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Journal of the American College of Nutrition, Vol. 18, No. 6, 602-613 (1999)
Published by the American College of Nutrition

Original Research

Effects of Dietary {omega}-3 and {omega}-6 Lipids and Vitamin E on Serum Cytokines, Lipid Mediators and Anti-DNA Antibodies in a Mouse Model for Rheumatoid Arthritis

Jaya T. Venkatraman, PhD, CNS and Wei-chia Chu, MS

Department of Physical Therapy, Exercise and Nutrition Sciences, State University of New York at Buffalo, 15 Farber Hall, Buffalo, New York


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Objective: Omega-3 ({omega}-3) fatty acid rich-fish oil (FO) and vitamin E (vit-E) may delay the progress of certain autoimmune diseases. The present study examined the mechanism of action of {omega}-3 and {omega}-6 lipids and vit-E on the serum cytokines and lipid mediators in autoimmune-prone MRL/lpr mice (a model for rheumatoid arthritis, RA). The lpr (lymphoproliferative) gene is overexpressed in these mice causing extensive lymphoproliferation, lupus-like symptoms and accelerated aging.

Methods: Weanling female MRL/lpr and congenic control MRL/++ mice were fed 10% corn oil (CO, {omega}6) or FO-based semipurified diets containing two levels of vitamin E (vit-E-75, I.U. and vit-E-500 I.U./Kg diet) for four months. At the end of the experiment, serum anti-DNA antibodies, cytokines and lipid mediators levels were determined.

Results: The appearance of enlarged lymph nodes was delayed in the mice fed FO, and the FO-500 IU vit-E diet offered further protection against enlargement of lymph nodes. The MRL/lpr mice exhibited significantly higher levels of serum anti-dsDNA antibodies. The FO-fed mice had significantly lower serum IL-6, IL-10, IL-12, TNF-{alpha}, PGE2, TXB2 and LTB4 levels compared with CO-fed mice. In mice fed 500 IU vit-E diets, the serum IL-6, IL-10, IL-12 and TNF-{alpha} levels were significantly lower and serum IL-1ß was significantly higher compared to 75 IU-vit-E-fed mice in CO/FO or both. The levels of anti-DNA antibodies, IL-4, IL-6, TNF-{alpha}, IL-10 and IL-12 were higher in the sera of MRL/lpr mice. The FO diet lowered the levels of these cytokines (except IL-4) and lipid mediators. Adding 500 IU of vit-E to the FO diet further lowered the levels of IL-6, IL-10, IL-12, and TNF-{alpha}.

Conclusion: It is clear from our observations that the beneficial effects of FO can be enhanced by the addition of 500 IU of vit-E in the diet. The FO diet containing 500 IU of vit-E may specifically modulate the levels of IL-6, IL-10, IL-12 and TNF-{alpha} and thereby may delay the onset of autoimmunity in the MRL/lpr mouse model. The observations from this study may form a basis for selective nutrition intervention based on specific fatty acids and antioxidants in delaying the progress of RA.

Key words: {omega}-3 lipids, fish oil, vit-E, cytokines, lipid mediators


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Defective regulation of inflammatory responses and disordered immune mechanisms are central to the pathological processes encountered in rheumatic diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). RA is an autoimmune disease characterized by progressive joint destruction and immobility. Prevention of cartilage erosion would be of great therapeutic benefit to RA patients. Among other factors, imbalances in pro- and antiinflammatory cytokines have been observed in RA and SLE. Various cytokines and chemokines have been implicated as important mediators of inflammation and joint destruction in RA and other inflammatory processes. In autoimmune diseases, an abnormal production of pro-inflammatory cytokines or a reduced inhibition of their action occurs and leads to an imbalance in the cytokine network [1]. Some of the cytokines have been linked to cartilage destruction in RA patients.

Treatment of autoimmune diseases with nutrition interventions has received considerable attention in the recent years. Clinical trials using {omega}-3 lipids on RA patients have been conducted by several investigators, with FO treatments seeming to ameliorate clinical symptoms. RA subjects who were taking FO supplements could eventually reduce the dosage of NSAIDs or even discontinue the use of medication [2]. The mechanism of action of {omega}-3 lipids and antioxidants in protecting against autoimmune disease is not well understood at the present time. Significant beneficial effects of dietary supplementation with {omega}-3 fatty acids have been reported in autoimmune-prone mice [3,4] and in patients with RA [5,6]. Nutritional intervention has been accepted as having a major therapeutic potential [7–9]. Incorporation of some fatty acids into tissues may modify inflammatory and immune reactions, and {omega}-3 lipids are found to have a potential therapeutic value for inflammatory diseases [10,11]. Nutritional intervention with marine lipids containing long chain {omega}-3 fatty acids (EPA, DHA), have been reported to significantly increase the life span and delay the onset of autoimmune disease in autoimmune-prone mice [12–14]. The beneficial effects have been attributed to increased EPA (20:5{omega}-3) and DHA (22:6{omega}-3) content and to markedly decreased arachidonic acid (20:4{omega}-6) in the subcellular membranes of the FO-fed animals.

In recent years several animal models have been developed which serve as ideal models to investigate the autoimmune phenomena involved in RA. MRL/lpr mice spontaneously develop massive lymphadenopathy with hypergammaglobulinemia, autoantibodies, high levels of acute phase proteins, arthritis and immune complex glomerulonephritis [15]. The two substrains of these mice (MRL/lpr and +/+ mice) offer a unique controlled model for investigating dietary and drug effects on autoimmune disease [16,17]. A single gene lymphoproliferative (lpr) disease model of spontaneous systemic autoimmunity has attracted considerable attention in recent years. Congenic MRL/lpr/ and MRL/++ mice differ in their incidence of anti-DNA and anticardiolipin autoantibodies, lymphoproliferative disease, immune complex glomerulonephritis and survival. These characteristics are linked closely with the regulatory imbalance of T cells and B cells that display an exceptional constellation of altered membrane markers [18,19]. Several studies have shown that MRL/lpr mice develop autoimmune renal disease and have a short life span [20,21]. In MRL mice, the mostly recessive lpr mutation (identified as Fas mutation) results in both the accumulation of abnormal T cells in lymphoid tissue and many features of generalized autoimmune disease, including immune complex glomerulonephritis.

It is evident from the literature that FO may delay the process of autoimmune disease by lowering the levels of specific proinflammatory cytokines and inflammatory lipid mediators. The present study was planned to examine the mechanism of action of {omega}-3 lipids and vit-E on autoimmune disease in a mouse model for RA. We have investigated the effects of feeding {omega}6 and {omega}-3 lipids in the presence of 75 IU and 500 IU of vit-E on the levels of serum pro- and anti-inflammatory cytokines in MRL/lpr mice. The primary goal of this study was to investigate whether addition of vit-E (75 and 500 IU/kg diet) to {omega}-3 dietary lipids provides additional protection against autoimmune disease in MRL/lpr mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSION
 REFERENCES
 
Experimental Animals and Diets
Weanling female MRL/lpr mice and MRL/++ mice (10/group) purchases from Jackson Laboratories (Bar Harbor, ME) were fed semipurified diets containing 10% (w/w) corn oil (ICN, Irvine, CA) or 10% odor-free menhaden FO (U.S. Department of Commerce, National Marine Fisheries Service, Charleston, NC) with low (75 IU/kg diet) and high levels (500 IU/kg diet) of vit-E (Table 1). Both dietary oils had equal levels of antioxidant supplements, 1.3 g/kg oil of D-{alpha}-tocopherol oil (ICN, Irvine, CA), 1.2 g/kg oil of {gamma}-tocopherol (U.S. Department of Commerce, National Marine Fisheries Service, Charleston, NC) and 1 g/kg oil of tertiary butylhydroxyquinone (TBHQ) (Dyets, Bethlehem, PA), as recommended by the National Institutes of Health (NIH) to prevent peroxidation during storage(Table 2). Fresh diet was provided daily and precautions were taken to prevent oxidation of lipids. The mice were maintained in plastic cages and a 12-hour light/12-hour dark cycle was followed. The body weights were recorded every two weeks, and the MRL/lpr mice were checked for enlarged lymph nodes. The investigators strictly followed National Institutes of Health guidelines as described in the guide for the care and use of laboratory animals. The animals were sacrificed at 4.5 months of age through cervical dislocation.


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  		Table 1. Composition of the Experimental Diets

 

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  		Table 2. Fatty Acid Composition of the Oils1

 
Collection of Blood and Preparation of Serum
Blood was collected and centrifuged at 10,000xg for 10 minutes at 4°C and clear sera were stored at -70°C for analyses of cytokines and anti-DNA antibodies. The kidneys were preserved in 10% buffered formalin for histological analysis.

Histology of Kidneys
Kidneys used for light microscopic examination were immediately fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 µm, stained with hematoxylin-eosin (H & E) using standard techniques and graded based on a semiquantitative scale.

Measurement of Serum Anti-dsDNA Antibodies
Serum anti-dsDNA antibodies were determined by ELISA as previously described [22]. Polyvinylchloride Immulon II 96-well microtiter plates were coated with 0.01% methylated-BSA in carbonated buffer (0.05 M, pH 9.6), sealed and incubated overnight at 4°C in a humid chamber to improve anti-dsDNA antigen binding. The following day, the liquid was aspirated. The plates were washed once with PBS-T, and anti-dsDNA antigens were added. Then the plates were sealed and incubated for 1.5 hours at 37°C. After aspirating the antigens, the plates were washed five times with PBS-T, and nonspecific sites were blocked with PBS 3%—BSA by incubating for 1.5 hours at 37°C. The assay was continued from this point as described for the cytokines below.

Determination of Cytokines in the Sera
The cytokine concentrations in the sera were determined by ELISA utilizing mouse cytokine kits purchased from Genzyme Diagnostics (Cambridge, MA). The kit protocols were followed. Briefly, Nunc-Maxisorb 96 well ELISA plates were coated with specific anti-mouse capture antibodies in a coating buffer and incubated at 4°C overnight. The following day, capture antibodies were aspirated, the plates were washed with PBS-T [phosphate-buffered saline (PBS—0.01 M, pH 7.3)—0.05% Tween-20]. The plates were incubated with 240 µL of blocking buffer [PBS (0.01 M, pH 7.3)—3% BSA] for two hours at 37°C. Blocking buffer was aspirated from the wells, and appropriately diluted samples or standards (final volume 100 µL) were added to test wells in duplicates and incubated overnight at 4°C. The wells were washed and the secondary antibodies were added and incubated for 60 minutes at 37°C. After washes, 100 µL detection reagent [Horseradish peroxidase-conjugated Streptavidin] was added and incubated for 15 minutes at 37°C. The wells were washed, and then 100 µL substrate [3, 3', 5, 5'-tetramethylbenzidine and hydrogen peroxide solution] was added to the wells. The reaction was then stopped by the addition of 50 µL of 1 M sulfuric acid. The intensity of the yellow color was read at 450 nm in a microplate reader (EL311s, Bio-Tek Instruments Inc., Winooski, VT). The mean absorbance of the standards and samples was calculated. Absorbance was corrected by subtracting background from the mean values of the samples and other standards. A standard curve was constructed to quantitate the concentration of specific cytokines in the samples.

Determination of Serum Lipid Mediators (Prostaglandin E2, Leukotriene B4 and Thromboxane B2)
Serum PGE2, TXB2 and LTB4 levels were determined by ELISA using kits purchased from Neogen Corporation (Lexington, KY). The kits were supplied with 96 well MaxiSorpTM Nunc microplates precoated with anti-PGE2, TXB2 or LTB4 antibodies. To extract PGE2 or TXB2 from the sera, 20 µL of methanol was added to 100 µL of serum, vortexed and centrifuged at 1000xg for five minutes, and the supernatants were collected. To extract LTB4 from the sample, 60 µL of serum was acidified to pH 3.5 with HCl. The lipid mediators were purified by passing them through C18 Sep-Pak® columns (Waters® Corporation, Milford, MA), eluting with methyl formate and evaporating under a stream of nitrogen. Appropriately diluted samples or standards (50 µL) and diluted PGE2-, LTB4- or TXB2-horseradish peroxidase (50 µL) were added to the test wells. The plates were incubated at room temperature for one hour, and the wells were washed with EIA wash buffer. Then 150 µL of substrate (TMB and hydrogen peroxide solution) were added. The reaction was stopped by adding 50 µL of 1 M HCl. The intensity of the yellow color was read at 450 nm in a microplate reader. Mean absorbance of the standards and samples were calculated. The corrected absorbance was calculated by subtracting background (mean O.D. of the zero ng/mL standard) from the mean values of the samples and other standards. A standard curve was constructed to quantitate the concentration of cortisol or estradiol in the samples.

Statistical Analysis
The values are presented in mean±SEM. Statistical analyses of the data were carried out using Statview 4.0/Super ANOVA package software (Abacus Concepts, Berkeley, CA). Data were analyzed by 3-way analysis of variance using strain, oil and vit-E as main factors. Where a significant F ratio was found P<0.05, Fisher’s PLSD (protected least significant difference) test was used to describe differences in the means among groups (p<0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSION
 REFERENCES
 
The effects of feeding CO and FO base-diets (at 10% level) containing low or high levels of vit-E for 4.5 months to MRL/lpr and MRL/++ mice on the body weights, serum anti-dsDNA antibodies, cytokines, and lipid mediators were determined.

Body Weights
The MRL/lpr mice developed lymphadenopathy and enlarged lymph nodes. The body weights and body weight gain of the mice fed CO and FO with low- and high-levels of vit-E (for 4.5 months) are presented in Figs. 1 and 2. Body weights were significantly lower in the MRL/lpr compared to the MRL/++ mice. In the control mice (MRL/++), the body weights were similar, except that the group fed CO-500 IU vit-E diet had lower body weights compared to the other MRL/++ groups. The body weight gain at 4.5 months was significantly higher when CO 500 IU vit-E and FO-500 IU vit-E diets were fed to the MRL/lpr and MRL/++ mice, with the exception of the CO-500 IU vit-E diet fed MRL/++ group. Though the food consumption was similar among all the groups, the MRL/lpr group fed CO-75 IU vit-E was unable to gain as much weight as the other groups. Though this observation is hard to explain, it is reasonable to speculate that there could be hormone/cytokine-related catabolic events occurring relatively earlier in these mice than in the MRL/lpr mice on the other diets. The enlarged lymph nodes were also higher in this group.



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  		Fig. 1. Effects of dietary lipids and vit-E on body weights of MRL/lpr and MRL/++ mice. Values are Mean±SEM, n=10 mice/group, CO: Corn oil: FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 


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  		Fig. 2. Effects of dietary lipids and vit-E on body weight gain of MRL/lpr and MRL/++ mice. Values are Mean±SEM, n=10 mice/group. Means with different superscripts are significantly different at p<0.05 as revealed by Fisher’s PLSD test; CO: Corn oil; FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 
As the lymphoproliferative (lpr) gene is overexpressed in the MRL/lpr mice, these mice suffer from lymphadenopathy and accelerated aging. The MRL/lpr mice suffered from enlarged lymph nodes. The body weight gain of the mice fed CO and FO with low- and high-levels of vit-E (for 4.5 mo) are presented in Fig. 1. Generally the body weight gain at 4.5 months was higher in groups fed diets containing high levels of vit-E, with the exception of the CO-500 IU Vit-E fed MRL/++ group. The body weight gain in the CO-75 IU vit-E diet fed MRL/lpr group was significantly lower than that of the other MRL/lpr groups. In the MRL/++ groups, the weight gain in mice fed the FO-500 IU vit-E diet was significantly higher compared to the CO-75 IU vit-E diet fed MRL/++ group.

When the MRL/lpr mice were nine weeks old, all the mice were surviving, but the mice fed the CO-75 IU vit-E and FO-500 IU vit-E diets exhibited enlarged lymph nodes (Table 3). Fifty percent of the mice in the CO-75 IU vit-E diet had enlarged lymph nodes at nine weeks, while those in the 500 IU vit-E diets did not exhibit visible signs of enlarged lymph nodes. By eleven weeks of age, only 90% of mice were surviving in the 75 IU vit-E groups, while 100% of the mice were surviving in the 500 IU vit-E groups. At thirteen weeks, most of the mice in the 75 IU vit-E groups had enlarged lymph nodes, while there were no visible signs of enlarged lymph nodes in the FO-500 IU vit-E diet group, suggesting {omega}-3 fatty acids containing 500 IU vit-E levels of vit-E may offer protection against enlargement of lymph nodes in the MRL/lpr mouse model.


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  		Table 3. Effects of Dietary Lipids and Vit-E Levels on Enlargement of Lymph Nodes and Survival in MRL/lpr Mice

 
Pathology of Kidneys
Compared to the MRL/++ mice, the kidneys of MRL/lpr mice exhibited mild pathology (Table 4). The glomeruli of the MRL/lpr mice had mild proliferation of mesengial segments and thickening of glomerular basement membrane, the vessels had mild lymphoid infiltrates, and there was 0 to 20% atrophy of the intestitium.


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  		Table 4. Effects of Dietary Lipids and Vit-E Levels on Histopathology of Kidneys in MRL/lpr Mice

 
Serum Anti-dsDNA Antibodies
The effect of CO-75 IU vit-E (for 4.5 months) on serum anti-dsDNA antibody titers is presented in Fig. 3a. The antibody titers were significantly lower in sera from CO-75 IU of vit-E diet fed MRL/++ mice than in the MRL/lpr mice. The effect of CO and FO diets containing 75 IU and 500 IU of vit-E on serum anti-dsDNA antibodies are presented in Fig. 3b. ANOVA revealed significant differences of serum anti-dsDNA antibodies levels between mouse strains (p<0.0001). The MRL/lpr mice had significantly higher levels of anti-dsDNA antibodies than MRL/++ mice did.



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  		Fig. 3. Effects of dietary lipids and vit-E on serum anti-dsDNA levels in MRL/lpr and MRL/++ mice. (3a. Titration curve, the differences between the two groups were statistically significant; 3b. Effects of strain and diets). Values are Mean±SEM, n=8–10 mice/group. Means with different superscripts are significantly different at p<0.05 as revealed by Fisher’s PLSD test; CO: Corn oil; FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 
Serum Cytokines
Interleukin-2 and Interferon-{gamma}.
The serum IL-2 levels ranged between 3.5 and 9 pg/mL (Fig. 4). Dietary oils, vit-E levels and strain had no significant effect on serum IL-2 levels. The serum IFN-{gamma} levels ranged between 30 and 100 pg/mL (Fig. 4). There was a significant effect of mouse strain (p<0.0345) on IFN-{gamma} levels. CO-75 IU vit-E diet fed MRL/++ mice had the highest levels of serum of IFN-{gamma} compared to other groups, except for the FO-500 IU vit-E diet fed MRL/++ mice. The FO-500 IU vit-E diet fed MRL/++ mice had significantly higher levels of serum IFN-{gamma} (p<0.0142) than FO-75 IU vit-E diet fed MRL/lpr mice.



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  		Fig. 4. Effects of dietary lipids and vit-E on serum IL-2 and IFN-g levels in MRL/lpr and MRL/++ mice. Values are Mean±SEM, n=10 mice/group. Means with different superscripts are significantly different at p<0.05; CO: Corn oil; FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 
Interleukin-4, Interleukin-10 and Interleukin-12.
The effects of dietary lipids and vit-E on the levels of IL-4, IL-10 and IL-12 are presented in Fig. 5. The levels of IL-4 in the serum ranged between 6 and 16 pg/mL. ANOVA revealed a significant effect of strain (p<0.0028) on the serum IL-4 levels. The serum IL-4 levels were higher in MRL/lpr mice than MRL/++ mice in all groups.



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  		Fig. 5. Effects of dietary lipids and vit-E on serum IL-4, IL-10 and IL-12 levels in MRL/lpr and MRL/++ mice. Values are Mean±SEM, n=8–10 mice/group. Means with different superscripts are significantly different at p<0.05; CO: Corn oil; FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 
The IL-10 levels ranged between 500 and 10,000 pg/mL in serum. Strain (p<0.0001), oil (p<0.0035) and vit-E levels (p<0.0149) had significant effects on serum IL-10 levels. MRL/lpr mice had higher levels of serum IL-10 than MRL/++ mice (7 to 12 fold). Both FO and diets containing 500 IU of vit-E also decreased serum IL-10 levels. In MRL/lpr mice, both FO and diets containing 500 IU vit-E also decreased serum IL-10 levels to levels similar to that of MRL/++ mice suggesting that FO-500 IU vit-E may attenuate the levels of IL-10 in MRL/lpr mice.

The serum IL-12 levels ranged between 1,800 and 17,000 pg/mL. Serum IL-12 levels were significantly higher (3 to 7 fold; p<0.0001) in MRL/lpr mice than in MRL/++ mice in all the groups. Both dietary oils (p<0.0001) and vit-E levels (p<0.0022) had significant effects on serum IL-12 levels. Both 500 IU of vit-E and FO were effective in significantly lowering the levels of proinflammatory cytokine IL-12. A significant interaction between dietary oils and mouse strain was observed (p<0.0029).

Interleukin-1ß, Interleukin-6 and Tumor Necrosis Factor-{alpha}.
The serum IL-1ß levels ranged between 25 and 150 pg/mL (Fig. 6). When serum IL-1ß levels were analyzed by ANOVA, a significant effect of vit-E levels (p<0.0074) was observed. Serum IL-1ß levels were lower in the groups fed 75 IU vit-E-based diets. The serum IL-1ß levels were higher in CO-500 IU vit-E diet fed MRL/lpr mice but were lower in all other groups of MRL/lpr mice. CO-500 IU vit-E diet fed MRL/lpr mice had the highest level of serum IL-1ß when compared to the other groups.



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  		Fig. 6. Effects of dietary lipids and vit-E on serum IL-1ß, IL-6 and TNF-{alpha} levels in MRL/lpr and MRL/++ mice. Values are Mean±SEM, n=8–10 mice/group. Means with different superscripts are significantly different at p<0.05; CO: Corn oil; FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 
The serum IL-6 levels ranged between 90 and 1200 pg/mL (Fig. 6). ANOVA revealed significant effects of dietary oils (p<0.0029), vit-E (p<0.0178), and strain (p<0.0062) on serum IL-6 levels. There was an interaction between strain and vit-E (p<0.0178). Serum IL-6 levels were higher in the MRL/lpr mice than in the MRL/++ group, while the significant differences could be found only in the CO-75 IU vit-E (10 fold) and FO-75 IU vit-E (6 fold) diet fed groups. CO-LE fed MRL/lpr mice had the highest serum IL-6 levels among all diet groups. Diets containing 500 IU of vit-E further lowered the levels of serum IL-6 in the MRL/lpr mice.

The serum TNF-{alpha} levels ranged between 70 and 600 pg/mL (Fig. 6). ANOVA revealed significant effects of dietary oil (p<0.0067), vit-E levels (p<0.002) and strain (p<0.0001) on serum TNF-{alpha} levels. MRL/lpr mice fed CO-75 IU vit-E diet had significantly higher levels of serum TNF-{alpha} than all the other groups, except for the FO-75 IU vit-E diet fed mice. CO-75 IU vit-E diet fed mice had the highest levels of serum TNF-{alpha}. Significant interactions were observed between oil and strain (p<0.006) and vit-E and strain (p<0.0025). The FO-500 IU vit-E diet lowered serum TNF-{alpha} levels similar to that of MRL/++ mice.

Serum Lipid Mediators (Prostaglandin E2, Leukotriene B4 and Tromboxane B2) levels
The effects of diets on serum PGE2, LTB4 and TXB2 levels are presented in Fig. 7. The serum PGE2 levels ranged between 45 and 150 ng/mL. The serum PGE2 levels were significantly lower in the groups fed FO (p<0.0295). There was also an interaction between vit-E and strain for serum PGE2 levels (p<0.0489). The serum LTB4 levels ranged between 95 and 220 ng/mL. ANOVA revealed a significant effect of dietary oils (p<0.04) on the serum LTB4 levels. The serum TXB2 levels ranged between 12 and 30 ng/mL. ANOVA revealed a significant effect of dietary oils (p<0.0069) on the serum TXB2 levels. The levels of PGE2, LTB4 and TXB2 were lower in MRL/lpr mice fed FO-500 IU vit-E than in MRL/lpr mice fed FO-75 IU vit-E, though the difference was not statistically significant.



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  		Fig. 7. Effects of dietary lipids and vit-E on serum lipid mediators (PGE2, LTB4 and TXB2) levels in MRL/lpr and MRL/++ mice. Values are Mean±SEM, n=8–10 mice/group. Means with different superscripts are significantly different at p<0.05; CO: Corn oil; FO: Fish oil; vit-E-75 IU and vit-E-500 IU refer to vit-E levels/kg diet.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSION
 REFERENCES
 
In the present study, we investigated the effects of FO containing two levels of vit-E (75 IU and 500 IU/kg diet) on serum cytokines, lipid mediators and anti-DNA antibodies in MRL/lpr mice. Data from this study indicated that both FO and FO plus 500 IU of vit-E delayed enlargement of lymph nodes in the MRL/lpr mice. The MRL/lpr mice had a very different cytokine and inflammatory mediators profile from that of the MRL/++ mice (congenic control). Several of these factors may contribute to the onset of autoimmune disease. The levels of anti-DNA antibodies, IL-4, IL-6, TNF-{alpha}, IL-10 and IL-12 were higher in the sera of the MRL/lpr mice. Feeding FO-based diet (containing 75 IU vit-E) lowered the levels of these cytokines, PGE2, LTB4 and TXB2. Increasing the level of vit-E to 500 IU in the FO diet further lowered the levels of IL-6, TNF-{alpha}, IL-10, IL-12 in the sera of the MRL/lpr mice, suggesting that adding 500 IU of vit-E may offer additional protection in delaying autoimmune disease in these mice.

Inflammation is a prominent feature of the autoimmune phenomena that lead to tissue injury. RA is associated with significant and consistent changes in serum levels of inflammatory cytokines and soluble receptors [23]. Overproduction of cytokines that activate inflammatory cytokines and a deficiency of cytokines that normally suppress the inflammation pathway have been associated with autoimmune disease. Type-1 cytokines such as IL-2, IFN-{gamma}, IL-12 promote cell-mediated immunity while type-2 cytokines such as IL-4, IL-5, IL-6, IL-10, IL-13 promote humoral immunity. Th1 and Th2 cells develop opposing patterns of responsiveness to the cytokines IL-12 and IFN-{gamma}, with Th1 retaining only IL-12 responsiveness [24]. The balance of Th1/Th2 cytokines is thought to have a role in the progression of many autoimmune diseases. Dietary FO may dramatically downregulate key immunoregulatory cytokines involved in autoimmune disease [22].

In the present study, serum IFN-{gamma} levels were higher in the sera of MRL/++ mice with the exception of the CO-500 IU vit-E diet fed MRL/++ group. It is difficult to explain the reasons for IFN-{gamma} levels’ being higher in MRL/++ mice, yet IL-12 levels in these mice were low. Though IL-12 is a very potent inducer of IFN-{gamma}, it is known that IL-12 is more potent in counteracting IL-10 production than IFN-{gamma} [25]. In our studies, IL-12 and IL-10 levels followed a similar trend, the levels were higher in MRL/lpr sera. Decreased levels and activities of IFN-{gamma} in the rheumatoid joint environment in vitro and in vivo have been reported [26]. IFN-{gamma} is an important mediator of the immune system, having profound effects on immune regulation, on the activities of T cells, B cells and NK cells and on inflammation. It is involved in the upregulation of the expression of MHC class II antigens in a wide variety of cells and accelerates development of autoimmunity in B/W mice [27]. Both increased production of IFN-{gamma} following consumption of {omega}-3 fatty acid [2] and no changes in IFN-{gamma} levels [28] have been reported.

It is not known whether decreased levels of IFN-{gamma} occurs due to a defective production or to a physiological downregulation of IFN-{gamma} in RA. Blood leukocytes of tested patients generated in vitro a reduced amount of Staphylococcus enterotoxin A-induced IFN-{gamma} and virus-induced acid-labile IFN-{gamma}, suggesting impaired functioning of T and B blood cells in autoimmune disease [29]. The production of IFN-{gamma} by peripheral blood lymphocytes in RA was lower than in healthy subjects [30]. Production of IFN may contribute to immunologic aberrations in autoimmune disease and also may protect the already compromised host from viral infections [31].

In the present study, we observed that serum IL-2 levels of the MRL mice were not affected by dietary oil, strain and vit-E levels. Though IL-2 is generally considered antiinflammatory, it could be proinflammatory especially in RA. There have been few animal studies on the effects of dietary lipids on lymphocyte-derived cytokines other than IL-2. Feeding rats on a diet containing 20% FOP decreased proportion of spleen and thymic lymphocytes bearing IL-2R (CD25) following Con-A stimulation [32]. FO is reported to decrease IL-2R expression following mitogenic stimulation [33]. Feeding FO to autoimmune-prone mice resulted in elevated levels of mRNA for IL-2, IL-4 and TGF-ß and reduced levels of mRNA for c-myc and c-ras in the spleen [22].

Our data suggest that dietary oil does not have a significant effect on serum IL-4 levels of MRL mice. Feeding CO- and FO-based diets did not alter IL-4 production by spleen cells of B/W mice [4]. These observations are similar to those reported earlier [22]. Minimal effects of dietary fat on production of IL-4, IL-10 and IFN-{gamma} have been reported [22,34]. IL-4 generally produced by T cells seems to exhibit a coordinated anti-inflammatory action [35]. In synoviocytes, IL-4 is reported to block IL-1-induced PGE2, but increase IL-6 production [36]. IL-4 acts in concert to induce activated B lymphocytes to grow, switch isotypes and, ultimately, differentiate into antibody-producing plasma cells. IL-4 is reported to inhibit the secretion of pro-inflammatory cytokines by monocytes/macrophages and neutrophils and enhance the presentation of antigens by monocytes/macrophages and dendritic cells.

In the present study, we observed higher levels of serum IL-4, IL-10 and IL-12 in MRL/lpr mice than in MRL/++ mice. The present study indicated that MRL/lpr mice had higher levels of IL-12 than MRL/++ mice. We observed that FO diet containing 500 IU of vit-E lowered the serum IL-12 levels. There is no information available on the effects of dietary lipids or antioxidants on IL-12 levels at the present time. Continuous treatment of lupus-prone B/W mice with anti-IL-10 antibodies is reported to substantially delay the onset of autoimmunity [37]. Our results showed that FO and 500 IU of vit-E lowered the serum IL-10 levels in MRL/lpr mice. IL-10 is produced by subsets of activated T cells, B cells and macrophages which mediate a variety of both immunostimulatory and immunosuppressive properties in the mouse and human in vitro [38]. IL-12 plays a pivotal role in cell-mediated inflammatory reactions. IL-12 has a crucial role in promoting proliferation and differentiation of Th1 cells [39] and is involved in inflammation and in the arthritic cytokine cascade. Although it is not entirely clear to what extent IFN-{gamma} mediates the effects of IL-12, it is known that IL-12 is more potent in counteracting IL-10 production than IFN-{gamma} [40].

The data from our study indicated that both FO and 500 IU of vit-E in the diet lowered the TNF-{alpha} and IL-6 levels in MRL/lpr mice, but not IL-1ß levels. The MRL/lpr mice had higher levels of serum IL-6 and TNF-{alpha} than MRL/++ mice. Overproduction of IL-1, IL-6 and TNF-{alpha} has been implicated in the pathogenesis of several inflammatory diseases such as RA. Generally high levels of IL-6 are detected in synovial fluid and in sera of patients with RA. The synovial membrane contains IL-1, TNF-{alpha}, TNF receptors and IL-6 [41–43], while IL-10 and TNF-ß are produced in RA joints [44,45]. Data from animal and human studies have demonstrated that vit-E has beneficial effects on symptoms of arthritis [46,47]. Feeding {omega}-3 PUFA-containing oils to rodents is reported to enhance production of TNF by macrophages [48] or decrease production [49] or have no effect [50]. FO feeding may decrease LPS-stimulated IL-6 production [51] by thioglycollate-elicited peritoneal macrophages. FO feeding may decrease IL-1ß mRNA levels in LPS or PMA-stimulated lymphocytes [52]; and decrease levels of IL-1ß and TNF-{alpha} mRNA levels [53].

FO may ameliorate clinical symptoms of RA patients by lowering the levels or proinflammatory cytokines, such as IL-1ß, IL-6 and TNF-{alpha}. These cytokines mediate the response of the host to inflammatory stimuli and can trigger cells to secrete other inflammatory mediators, including other cytokines such as the T lymphocyte products IFN-{gamma} and IL-2 and lipid mediators such as PGs, LTs and platelet-activating factor [54]. The cartilage destructive process is mainly IL-1 driven [55]. In addition to TNF-{alpha} and IL-1 action, the destructive process appears to be under the control of mediators such as IL-6 and IL-10. It is suggested that IL-1-{alpha}, EGF and TGF-ß may be important in modulating the contribution of the intracellular and extracellular route of collagen breakdown.

Patients taking dietary supplements of FO exhibit improvements in clinical parameters of disease activity from baseline, including the number of tender joints, and these improvements are associated with significant decreases in levels of IL-1ß from baseline [56]. Some patients who were taking FO supplements were able to discontinue NSAIDs without experiencing a disease flare. The anti-inflammatory effect of {omega}-3 fatty acids could, in part, be explained by their ability to decrease the production of proinflammatory cytokine [57].

The levels of PGE2, LTB4 and TXB2 were lower in MRL/lpr mice fed FO-500 IU vit-E than in MRL/lpr mice fed FO-75 IU vit-E, though this was not statistically significant. Our observations [58,59] regarding FO’s lowering the levels of PGE2, LTB4 and TXB2 are similar to those reported by other investigators. As cytokine production is regulated by eicosanoids and, since dietary lipids effect eicosanoid production, it is possible that dietary lipids containing {omega}-3 PUFA will affect cytokines production [22,60].

Anti-dsDNA antibodies are known to serve as prognostic markers for the onset and severity of renal disease. Marine {omega}-3 fatty acids and vit-E might exert some protection against autoimmune disease [4,22]. In the present study, the MRL/lpr mice had high levels of serum anti-dsDNA antibodies, and FO or 500 IU of vit-E had no effect on serum anti-dsDNA antibody levels. FO is reported to lower serum anti-DNA antibody levels in NZB/NZW mice [22]. {omega}-3 fatty acids may offer protection against autoimmune disease through several mechanisms. The exact mechanism or mechanisms involved in delaying autoimmune disease by FO are still not clear, although several possibilities have been suggested [7,8,61]. In autoimmune diseases, an abnormal production of proinflammatory cytokines, or a reduced inhibition of their actions, may lead to an imbalance [1]. They may alter the production of mediators (eicosanoids, cytokines, nitric oxide) involved in communication between cells of the immune system and to alter the expression of key cell-surface molecules involved in the direct cell-to-cell contact (adhesion molecules). The production of cytokines may be regulated by eicosanoids-dependent or eicosanoid-independent manner. One such mechanism could be through regulating expression of key genes involved in immune cell functioning and in the production of immune cell-derived mediators.

Changing the fatty acid composition of the diet markedly alters the phospholipid and diacylglycerol molecular species compositions of lymphocytes [62] and macrophages [63]. Lymphocytes and other immune and inflammatory cells contain many transcription factors, various oncogene products, steroid hormone receptors and specific nuclear factors such as NF-IL-2, NF-IL-6 and NF-ICAM-1. NF{kappa}B plays a key role in inducing production of many key mediators within the immune system, such as regulating the synthesis of cytokines, including IL-1, IL-2, IL-6, TNF-{alpha} and IFN-ß, of cytokine receptors including IL-2R, of adhesion molecules, of enzymes involved in mediator generation, such as inducible NO synthase, and of a range of acute-phase proteins.

The observations from this study suggest that FO may be providing protection against autoimmune disease by decreasing the levels of serum IL-6, IL-10, IL-12, TNF-{alpha} and proinflammatory lipid mediators. Adding 500 IU of vit-E to FO diet offered additional protection by lowering the levels of IL-6, IL-10, IL-12 and TNF-{alpha}. It is not clear at this point how these cytokines may be accelerating lymphoproliferative disease in the MRL/lpr mice. It is reasonable to speculate that the mechanism of action of both FO and vit-E may be at the level of altering gene expression and altering the levels of mRNA for these cytokines. Alternatively it may be altering the activity of nuclear factors which are important for cytokines. FO may also be exerting its effects through altering signal-related mechanisms. Further research is required in this area to explore the action of FO and vit-E in protecting against this autoimmune phenomena.


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSION
REFERENCES
 
Data from the present study indicate that the MRL/lpr mice had higher levels of serum pro-inflammatory cytokines (IL-4, IL-6, IL-10, IL-12 and TNF-{alpha}) than MRL/++ mice. Both FO and 500 IU of vit-E significantly reduced the levels of these cytokines. In addition, FO also lowered the levels of PGE2, TXB2 and LTB4 in sera of MRL/lpr mice. It is clear from our observations that FO and vit-E are beneficial in modulating the levels of specific cytokines and, thereby, may affect the immune system and the onset of autoimmunity. In the MRL/lpr mouse model, the lpr gene is overexpressed at very early stages of life. The role of dietary intervention with {omega}-3 lipids and vit-E in delaying the early expression of the lpr gene remains to be investigated. It is evident from the present study that 500 IU of vit-E in the FO diet may have additional benefits in delaying enlargement of lymph nodes and decreasing abnormal levels of specific proinflammatory cytokines in the MRL/lpr mice. Studies in the future should address the role of individual {omega}-6 and {omega}-3 fatty acids on autoimmune disease, the role of FO and vit-E ligand in apoptosis-related mechanisms and molecular aspects of the pro- and antiinflammatory cytokines, the role of cytokine receptors, soluble receptors, inhibitors and oncogenes in the MRL/lpr mouse model. The observations from this study may form the basis for future studies on selective nutritional interventions based on specific fatty acids and antioxidants in delaying the progress of autoimmune diseases, particularly in RA patients.


   ACKNOWLEDGMENTS
 
This research was funded by National Institute of Arthritis and Musculoskeletal and Skin Disorders grant IR15AR/AI43517. Wei-chia Chu was recipient of Mark Diamond Research Funds for graduate research. The authors wish to acknowledge Dr. David Pendergast, Professor, Department of Physiology and Biophysics, SUNY at Buffalo, for reviewing the manuscript.


   FOOTNOTES
 
Presented in part at the Experimental Biology ’98 meeting in San Francisco, CA.

Abbreviations: CO=corn oil; FO=fish oil, IFN-{gamma}=interferon-{gamma}, IL=interleukin, LTB4=leukotriene B4, lpr=lymphoproliferative, NSAIDS=non-steroidal antiinflammatory drugs, PGE2=prostaglandin E2, RA=rheumatoid arthritis, SLE=systemic lupus erythematosus, TNF-{alpha}=tumor necrosis factor-{alpha}, vit-E=vitamin E.

Address reprint requests to: Jaya T. Venkatraman, Ph.D., Associate Professor, Nutrition Program, Department of Physical Therapy, Exercise and Nutrition Sciences, State University of New York at Buffalo, 15 Farber Hall, Buffalo, NY 14214.

Received November 1, 1998. Accepted July 1, 1999.


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