fish oil supplementation inhibits tumor necrosis factor- (TNF-) production in mice and human subjects; however, the mechanisms remain unclear. Nuclear factor-B (NF-B) is a transcription factor that plays an important role in controlling the expression of pro-inflammatory genes including TNF-. Activation of NF-B has been shown to mediate the maximal expression of TNF- in human monocytes. NF-B is kept in an inactive form in the cytoplasm by IB, the inhibitory subunit of NF-B complex. Phosphorylation and subsequent degradation of IB lead to NF-B activation.

Objectives: The effect of eicosapentaenoic acid (EPA), a major n-3 fatty acid in fish oil, on the lipopolysaccharide (LPS)-induced expression of TNF- and activation of NF-B were investigated. The mechanism underlying EPA modulation of NF-B activation was also studied.

Methods: Human monocytic THP-1 cells were pre-incubated with EPA and stimulated with LPS. The levels of secreted TNF- were determined by ELISA. The DNA binding activity of NF-B was analyzed by EMSA. The degradation and phosphorylation of IB- were examined by Western blot analysis.

Results: TNF- production and expression induced by LPS were significantly decreased in cells pre-incubated with EPA. LPS-induced NF-B activation, translocation of p65 subunit to the nucleus, phosphorylation and degradation of IB- were partially prevented by EPA.

Conclusions: The results suggest that suppression of the TNF- expression by EPA is partly attributed to its inhibitory effect on NF-B activation. EPA appears to prevent NF-B activation by preventing the phosphorylation of IB-.

Key words: eicosapentaenoic acid, fish oil, LPS, TNF-, NF-B, IB-

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
Eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid mainly found in fish oil. Supplementation of fish oil or n-3 fatty acids such as EPA has been shown to alleviate the symptoms of a series of autoimmune, atherosclerotic and inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease [1–3]. Blocking the production of pro-inflammatory mediators including cytokines and eicosanoids has been considered as one of the mechanisms that contribute to the beneficial effects of n-3 fatty acids.

Tumor necrosis factor- (TNF-), a cytokine produced primarily in monocytes and macrophages, is one of the principal mediators of inflammation [4]. It has been implicated in the pathobiology of many human diseases including AIDS, rheumatoid arthritis, cancer cachexia, diabetes, inflammatory bowel disease and multiple sclerosis [5–7]. Suppression of elevated TNF- levels has been proposed as one of the strategies to slow down the progression of these diseases [5]. Lipopolysaccharide (LPS), a surface component of gram-negative bacteria released upon host infection, is a major mediator of tissue injury and shock. LPS is one of the strongest stimuli that induce TNF- production, and the adverse effects of LPS are partly mediated by TNF- [8–10].

The promoter region of TNF- gene contains several potential regulatory elements including B. Binding of the transcription factor, nuclear factor-B (NF-B), to B site has been shown to mediate the maximal LPS-induced TNF- expression in human monocytic cells [11–15]. NF-B, a ubiquitous transcription factor, is kept in an inactive form in the cytoplasm by IB, the inhibitory subunit of NF-B complex [16]. IB is phosphorylated by IB kinase (IKK) in response to external stimuli [17,18]. Phosphorylation and subsequent ubiquitination of IB lead to its degradation. The released NF-B moves to the nucleus, binds to target DNA elements and activates the transcription of target genes [19–21]. Because inappropriate activation of NF-B is associated with a wide range of human diseases including septic shock, inflammatory bowel disease, autoimmune arthritis, asthma, AIDS, glomerulonephritis, lung fibrosis, atherosclerosis and cancer [22,23], NF-B has been considered as an important target for therapeutic intervention.

N-3 fatty acid supplementation has been reported to decrease TNF- production in human and mice, yet the mechanisms remain unclear [24–26]. Previous studies in our laboratory have shown that fish oil supplementation inhibited TNF- production as well as NF-B activation induced by virus infection in a mouse model of AIDS [27,28]. Therefore, EPA may decrease TNF- expression by inhibiting the activation of NF-B. The objective of the present study was to investigate the effect of EPA on LPS-induced NF-B activation in order to elucidate the mechanisms underlying the modulation of TNF- expression by EPA. The effects of EPA on LPS-induced NF-B DNA binding activity, B directed gene expression and phosphorylation of IB- were investigated in human monocytic cell line THP-1. These cells display many characteristics similar to human monocytes and have long been used to study the expression of TNF- [11,14,29].

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
Materials
Sodium salt of cis-5, 8, 11, 14, 17-eicosapentaenoic acid and LPS (Escherichia Coli 0111:B4) were purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS), penicillin and streptomycin were purchased from Life Technologies (Grand Island, NY). Plasmids used for transfection, pNF-B-luciferase and pSV-ß-galactosidase were gifts from Dr. Fajun Yang. Antibody for phosphorylated IB- was purchased from New England Biolab (Beverly, MA). Antibody for p65 was purchased from Rockland (Gilbertsville, PA). Other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were purchased from Sigma unless they are stated otherwise.

Cell Culture
THP-1 cell line was kindly provided by Dr. John S. Thompson, Department of Internal Medicine, University of Kentucky. Cells were maintained in RMPI 1640 medium supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS), as suggested by ATCC, in a 5% CO₂ humid atmosphere at 37°C. EPA sodium salt was dissolved in RPMI 1640 medium with 5% FBS to make a stock solution. Cells with a density of 1 x 10⁶ were incubated with 60 µM of EPA and stimulated with LPS.

Enzyme-Linked Immunosorbent Assay (ELISA)
TNF- was determined by using a commercial ELISA kit (Biosource International, Camarillo, CA) following the procedure provided. The color developed was measured by a microplate reader (Bio-kinetic Reader EL312, Bio-Tek instruments Inc.). The concentrations of TNF- were read from the standard curve.

Quantification of mRNA
Total RNA was isolated by using a commercial kit (Qiagen, Valencia, CA) following the protocol provided. Levels of TNF- mRNA and ß-actin mRNA were determined by using commercial kits (Endogen, Woburn, MA) following the procedures described by Van Arsdell et al. [30]. First, two target-specific oligonucleotide probes were hybridized to a splice junction present in the mRNA of interest. The 3'-end of the invader probe overlapped the region of the signal probe that hybridized to the target mRNA. The overlapping nucleic acid complex was recognized by an enzyme called Cleavase VII that cleaved the signal probe and released its 5' fragment, which was then captured on a streptavidin-coated plate via a biotinylated capture oligo. The complex formed by the capture oligo and signal probe fragment generated a primer-template substrate for DNA polymerase, which extended the signal probe fragment with fluorescein-dUTP. The fluorescein-dUTP was detected by an anti-fluorescein alkaline phosphatase-conjugated antibody and a chemifluorescent substrate. The number of target mRNA transcript in the sample was determined by comparing the fluorescent signal generated by the sample to a standard curve.

Cytosolic and Nuclear Extracts
Cytosolic and nuclear extracts were prepared as described by Finto et al. [31]. Cells were washed in cold phosphate-buffered saline and resuspended in 500 µL of cold buffer (10 mM HEPES, 2 mM MgCl₂, 0.1 mM EDTA, 10 mM KCl, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/mL leupepstatin, 1 µg/mL pepstatin and 1 µg/mL leucine thiol, 0.1% IgePal CA 630, pH 7.9) and left on ice for 30 minutes. The samples were then mixed and centrifuged at 5,000 rpm for 30 minutes. The supernatant (cytosolic extract) was kept at -70°C. The pellet was resuspended in 150 µL of cold saline buffer [20 mM HEPES, 50 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 300 mM NaCl, 25% (w/v) glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/mL leupepstatin, 1 µg/mL pepstatin and 1 µg/mL leucine thiol, 0.2% IgePal CA 630, pH 7.9] and left on ice for 1 hour. After centrifuging at 15,000 rpm for 15 minutes at 4°C, the supernatant (nuclear extract) containing the nuclear proteins was collected and stored at -70°C.

Western Blot Analysis
Protein in the sample was first resolved by SDS-polyacrylamide gel electrophoresis, then transferred electrophoretically to nitrocellulose membrane and subsequently incubated with the primary antibody. For detection, the nitrocellulose filter was incubated with a horseradish peroxidase coupled secondary antibody, followed by an enhanced chemiluminescence substrate reaction using ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Equal loading and transferring were determined by staining the membranes with 0.1% Ponceau S. solution.

Electrophoretic Mobility Shift Assay (EMSA)
DNA binding activity of NF-B was characterized by EMSA [28]. Nuclear extracts containing 5 to 10 µg of protein were incubated for 30 minutes at room temperature with 0.2 ng of ³²P-labeled oligonucleotide probe, 10 mm Tris pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM MgCl₂, 1 mM EDTA, 4% glycerol (w/v) and 0.08 mg/ml sonicated salmon sperm DNA. The oligonucleotide probes used in the EMSA contained consensus-binding sequence for NF-B, 5'-AGT TGA GGG GAC TTT CCC AGG C-3'. DNA-protein complex with a total volume of 20 µL was resolved on a nondenaturing 6% (v/v) polyacrylamide gel and run for about one hour at 200 V in 0.25 X TBE (2.5 mM Tris, 2.5 mM H₃BO₃, 2 mM EDTA, pH 8.5). The gel was then dried and autoradiographed using Kodak X-ray film. For competition assays, unlabeled probes were added in excess (50 times) in the binding buffer. The mutant oligonucleotide, 5'-AGT TGA GGC GAC TTT CCC AGG C-3', has a "G""C" substitution that does not form a DNA-NF-B protein complex [16]. Probes were labeled with [³²P]dATP. The labeled probes were purified using Nick spin columns from Amersham Pharmacia Biotech (Piscataway, NJ). Antibodies were added in the reaction buffer for the supershift assay.

Transfection
THP-1 cells were transfected with pNF-B-Luc that contains a firefly luciferase gene as the reporter gene. The efficiency of transfection was monitored by co-transfection of cells with a pSV-ß-galactosidase control vector. Transient transfection was performed using DEAE-Dextran. THP-1 cells were incubated in 1 ml of serum-free medium containing 5 µL of DEAE-Dextran reagent and 4 µg of plasmid DNA at 37°C with 5% CO₂. RPMI 1640 medium supplemented with 10% FBS was added after 30 minutes. Cells pretreated with or without EPA were stimulated with LPS for 6 hours. The cell extracts were prepared using the reporter lysis buffer (Promega, Madison, WI) and stored at -70°C before analyzing the enzyme activity. The activities of luciferase and ß-galactosidase were determined by using the assay buffers purchased from Promega (Madison, WI) following the protocols provided.

Statistical Analysis
Data were analyzed using one-way or two-way ANOVA by SAS program. When analysis of variance indicated significant differences, the treatment means were compared in pairs using Fisher’s least significant difference procedure [32]. Statistical probability of p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
EPA Prevents LPS-Induced TNF- Production
THP-1 cells were pre-incubated with or without EPA before being stimulated by LPS at various concentrations. Without LPS stimulation, no TNF- production was detected in either the control or EPA pre-treated cells. Addition of LPS from 0.05 to 1.00 µg/ml to cell culture increased TNF- production in a concentration-dependent manner. Pretreatment with 60 µM EPA significantly decreased LPS-induced TNF- production at each LPS concentration (Fig. 1). TNF- production was decreased to similar extent by EPA pre-treatment when cells were stimulated with LPS for various time periods (6, 12 and 24 hours) (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 1. EPA prevents LPS-induced production of TNF-. THP-1 cells were stimulated with graded concentrations of LPS for 6 hours after pre-incubation with or without 60 µM of EPA for 24 hours. Cells were collected and TNF- levels were determined in cell culture supernatant by ELISA. Mean ± S.E., n = 3. *Significantly different from LPS group (p < 0.05). Data are representative of three experiments.

EPA Prevents LPS-Induced TNF- Expression

View larger version (17K): [in this window] [in a new window]   Fig. 2. EPA prevents LPS-induced mRNA levels of TNF-. Cells were stimulated with 0.2 µg/ml of LPS after pre-incubation with or without 60 µM of EPA for 24 hours. TNF- and ß-actin mRNA levels were determined at various time points after LPS was added. Mean ± S.E., n = 2. *Significantly different from LPS group (p < 0.05). Data are representative of two experiments.

Effect of EPA on Stability of TNF- mRNA

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effect of EPA on stability of TNF- mRNA. THP-1 cells were stimulated with 0.2 µg/mL of LPS after pre-incubation with or without 60 µM of EPA for 24 hours. Actinomycin D was added to the medium after cells were stimulated with LPS for 90 minutes. Cells were collected at various time points after addition of actinomycin D, and mRNA levels of TNF- and ß-actin were determined. Relative amount of mRNA (TNF-/ß-actin) at time 0 of actinomycin D addition was used as 100%. Mean ± S.E., n = 3. There was no significant difference between the means of two groups.

EPA Prevents LPS-Induced NF-B DNA Binding Activity

View larger version (48K): [in this window] [in a new window]   Fig. 4. EPA prevents LPS-induced NF-B activation. (A) THP-1 cells pre-incubated with or without 60 µM of EPA for 24 hours were stimulated with 0.2 µg/mL of LPS for 45 minutes. Nuclear extracts were prepared and analyzed by EMSA. Data are representative of three experiments. (B) The competition assay was carried out by incubating nuclear extracts prepared from LPS-stimulated THP-1 cells with labeled probes (1), labeled probes plus excess unlabeled probes (2), or labeled probes plus excess mutated oligonucleotide (3). The mutated oligonucleotide has a "G""C" substitution in the NF-B/Rel DNA binding motif as described in "Material and Methods". (C) Supershift assay of NF-B. Antibodies of p65, p50 or control IgG was incubated with nuclear extracts of LPS-stimulated THP-1 cells and EMSA was performed.

EPA Prevents LPS-Induced Translocation of P65 Subunit of NF-B to Nucleus

View larger version (18K): [in this window] [in a new window]   Fig. 5. EPA prevents LPS-induced translocation of p65 subunit of NF-B to nucleus. THP-1 cells pre-incubated with or without 60 µM of EPA were stimulated with 0.2 µg/mL of LPS for various time periods. Nuclear extracts were prepared and analyzed by Western blot. Data are representative of three experiments.

EPA Prevents B-Directed Luciferase Expression Induced by LPS

View larger version (23K): [in this window] [in a new window]   Fig. 6. EPA prevents LPS-induced expression of B-directed luciferase. THP-1 cells transfected with pNF-B-Luc were pre-incubated with or without 60 µM of EPA for 24 hours before stimulation with 0.2 µg/ml of LPS for 6 hours. The efficacy of transfection was monitored by co-transfection of the cells with pSV-ß-galactosidase control vectors. Mean ± S.E., n = 6. Means with different letters are significantly different (p < 0.05). Data are representative of three experiments.

EPA Prevents LPS-Mediated Phosphorylation of IB- and Degradation of IB-

View larger version (27K): [in this window] [in a new window]   Fig. 7. EPA prevents LPS-induced IB- phosphorylation and degradation. THP-1 cells pre-incubated with or without 60 µM of EPA for 24 hours were stimulated with 0.2 µg/mL of LPS for various time periods. Cytosolic extracts were prepared and analyzed by Western Blot using antibodies specifically recognize IB- (A) and phosphorylated IB- (B). Data are representative of three experiments.

	DISCUSSION

Our results showed that EPA supplementation decreased the levels of LPS-induced TNF- mRNA as well. The AU-rich sequence at 3'-untranslated region of TNF- mRNA has been suggested to play a role in the destabilization of TNF- mRNA [36,37]. It is possible that EPA treatment promotes this destabilization and decreases the steady-state mRNA levels of TNF- by increasing TNF- mRNA degradation. Our result that EPA did not affect the stability of TNF- mRNA indicates that EPA pretreatment most likely decreased mRNA levels of TNF- by inhibiting its formation rather than increasing its degradation. Therefore, EPA may modulate TNF- expression at the transcriptional level.

NF-B is one of the transcription factors that are critical for LPS-induced TNF- expression [11–15]. We previously observed that NF-B DNA binding activity induced by murine leukemia virus infection was suppressed by supplementing fish oil to mice [28]. In this study we observed that LPS-induced DNA binding activity of NF-B was decreased in THP-1 cells pre-incubated with EPA. Similar effects of EPA were reported in mouse marcophage cell line, RAW 264.7 cells [38]. The result in the present study that LPS-induced expression of B-directed luciferase was decreased by pre-incubating cells with EPA further supports that EPA prevents LPS-induced NF-B activation. Recently, suppressed NF-B activation in mice fed a diet enriched with n-3 fatty acids was reported [39]. These results demonstrated that EPA might prevent the expression of TNF- partly by blocking the activation of NF-B.

NF-B is kept in an inactive form by the inhibitory subunit IB in most cell types [16]. Phosphorylation of IB by IKKs leads to its ubiquitination and subsequent degradation, resulting in the translocation and activation of NF-B [17–21]. Our results demonstrated that THP-1 cells pre-incubated with EPA had significantly lower levels of phosphorylated IB- upon LPS stimulation. However, EPA was only able to slightly prevent the decrease of IB levels. First, the amount of IB- is much more abundant compared to the amount of phosphorylated IB-, therefore, it is more difficult to observe the difference providing that EPA is a weak modulator of NF-B activation. Secondly, the IB- levels shown in Fig. 7A represents the total amount of pre-existed and newly synthesized IB-. Since IB- gene is up-regulated by the activation of NF-B (40,41), a decrease in NF-B activation by EPA may result in a slow regeneration of IB- that diminishes the difference of IB- levels between the control cells and EPA-treated cells.

In summary, the results demonstrated that the prevention of LPS-induced NF-B activation by EPA was associated with the prevention of IB- phosphorylation and degradation. Therefore, EPA may suppress NF-B activation through inhibiting the phosphorylation of IB. To clarify the mechanisms underlying EPA modulation of IB- phosphorlation, it will be necessary to investigate the effects of EPA on IKK activity. Since the transcriptional activity of NF-B is also regulated by the phosphorylation of p65 [42,43], it would be interesting to find out whether EPA directly modulates NF-B activity by altering the phosphorylation of the NF-B subunit.

The molecular pathways affected by EPA, which leads to its inhibition on NF-B activation, remain to be understood. EPA may exert its effect on NF-B activation via blocking upstream signals leading to IKK activation. In fact, it has been reported that CD14 expression stimulated by LPS is down-regulated by EPA [44]. Previous findings in our laboratory demonstrated that supplementing fish oil to mice restored the tissue antioxidant defense systems that were suppressed by infection with murine leukemia virus [45]. Numerous studies have shown that NF-B is a redox-sensitive transcription factor [46,47]. A low concentration of hydrogen peroxide can activate NF-B and various antioxidants can prevent NF-B activation [48–50]. LPS stimulation of cells can cause a 6–10 fold increase in the release of reactive oxygen species (ROS) including superoxide and hydrogen peroxide [51,52]. In a CD14-independent pathway, Rac1-dependent ROS formation is shown to mediate LPS-induced IKK activation and induction of NF-B [51]. Pre-treating cells with antioxidants reduces LPS-induced NF-B transcription activity [51,52] and the activities of upstream Rac1 and protein tyrosine kinases [53,54]. Therefore, it is possible that EPA may prevent NF-B activation and TNF- production via reducing ROS production induced by LPS.

This study focused on the effect of EPA on the activation of NF-B, one of the key transcriptional factors that regulate LPS-induced TNF- expression. Since the expression of TNF- involves multiple transcriptional factors, such as activator protein-1 (AP-1) and cyclic AMP responsive element binding protein (CREB) [11], the possibility that EPA may affect the activities of other transcriptional factors cannot be excluded.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
The B sites have been found in the promoters and enhancers of numerous genes including cytokines, acute phase proteins and adhesion molecules [19,20]. Inappropriate regulation of NF-B is linked to a wide range of human diseases including septic shock, atherosclerosis, autoimmune arthritis, asthma, glomerulonephritis, and lung fibrosis [22,23]. The results suggest that EPA prevents the LPS-induced TNF- expression by preventing the activation of NF-B, and that EPA prevents NF-B activation through decreasing phosphorylation of IB-. It has been suggested that diets with increased ratio of n-3 to n-6 PUFA may provide a condition favorable to effective drug treatment, since current Western diet with low ratio may be suboptimal for the effective drug therapies that require inhibition of pro-inflammatory cytokine such as TNF- [34]. Such a community-wide strategy may decrease the emergence of inflammatory diseases and thrombic episodes as well as decrease their severity [34]. Thus, EPA or fish oil may have its special value as a food component or supplement for prevention or dietary intervention of many inflammatory diseases.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by KYAES Project No. 010006, Paper No. 02-10-47.

Received December 23, 2002. Accepted August 12, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSIONS ACKNOWLEDGMENTS REFERENCES

 

1. Alexander JW: Immunonutrition: The role of -3 fatty acids. Nutrition14 :627 –633,1998 .[Medline]
2. Belluzzi A, Boschi S, Brignola C, Munarini A, Cariani G, Miglio F: Polyunsaturated fatty acids and inflammatory bowel disease. Am J Clin Nutr71 :339S –342S,2000 .[Abstract/Free Full Text]
3. Kremer JM: N-3 fatty acids supplements in rheumatoid arthritis. Am J Clin Nutr71 :249S –251S,2000 .
4. Bemelmans MHA, van Tits LJH, Buurman WA: Tumor necrosis factor: Function release and clearance. Crit Rev Immunol16 :1 –11,1996 .[Medline]
5. Tracey KJ, Cerami A: Tumor necrosis factor: A pleiotropic cytokine and therapeutic target. Ann Rev Med45 :491 –503,1994 .[Medline]
6. Dinarello CA, Cannon JG, Wolff SM, Bernheim HA, Beutler B, Cerami A, Figari IS, Palladino Jr MA, O’Connor JV: TNF is an endogenous pyrogen and induces production of IL-1. J Exp Med163 :1433 –1450,1986 .[Abstract/Free Full Text]
7. Kollias G, Douni E, Kassiotis G, Kontoyiannis D: The function of tumor necrosis factor and receptors in models of multi-organ inflammation, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Ann Rheum Dis58 :131 –139,1999 .
8. Morrison DC, Ryan JL: Endotoxins and disease mechanisms. Ann Rev Med38 :417 –432,1987 .[Medline]
9. Raetz CR, Ulevitch RJ, Wright SD, Sibley CH, Ding A, Nathan CF: Gram-negative endotoxin: An extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J5 :2652 –2660,1991 .[Abstract]
10. Morrison DC, Ryan JF: Bacterial and host responses. Adv Immunol28 :293 –450,1979 .[Medline]
11. Yao J, Mackman N, Edgington TS, Fan ST: Lipopolysaccharide induction of tumor necrosis factor- promoter in human monocytic cells. J Biol Chem272 :177795 –177801,1997 .
12. Shakhov AN, Collart MA, Vassalli P, Nedospasov SA, Jongeneel CV: KappaB-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor-alpha gene in primary macrophages. J Exp Med171 :35 –47,1990 .[Abstract/Free Full Text]
13. Ziegler-Heitbrock HW, Sternsdorf T, Liese J, Belohradsky B, Weber C, Wedel A, Schreck R, Bauerle P, Strobel: Pyrrolidine dithiocarbamate inhibits NF-kappaB mobilization and TNF production in human monocytes. J Immunol151 :6986 –6993,1993 .[Abstract]
14. Trede NS, Tsytsykova AV, Chatila T, Goldfeld AE, Geha RS: Transcriptional activation of the human TNF-alpha promoter by superantigen in human monocytic cells: Role of NF-kappaB. J Immunol155 :902 –908,1995 .[Abstract]
15. Udalova IA, Knight JC, Vidal V, Nedospasov SA, Kwiatkowski D: Complex NF-B interactions at the distal tumor necrosis factor promoter region in human monocytes. J Biol Chem273 :21178 –21186,1998 .[Abstract/Free Full Text]
16. Lenardo MJ, Baltimore D: NF-B: A pleiotropic mediator of inducible and tissue specific gene control. Cell58 :227 –229,1989 .[Medline]
17. Verma IM, Stevenson J: IB kinase: Beginning, not the end. Proc Natl Acad Sci USA94 :11758 –11760,1997 .[Free Full Text]
18. DiDonato JA, Hayakawa M, Rothwarf DM, Zandi E, Karin M: A cytokine-responsive IB kinase that activates the transcription factor NF-B. Nature388 :548 –554,1997 .[Medline]
19. Sha WC: Regulation of immune responses by NF-B/Rel transcription factors. J Exp Med187 :143 –146,1998 .[Free Full Text]
20. Baldwin Jr AB: The NF-B and IB proteins: New discoveries and insights. Ann Rev Immunol14 :649 –681,1996 .[Medline]
21. Baeuerle PA, Baltimore D: Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappaB transcription factor. Cell53 :211 –217,1988 .[Medline]
22. Grossmann M, Nakamura Y, Grumont R, Gerondakis S: New insights into the roles of ReL/NF-kappaB transcription factors in immune function, hemopoiesis and human disease. Int J Biochem Cell Biol10 :1209 –1219,1999 .
23. Chen F, Castranova V, Shi X, Demers LM: New Insight into the role of nuclear factor-B, a ubiquitous transcription factor in the initiation of diseases. Clin Chem45 :7 –17,1999 .[Abstract/Free Full Text]
24. Renier G, Skamene E, DeSanctis J, Radzioch D: Dietary n-3 polyunsaturated fatty acids prevent the development of atherosclerotic lesions in mice: Modulation of macrophage secretory activities. Arterioscler Thromb13 :1515 –1524,1993 .[Abstract/Free Full Text]
25. Meydani SN, Endres S, Woods MM, Goldin BR, Soo C, Morrill-Labrode C, Dinarello CA, Gorbach SL: Oral n-3 fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: Comparison between young and old women. J Nutr121 :547 –555,1991 .
26. Endres S, Ghorbani R, Kelley VE, Georgilis K, Lonnemann G, van der Meer JW, Cannon JG, Rogers TS, Klempner MS, Weber PC: The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the synthesis of IL-1 and TNF by mononuclear cells. New Engl J Med320 :265 –271,1989 .[Abstract]
27. Xi S, Cohen DA, Chen LH: Effects of fish oil on cytokines and immune functions of mice with murine AIDS. J Lipid Res39 :1677 –1687,1998 .[Abstract/Free Full Text]
28. Xi S, Cohen D, Barve S, Chen LH: Fish oil suppressed cytokines and nuclear factor-B induced by murine AIDS virus infection. Nutr Res21 :865 –878,2001 .
29. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K: Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer26 :171 –176,1980 .[Medline]
30. Van Arsdell SW, Murphy KP, Pazmany C, Erickson D, Burns C, Moody MD: Explore mRNA assays for quantification of IL-1ß and TNF- mRNA in lipopolysaccharide-induced mouse macrophages. Biotechniques28 :1220 –1225,2000 .[Medline]
31. Finco TS, Beg AA, Baldwin Jr AS: Inducible phosphorylation of IB is not sufficient for its dissociation from NF-B and is inhibited by protease inhibitors. Proc Natl Acad Sci USA91 :11884 –11888,1994 .[Abstract/Free Full Text]
32. Snedecor AA: "Statistical methods," 7th ed. Ames, IA: Iowa State Press,1974 .
33. Yaqoob P, Calder P: Effects of dietary manipulation upon inflammatory mediator production by murine macrophages. Cell Immunol163 :120 –128,1995 .[Medline]
34. Caughey GE, Mantzioris E, Gibson RA, Cleland LG, James MJ: The effect on human tumor necrosis factor- and interleukin-1ß production of diets enriched in n-3 fatty acids from vegetable oil or fish oil. Am J Clin Nutr63 :116 –122,1996 .[Abstract/Free Full Text]
35. Zhao Y, Chen LH: N-3 fatty acids/eicosanoids and inflammatory responses. In Huang YS, Li SJ, Huang PC (eds): "Essential Fatty Acids and Eicosanoids: Invited Papers from the Fifth International Congress." Champaign, IL: AOCS Press, pp219 –226,2003 .
36. Zuckerman SH, Evans GF, Guthrie L: Transcriptional and post-transcriptional mechanisms involved in the differential expression of LPS-induced IL-1 and TNF mRNA. Immunology73 :460 –465,1991 .[Medline]
37. Carballo E, Lai WS, Blackshear PJ: Feedback inhibition of macrophage tumor necrosis factor- production by tristetraprolin. Science281 :1001 –1005,1998 .[Abstract/Free Full Text]
38. Lo C, Chiu K, Fu M, Lo R, Helton S: Fish oil decreases macrophage tumor necrosis factor gene transcription by altering the NF-B activity. J Surg Res82 :216 –221,1999 .[Medline]
39. Jolly CA, Muthukumar A, Avula CP, Troyer D, Fernandes G: Life span is prolonged in food-restricted autoimmune-prone (NZB x NZW)F(1) mice fed a diet enriched with n-3 fatty acids. J Nutr131 :2753 –2760,2001 .[Abstract/Free Full Text]
40. Chiao PJ, Miyamoto S, Verma IM: Autoregulation of IkappaB alpha activity. Proc Natl Acad Sci USA91 :28 –32,1994 .[Abstract/Free Full Text]
41. Scott ML, Fujita T, Liou HC, Nolan GP, Baltimore D: The p65 subunit of NF-kappa-B regulates I kappa-B by two distinct mechanisms. Genes Dev7(7A) :1266 –1276,1993 .
42. Wang D, Baldwin Jr AS. Activation of nuclear factor-kappaB-dependent transcription by tumor necrosis factor-alpha is mediated through phosphorylation of RelA/p65 on serine 529. J Biol Chem273 :29411 –29416,1998 .[Abstract/Free Full Text]
43. Sizemore N, Lerner N, Dombrowski N, Sakurai H, Stark GR: Distinct roles of the IkappaB kinase alpha and beta subunits in liberating nuclear factor-kappaB from IkappaB and in phosphorylating the p65 subunit of NF-kappaB. J Biol Chem277 :3863 –3869,2002 .[Abstract/Free Full Text]
44. Chu AJ, Walton MA, Prasad JK, Seto A: Blockade by polyunsaturated n-3 fatty acids of endotoxin-induced monocytic tissue factor activation is mediated by the depressed receptor expression in THP-1 cells. J Surg Res87 :217 –224,1999 .[Medline]
45. Xi S, Chen LH: Effects of dietary fish oil on tissue glutathione and antioxidant defense enzymes in mice with murine AIDS. Nutr Res20 :1287 –1299,2000 .
46. Schreck R, Albermann K, Baeuerle PA: Nuclear Factor-B: An oxidative stress-responsive transcription factor of eukaryotic cells [Review]. Free Rad Res Comm17 :221 –237,1992 .[Medline]
47. Gius D, Botero A, Shah S, Curry HA: Intracellular oxidation/reduction status in the regulation of transcription factors NF-B and AP-1. Toxicol Letters106 :93 –106,1999 .[Medline]
48. Schreck R, Rieber P, Baeuerle PA: Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-B transcription factor and HIV-1. EMBO J10 :2247 –2258,1991 .[Medline]
49. Suzuki YJ, Packer L: Inhibition of NF-B activation by vitamin E derivatives. Biochem Biophys Res Comm193 :277 –283,1993 .[Medline]
50. Meyer M, Schreck R, Baeuerle PA: H₂O₂ and antioxidants have opposite effects on activation of NF-B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J12 :2005 –2015,1993 .[Medline]
51. Sanlioglu S, Williams CM, Samavati L, Butler NS, Wang G, McCray Jr PB, Ritchie TC, Hunninghake GW, Zandi E, Engelhardt JF: Lipopolysaccharide induces Rac1-dependent reactive oxygen species formation and coordinates tumor necrosis factor-alpha secretion through IKK regulation of NF-kappaB. J Biol Chem276 :30188 –30198,2001 .[Abstract/Free Full Text]
52. Schreck R, Meier B, Mannel DN, Droge W, Baeuerle PA: Dithiocarbamates as potent inhibitors of nuclear factor-B activation in intact cells. J Exp Med175 :1181 –1194,1992 .[Abstract/Free Full Text]
53. Hsu HY, Wen MH: Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J Biol Chem277 :22131 –22139,2002 .[Abstract/Free Full Text]
54. Geng Y, Zhang B, Lotz M: Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes. J Immunol151 :6692 –6700,1993 .[Abstract]

This article has been cited by other articles:

				E. Higashihara, K. Nutahara, S. Horie, S. Muto, T. Hosoya, K. Hanaoka, K. Tuchiya, K. Kamura, K. Takaichi, Y. Ubara, et al. The effect of eicosapentaenoic acid on renal function and volume in patients with ADPKD Nephrol. Dial. Transplant., September 1, 2008; 23(9): 2847 - 2852. [Abstract] [Full Text] [PDF]

				K. Tanaka, Y. Ishikawa, M. Yokoyama, H. Origasa, M. Matsuzaki, Y. Saito, Y. Matsuzawa, J. Sasaki, S. Oikawa, H. Hishida, et al. Reduction in the Recurrence of Stroke by Eicosapentaenoic Acid for Hypercholesterolemic Patients: Subanalysis of the JELIS Trial Stroke, July 1, 2008; 39(7): 2052 - 2058. [Abstract] [Full Text] [PDF]

				M. M. Diaz Encarnacion, G. M. Warner, C. E. Gray, J. Cheng, H. K. H. Keryakos, K. A. Nath, and J. P. Grande Signaling pathways modulated by fish oil in salt-sensitive hypertension Am J Physiol Renal Physiol, June 1, 2008; 294(6): F1323 - F1335. [Abstract] [Full Text] [PDF]

				J. K. Kiecolt-Glaser, M. A. Belury, K. Porter, D. Q. Beversdorf, S. Lemeshow, and R. Glaser Depressive Symptoms, omega-6:omega-3 Fatty Acids, and Inflammation in Older Adults Psychosom Med, April 1, 2007; 69(3): 217 - 224. [Abstract] [Full Text] [PDF]

				M. S. Rolph, T. R. Young, B. O. V. Shum, C. Z. Gorgun, C. Schmitz-Peiffer, I. A. Ramshaw, G. S. Hotamisligil, and C. R. Mackay Regulation of Dendritic Cell Function and T Cell Priming by the Fatty Acid-Binding Protein aP2 J. Immunol., December 1, 2006; 177(11): 7794 - 7801. [Abstract] [Full Text] [PDF]

				K. J. Auborn Can Indole-3-Carbinol-Induced Changes in Cervical Intraepithelial Neoplasia Be Extrapolated to Other Food Components? J. Nutr., October 1, 2006; 136(10): 2676S - 2678S. [Full Text] [PDF]

				A. A. Farooqui, W.-Y. Ong, and L. A. Horrocks Inhibitors of Brain Phospholipase A2 Activity: Their Neuropharmacological Effects and Therapeutic Importance for the Treatment of Neurologic Disorders Pharmacol. Rev., September 1, 2006; 58(3): 591 - 620. [Abstract] [Full Text] [PDF]

				P. C Calder n-3 Polyunsaturated fatty acids, inflammation, and inflammatory diseases Am. J. Clinical Nutrition, June 1, 2006; 83(6): S1505 - 1519S. [Abstract] [Full Text] [PDF]

				Q. Jia, H.-R. Zhou, Y. Shi, and J. J. Pestka Docosahexaenoic Acid Consumption Inhibits Deoxynivalenol-Induced CREB/ATF1 Activation and IL-6 Gene Transcription in Mouse Macrophages J. Nutr., February 1, 2006; 136(2): 366 - 372. [Abstract] [Full Text] [PDF]

				C. E. Loscher, E. Draper, O. Leavy, D. Kelleher, K. H. G. Mills, and H. M. Roche Conjugated Linoleic Acid Suppresses NF-{kappa}B Activation and IL-12 Production in Dendritic Cells through ERK-Mediated IL-10 Induction J. Immunol., October 15, 2005; 175(8): 4990 - 4998. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhao, Y. Articles by Chen, L. H. Search for Related Content PubMed PubMed Citation Articles by Zhao, Y. Articles by Che