![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
,**2
*
Department of Food Science and Human Nutrition,
Department of Microbiology and Molecular Genetics, and
**
Institute for Environmental Toxicology, Michigan State University, East Lansing, MI 48824
2To whom correspondence should be addressed. E-mail: pestka{at}pilot.msu.edu.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • fish oil • (n-3) polyunsaturated fatty acids • IgA nephropathy • kidney • trichothecene • vomitoxin • mice
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
,2
). The spectrum of (n-3) PUFA activities is broad and includes suppression of lymphocyte proliferation, cytotoxic T lymphocyte activity, natural killer cell activity, macrophage-mediated cytotoxicity, neutrophil/monocyte chemotaxis, major histocompatibility complex II expression and antigen presentation. Evidence that these specific cellular effects indeed affect immune function in vivo is reflected in experimental attenuation of acute inflammatory responses, cytokine responses to endotoxin, delayed-type hypersensitivity, graft vs. host/host vs. graft responses and allograft rejection by (n-3) PUFA. The mechanistic basis for the immune effects of (n-3) PUFA may relate to their capacity to modulate cytokine and inflammatory mediator profiles, cell-to-cell contact, signal transduction and apoptosis in immune cell populations (1
–3
).
The observation that (n-3) PUFA attenuate untoward immune responses has led to consideration of their use in therapeutic regimens for human autoimmune and inflammatory diseases. In support of this contention, epidemiologic studies have shown that consumption of FO correlates with very low incidences of autoimmune and inflammatory disorders as well as decreased cardiovascular disease (4
). The efficacy of FO supplementation in several animal models has been demonstrated by diminished joint inflammation in collagen-induced arthritis (5
), by depressed proteinuria and autoantibodies as well as increased life span in murine models of systemic lupus arthritis(6
,7
) and by reduced inflammation in rat colitis models (8
). Several clinical trials suggest benefits of FO ingestion in reducing symptoms in autoimmune and inflammatory diseases. Specifically, FO reduces the severity of rheumatoid arthritis (9
), immune-related skin disease (10
), multiple sclerosis (11
) and systemic lupus erythmatosous(12
). One disease in which FO holds particularly great promise is immunoglobulin A nephropathy (IgAN).
Human IgAN is the most common type of glomerulonephritis in the world (13
). The diagnostic hallmark of this autoimmune disease is diffuse mesangial deposition of IgA in the kidney glomerulus. Approximately 150,000 people in the United States have been diagnosed with IgAN with nearly 4000 new cases occurring each year (14
). Between 20 and 40% of these will develop progressive renal failure over a 25 y period from the time of initial diagnosis, with 1–2% of adult patients entering end-stage renal failure yearly and requiring hemodialysis or kidney transplantation (15
,16
). Development of hypertension, persistent hematuria, proteinuria and impaired renal function at diagnosis are associated with rapidly progressive IgAN (17
). IgAN accounts for up to half of glomerulopathies in Japan where yearly urinalyses are run on all school children and adolescents (18
,19
). In European countries, this rate is estimated to range from 20 to 35% and in North America from 5 to 10%. These estimated rates are strongly influenced by the kidney biopsy policies of the various countries (i.e., higher biopsy rates in Japan correlate with higher reported IgAN incidence). Etiologic factors associated with IgAN include prior mucosal infections (13
,20
), genetic predisposition (21
), diet (22
) and environmental agents such as mycotoxins (23
,24
).
At the present time, there is no consensus on how to best treat human IgAN because corticosteroids, cyclosporine, anticoagulants, antiplatelet drugs and phenytoin have negligible or questionable outcomes (25
). Donadio (26
) proposed that dietary supplementation with FO may benefit patients with immune-related renal diseases including IgAN, lupus nephritis and cyclosporine-induced nephrotoxicity. In support of this hypothesis, Holmon et al. (27
) found that the level of (n-3) PUFA in plasma phospholipids was lower in IgAN patients than controls. Furthermore, Wakai et al. (28
) reported in a Japanese case control study that dietary (n-3) PUFA were negatively associated with the risk of IgAN. Definitive support of Donadio’s hypothesis comes from a multicenter, placebo-controlled, randomized trial of FO in IgAN in which the Mayo Nephrology Collaborative Group (15
,17
) reported that supplementation of patients with daily doses of 12 g of FO containing 1.9 g of eicosapentaenoic acid [20:5(n-3), EPA] and 1.4 g of docosahexaenoic acid [22:6(n-3), DHA] resulted in a significant reduction in the development of end stage renal failure over a 6.4-y period. In similar fashion, other randomized clinical trials evaluating efficacy of FO in treating IgAN showed benefits (29
,30
). There is some controversy over these findings because at least two other earlier FO trials showed no benefits from (n-3) PUFA ingestion (31
,32
). The lack of effect might relate to inherent problems with population size, time of follow-up, lack of hypertension control or stage of disease studied (17
). Thus, although dietary FO supplementation appears to be a promising therapeutic regimen for IgAN, further research is warranted both to establish the mechanistic basis for these effects and to determine the optimal dosing regimens for EPA and DHA. Some of these efforts might best be approached through an animal model.
A number of experimental animal models have been developed to elucidate pathogenetic mechanisms of IgAN (33
). Approaches have included injection of IgA immune complexes (IgA-IC), mucosal immunization, viral infection and use of mice genetically prone to mesangial IgA deposition. While evaluating the immunotoxic potential of vomitoxin (VT, deoxynivalenol), a trichothecene mycotoxin produced by Fusarium and a food contaminant, our laboratory discovered that mice fed experimental diets containing this mycotoxin developed the early characteristic features of human IgAN including elevated serum polymeric IgA and IgA-IC as well as mesangial IgA deposition (34
,35
). These effects persisted for up to 3 mo after removal of VT from diet (36
). Concurrent with these effects, VT-exposed mice exhibit increased numbers of membrane IgA+ cells and IgA-secreting cells in Peyer’s patches and spleens (37
–39
), thus suggesting that this mycotoxin promotes excessive IgA secretion. The mechanisms underlying this model have been extensively studied by our group and appear to involve transcriptional and post-transcriptional regulation of cytokine gene expression. These cytokines promote differentiation of IgA-secreting cells and, ultimately, systemic overproduction of IgA (40
).
In view of the suggested efficacy of treating human IgAN with FO supplementation, it appeared reasonable to evaluate this modality in the VT. The purpose of this study was to test the hypothesis that replacing corn oil (CO) with FO in the rodent diet can suppress VT-induced elevation of serum IgA, serum IgA-IC and kidney mesangial IgA deposition in mice. The results suggest that diets containing FO attenuate IgA dysregulation in this model, thus providing a new window for studying the underlying mechanisms by which (n-3) PUFA consumption might suppress IgAN at its earliest stages.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
All chemicals (reagent grade or better) were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. The VT used in this study was produced in Fusarium graminearum R6576 cultures and purified by the water-saturated silica gel chromatography method of Witt et al. (41
). Purity of VT was verified by a single HPLC peak occurring at 224 nm. Concentrated toxin solutions were handled in a fume hood. Labware that was contaminated with mycotoxin was detoxified by soaking for >1 h in 100 mL/L sodium hypochlorite (42
). Purified VT was added to powdered diets as described previously (35
).
Animals.
Male B6C3F1 mice (8–10 wk old) were obtained from Charles River (Portage, MI). Mice were housed singly in environmentally protected cages which consisted of a transparent polycarbonate body with a filter bonnet, stainless steel wire lid and a layer of heat-treated hardwood chips. The mice were allowed to acclimate for at least 7 d to their new housing, regulated temperature (25°C), feed, 12-h light:dark cycle and to a negative-pressure ventilated area before feeding regimens began. All animal handling was conducted in accordance with guidelines established by the National Institutes of Health. Experiments were designed to minimize numbers of animals required to adequately test the proposed hypothesis and were approved by Michigan State University Laboratory Animal Research Committee.
Study 1 diet and experimental design.
The first mouse diet was predicated on the original formulation (AIN-76A) recommended by the American Institute of Nutrition for rodent feeding studies (43
). The basal diet consisted of the following ingredients per kg: 35 g AIN-76 mineral mix, 10 g AIN-76 vitamin mix, 200 g casein, 150 g cornstarch, 50 g cellulose, 15 g L-cystine, 2 g choline bitartrate, which were obtained from Dyets (Bethlehem, PA) and 500 g sucrose, which was obtained from a local commercial source. Basal diets were amended to yield four experimental diets containing the following (per kg): 1) 50 g CO, (Gordon Food Service, Grand Rapids, MI); 2) 50 g CO, and 10 mg VT; 3) 50 g CO, 10 mg VT, and 9 mg tert-butylhydroquinone (TBHQ); and 4) 5 g CO, 45 g menhaden FO (already containing 200 mg/kg TBHQ as an antioxidant; Dyets) and 10 mg VT. The fatty acid compositions of the CO- and FO-diets are shown in Table 1
.
|
Study 2 diet and experimental design.
The second diet was based on the AIN-93G formulation (44
) and consisted of the following ingredients (per kg): 35 g AIN-93G mineral mix, 10 g AIN-93 vitamin mix, 200 g casein, 397.5 g cornstarch, 132 g Dyetrose (dextrinized cornstarch), 50 g cellulose, 3 g L-cystine, 2.5 g choline bitartrate, 14 mg TBHQ, which were purchased from Dyets, and 100 g sucrose, which was obtained from a local commercial source. CO and menhaden FO (each already containing 200 mg/kg of TBHQ) from Dyets, were used to amend the basal diet to yield three experimental diets containing the following (per kg): 1) 70 g CO; 2) 70 g CO and 10 mg VT; and 3) 10 g CO, 60 g FO and 10 mg VT. The fatty acid compositions of these CO- and FO-containing diets are shown in Table 1
. Mice were fed for 20 wk and body weight monitored daily. Animals were bled at intervals and serum analyzed for IgA, IgG and IgM. After bleeding at wk 20, mice were killed by cervical dislocation. Spleens and Peyer’s patches were removed aseptically for preparing cell cultures. Kidneys of each mouse were removed for immunohistochemical examination.
Measurement of immunoglobulins and IgA-IC.
IgA, IgG and IgM were measured in serum by capture ELISA(35
) using mouse immunoglobin reference serum (Bethyl Laboratories, Montgomery, TX), goat anti-mouse IgA, G and M (heavy chain specific) and peroxidase-conjugated goat IgG fraction to mouse IgA, IgG, IgM (Organon Teknika, West Chester, PA). For detection of IgA-IC, diluted serum samples were precipitated using 70 g/L polyethylene glycol (PEG 6000; Sigma) (45
). IgA in precipitate was redissolved in PBS and quantified by ELISA.
Assessment of kidney mesangial IgA deposition.
At experiment termination, kidneys of each euthanized mouse were removed, cut in half and immediately frozen in liquid nitrogen. Each kidney was sectioned to 7 µm with a cryostat (Reichert-Jung, Cambridge Instruments, Buffalo, NY) and stained for IgA deposition with fluorescein isthiocyanate–labeled goat anti-mouse IgA (Sigma) as previously described (46
). Sections from each animal were viewed under a Nikon Labophot epifluorescence microscope through a Sony (Tokyo, Japan) imaging system consisting of CCD Video Camera DXC-151A and a PVM 13442Q Trinitron Video Monitor. Ten (10) glomeruli from each section were randomly selected and the image captured on a microcomputer using a Snappy Video System (Play Incorporated, Rancho Cordova, CA). Mean fluorescence intensity was determined in polygons encircling the glomeruli using UTHSCSA Image Tool Software V 1.2 (available via anonymous FTP at ftp://maxrad6.uthscsa.edu). This system generates a quantitative value from an encircled immunofluorescent stained glomerulus in a frozen frame and calculates the average brightness for the circled area based on each pixel of the screen included in the circle. The pixels in the circled area were measured on a grayness scale that ranged from 0 (black) to 255 (white).
Cell cultures.
Spleen and Peyer’s patches were teased apart in harvest buffer consisting of 0.01 mol/L PBS, pH 7.4 containing 20 mL/L heat inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY), 1 x 105 U/L penicillin and 100 mg/L streptomycin. Tissues were passed through a sterile 100-mesh stainless screen in the same buffer and cell suspensions held on ice for 10 min to allow settling of tissue particles. Supernatant was removed following centrifugation at 450 x g for 10 min. Erythrocytes were lysed for 3 min at room temperature in 0.02 mol/L Tris buffer (pH 7.65) containing 0.14 mol/L ammonium chloride. Cells were centrifuged, resuspended in RPMI-1640 medium supplemented with 100 mL/L FBS, 1 mmol/L sodium pyruvate, 1 x 105 U/L penicillin, 100 mg/L streptomycin, 0.1 mmol/L nonessential amino acid and 50 µmol/L 2-mercaptoethanol and then counted using a hemacytometer (American Optical, Buffalo, NY). Cells (2 x 108/L) from individual mice were cultured separately in 1 mL of medium in flat-bottomed 24-well tissue culture plates (Fisher Scientific, Corning, NY) at 37°C under 70 g/L CO2 in a humidified incubator. Supernatants were collected at 5 d and stored in aliquots at -20°C until analysis for IgA.
Statistics.
Data were analyzed using the Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). Data were subjected to one-way ANOVA and pairwise comparisons made by Bonferroni or Student-Newman-Keuls methods. If data did not meet the normality assumption, they were subjected to Kruskal-Wallace ANOVA on Ranks and pairwise comparisons made by Dunn’s or Student-Newman-Keuls methods. Differences were considered significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Because all previous studies of VT-induced IgAN from our laboratory used the AIN-76A diet, the same formulation was used for an initial evaluation of the ameliorative effects of dietary FO on this model. In addition, because the FO in Study 1 contained 200 mg/kg TBHQ, an additional CO + VT group was included in which the diet was amended with 9 mg/kg TBHQ (equivalent to final TBHQ concentration in FO diet) to control for possible modulation of the model by this antioxidant.
Feed refusal and reduced weight gain are two characteristic effects of VT found in previous feeding experiments with all mammalian species (47
). Consistent with these earlier studies, dietary VT caused marked feed refusal (Fig. 1
). However, no differences were observed among the VT + CO, VT + FO + TBHQ or VT + CO + TBHQ groups, suggesting that diet modulation did not attenuate or potentiate the characteristic anorectic response to VT by these mice. Consistent with feed consumption data, VT-fed groups of mice gained less weight than did their corresponding control (Fig. 2A
). These effects were significant both midway (wk 6) (Fig. 2
B) and at the end (wk 12) (Fig. 2
C) of the experiment. Weight gain of mice fed CO + VT + TBHQ or FO + VT did not differ from those fed CO + VT. Thus, although the presence of VT in the diet reduced both feed consumption and weight gain, these effects were the same among all three dietary treatments containing VT.
|
|
). THBQ supplementation reduced the elevated serum IgA response in mice fed CO + VT at wk 4 but not at wk 8 or 12. In contrast, FO + VT treatment significantly attenuated IgA increases compared with the CO + VT group at wk 4, 8 and 12. Serum IgA-IC concentrations were 2-, 1-, and 0.3-fold higher in mice fed for 12 wk with CO + VT, CO + VT + TBHQ or FO + VT, respectively, (P < 0.05, Fig. 3
B). THBQ supplementation suppressed the serum IgA-IC response in mice fed CO + VT. FO + VT treatment significantly attenuated IgA-IC concentrations compared with both CO + VT and CO + VT + TBHQ groups. The results suggested that replacement of CO with FO could impair VT-induced IgA and IgA-IC elevation, whereas addition of the antioxidant TBHQ alone had marginal or no effects.
|
) compared with mice fed CO (Fig. 4
D). Mesangial IgA fluorescence was slightly reduced in mice fed CO + VT + TBHQ (Fig. 4
B) but more markedly suppressed in mice fed FO + VT (Fig. 4
C). Image analysis of multiple sections from each mouse quantitatively confirmed these qualitative interpretations (Fig. 5
). The results suggested that diet containing FO could attenuate mesangial IgA accumulation and that this effect was not wholly dependent on TBHQ.
|
|
Study 1 indicated that the FO diet could attenuate induction of VT-IgAN and that TBHQ may contribute in part to this effect. A limitation of this study was the use of a generic corn oil, which may have contained unknown amounts of antioxidants. A second study was therefore conducted to verify the effects of FO on VT-induced IgAN and control for TBHQ content. CO and FO employed in Study 2 were purchased from the same supplier and contained identical TBHQ levels, thus eliminating the need for the additional CO + TBHQ treatment group used in Study 1. Two other modifications in Study 2 were as follows: 1) use of AIN-93G diet formulation, which has improved nutritional composition relative to AIN-76 (44
) and 2)increase of minimum corn oil concentration for all diets to 10 g/kg to ensure optimal (n-6) fatty acid intake.
As observed in Study 1, VT impaired body weight increases throughout the feeding period (Fig. 6A
) and these effects were significant at wk 10 and 20 (Fig. 6
B and C). There was no difference in body weight gain between mice fed CO + VT and those fed FO + VT.
|
). Serum IgA concentrations in the CO + VT and FO + VT groups did not differ at wk 4 and 8; they were significantly different at wk 12 through 20. Serum IgA-IC followed similar trends with concentrations that were significantly higher in all VT-treated groups (Fig. 7
B). IgA-IC concentrations in the CO + VT group were consistently but not significantly (P = 0.05) higher than the FO + VT group. Thus, as in Study 1, the FO diet markedly attenuated VT-induced serum IgA elevation, but this was less apparent for IgA-IC.
|
). Therefore, IgA production by ex vivo cultures of these two organs was assessed to determine the effects of FO ingestion. Spleen (Fig. 8A
) and Peyer’s patch (Fig. 8
B) cultures from CO + VT groups produced significantly more IgA than did their corresponding CO groups. Supernatant IgA levels in spleen cultures from the FO + VT group were significantly higher than those of the CO group but were significantly lower than those of the CO + VT group (Fig. 7
A). Supernatant IgA levels in Peyer’s patch cultures from the FO + VT group were not significantly different from the CO group or the CO + VT group (Fig. 8
B). The results suggested that the FO diet impaired VT-enhanced ex vivo production of IgA and that this was more prominent in spleen than in the Peyer’s patch cultures.
|
). Mean glomerular fluorescent intensities for CO + VT and FO + VT groups were 3- and 1.3- fold greater than that of the CO group (P < 0.05). The mean intensity of the FO + VT group was lower than that of the CO + VT group. Thus, as found in Study 1, dietary FO attenuated IgA deposition in kidney.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
–36
,48
,49
), which mimic aspects of the IgA deposition in human IgAN. Our data showing that diet containing FO can attenuate VT-induced elevations in IgA, IgA-IC, mesangial IgA deposition and ex vivo IgA production in the mouse represent unique and important observations that provide a novel window for improved understanding of how this nutritional modality might affect early development of IgAN. From a theoretical perspective, this model might provide insight into specific mechanisms by which (n-3) PUFA can attenuate IgAN as well as other autoimmune and inflammatory disease processes. From a practical standpoint, the model described herein may help answer questions relative to the most efficacious types of (n-3) PUFA, the optimal dose regimens and (n-3):(n-6) PUFA ratios, as well as the duration and frequency of (n-3) PUFA consumption to achieve maximum benefit in treating overactive IgA responses and, perhaps, ultimately IgAN.
The concentrations of FO employed here were based on those used in previous studies of immune function (1
–3
). A limitation of this study is that lipid incorporation into plasma membranes of lymphoid tissue was not measured. However, feeding periods ranging from 5 to 8 wk and employing FO have been shown to be sufficient to alter immune function and inflammatory responses (50
–53
). Thus, the 12- to 20-wk feeding periods employed in this study should have been adequate to ensure incorporation of (n-3) PUFA into cell membranes of lymphoid tissue. Nevertheless, future studies should include analytical data on fatty acid composition in leukocytes after the feeding of FO or purified (n-3) PUFA.
Another issue that might be raised concerning this model relates to the well-known anorectic effects of VT (47
). The 10 mg/kg VT dietary level employed here was selected because it was optimal for inducing IgA dysregulation in male mice (54
). This dose level did not cause losses from starting mouse body weight nor did it cause outward signs of ill health such as ruffled fur or lack of activity. Furthermore, previous studies with pair-fed controls have shown that reduced feed intake will impair weight gain as well as depress serum IgA (34
). Thus, the anorectic effect of VT, per se, is not the underlying cause for elevated serum and mesangial IgA. Finally, none of the dietary treatments employed for the groups fed VT resulted in differences in weight gain. This finding suggests that differences in IgA parameters found between mice fed CO and FO were not attributable to changes in intake or efficiency of nutrient utilization.
Although both studies yielded generally similar results, the FO-containing AIN-76A diet (Study 1) appeared somewhat more effective than the FO-containing AIN 93G (Study 2) relative to latency to observed attenuation of serum IgA elevation (4 wk vs. 12 wk) and significant reduction of IgA-IC. The latter difference may result in part from the higher variability in IgA-IC responses encountered in Study 2. A second possible cause may be the slightly higher (n-3):(n-6) PUFA ratio employed in Study 1 compared with Study 2 (4 vs. 3, respectively, based on Table 1
). Third, the lower efficacy in Study 2 may relate to the uniform inclusion of TBHQ in CO and FO, a compound that was verified to have attenuating effects in Study 1. Further study using purified EPA and DHA is thus warranted to examine the effects of specific (n-3) PUFA, (n-3) PUFA doses and (n-3):(n-6) PUFA ratio on VT-induced IgAN.
A notable difference observed between the studies was that serum IgA-IC concentrations in Study 2 were 5–10 times those observed in Study 1. Previous studies with VT and AIN 76A have yielded IgA-IC concentrations on the same order of magnitude as seen here in Study 1, whereas other preliminary studies with VT and AIN 93G in our laboratory (data not shown)have resulted in higher IgA-IC levels similar to Study 2. It is possible that differences in the composition of AIN 76A and AIN 93G are responsible for these observed dissimilarities and resolution of the causative factor would require additional studies targeting the role of specific components. Nevertheless, significant VT-induced increases in IgA-IC were observed in both studies, thus allowing us to evaluate the efficacy of FO treatment.
The presence of antioxidants in FO and, ultimately, in an experimental diet can be a potential factor in the development and outcome of a chronic disease model such as that described here (55
). Notably, 
-tocopherol has been added to some FO and suggested to contribute to the effects of these preparations. Dietary -tocopherol supplementation can attenuate glomerular hypertrophy, fibrogenic cytokine mRNA expression, hematuria and proteinuria in rats in which IgAN was induced by chronic oral immunization with bovine 
-globulin (56
,57
). In this latter model, -tocopherol–stripped FO had no effect unless it was supplemented with -tocopherol (57
). The FO used in this study was not supplemented with -tocopherol. Based on the supplier’s data, the level of natural -tocopherol in FO was 100 mg/kg and in CO was 200 mg/kg in Study 2. Because the recommended AIN-93G formulation contains 75 mg/kg of -tocopherol as a supplement (44
), the final FO and CO diets contained 
82 and 89 mg/kg of -tocopherol. Thus it is unlikely that -tocopherol contributed to differences in the response of our model to FO and CO diets. Relative to other antioxidants, supplementation of the CO diet with TBHQ alone did decrease some of the parameters associated with VT-induced IgAN. However, these effects were not of the same magnitude as for the FO diet in Study 1; furthermore, FO was effective in Study 2 in which both FO and CO contained equivalent TBHQ levels. It would be of interest in future studies to explore in detail the effects of -tocopherol and other antioxidants in this model by including these components at higher concentrations that might be expected to cause an in vivo effect.
It should be noted that the VT model mimics IgA deposition observed in human IgAN but does not produce complement activation or progressive renal disease during the 12- to 20-wk period employed here. Although the suggested premise for using FO in human IgAN is that (n-3) PUFA downregulate such inflammatory sequelae in the kidney during later stages of the disease (17
), our results nevertheless suggest that the (n-3) PUFA may also yield benefits much earlier in the disease process. Harper and Savage (16
) stated that the fundamental abnormality in IgAN lies within the IgA system, and not the kidney, because IgA deposition in IgAN patients reappears after renal transplantation. Several lines of evidence support the possibility that IgAN is an immune complex disease. These include the following: 1) the correlation between circulating IgA-IC and disease activities in IgAN patients; 2) high-molecular-weight IgA from serum and renal eluates of IgAN patients that contain dimeric IgA and activated complement; and 3) experimental rodent studies in which IgAN is induced with injected IgA-IC (19
,33
). An overly robust IgA response to mucosal infections and dietary antigens in terms of quantity, size (primarily polymeric) and glycosylation status is believed to contribute to the amount and pathogenicity of IgA-IC in IgAN (18
,19
). Upon recurrent accumulation of these IgA-IC in the kidney, a spectrum of inflammatory events is initiated, leading later to glomerulonephritis and, over the long term, kidney failure. The observation here that FO inhibits overactive IgA production, IgA-IC development and mesangial IgA deposition indicates that this nutritional regimen could act at early stages of the disease before the occurrence of frank kidney injury. Thus, besides providing benefits for persons at later stages of IgAN as demonstrated in clinical studies (15
,17
,29
,30
), (n-3) PUFA may have prophylactic value for persons at high risk for this disease due to family history or have therapeutic value for persons who exhibit early clinical signs.
The specific causes for overactive IgA responses in IgAN are not known, but may involve prior respiratory and gut infections (13
,19
), genetic predisposition (21
,58
), diet(22
) and/or environmental agents (23
,24
). Several cytokines modulate B-cell activation, class-switching, proliferation and terminal differentiation to IgA-producing plasma cells (59
). Relative to mucosal IgA immunity, IL-6 appears to be most critical based on production of this cytokine in the gut by macrophages, T cells and other cells and because of its effects on IgA-committed B cells in vitro (60
). Furthermore, the capacity to mount sustained IgA responses is completely restored upon administration of a recombinant vaccinia virus encoding interleukin (IL)-6 to IL-6–deficient mice. IL-6 may also contribute to the induction and immunopathologic sequelae associated with human IgAN. IgAN patients have been reported to have high urinary IL-6 activity (61
,62
), and peripheral blood cells from these patients also express abnormally high levels of IL-6 mRNA (63
).
Consistent with the above discussion, recent cellular and molecular studies on possible mechanisms by which VT potentiates IgA production highlight a significant role for IL-6. Peyer’s patches and splenic lymphocytes isolated from mice exposed to VT exhibit heightened capacity to secrete IgA that mimics the specificities found for IgA in human IgAN patients (34
–36
,64
67
).The underlying biochemical mode of action of VT and other trichothecenes relates to their capacity to bind ribosomes and inhibit translation (40
). Although high concentrations of trichothecenes completely shut down translation and induce apoptosis, these compounds, when at subinhibitory concentrations, can potentiate expression of cytokines by elevating transcription and increasing mRNA stability (68
–70
). Of several cytokines upregulated by VT in vivo, IL-6 appears to be most important in promoting polyclonal expansion of IgA secreting cells based on the following: 1) the kinetics and magnitude of the IL-6 response relative to other cytokines (71
); 2) ex vivo cell reconstitution studies (65
); and 3) antibody neutralization studies (72
). Most importantly, IL-6 deficient mice are resistant to VT-induced IgA nephropathy (73
).
The strong linkage of IL-6 to VT-induced IgA production makes this cytokine an attractive target for (n-3) PUFA. In support of this contention, it is well recognized that proinflammatory cytokine expression is attenuated extensively in tissues from animals and humans fed (n-3) PUFA. For example, Sadeghi et al. (52
) showed that prior feeding of FO reduced serum IL-1ß, IL-6 and tumor necrosis factor- levels in endotoxin-treated mice compared with those fed oils containing high n-6 PUFA levels. Chandrasekar and Fernandes (74
) demonstrated in a murine autoimmune model that fish oil–fed mice had undetectable levels of IL-1ß, IL-6 or TNF- mRNA in the kidney, whereas these proinflammatory cytokines mRNAs were expressed in kidneys of mice fed corn oil. Fish oil ingestion decreases ex vivo proinflammatory cytokine production by rodent and human mononuclear cells (52
,75
). Future work will therefore focus on the role of IL-6 in FO-suppression of VT-induced IgA nephropathy and on the capacity of (n-3) PUFA to interfere with upstream mechanisms of IL-6 gene upregulation.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: CO, corn oil; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; FBS, fetal bovine serum; FO, fish oil; IgA-IC, IgA immune complex; IgAN, IgA nephropathy; IL, interleukin; PUFA, polyunsaturated fatty acids; TBHQ, tert-butylhydroquinone; VT, vomitoxin.
Manuscript received 7 September 2001. Initial review completed 13 November 2001. Revision accepted 28 November 2001.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Wu, D. & Meydani, S. N. (1998) n-3 polyunsaturated fatty acids and immune function. Proc. Nutr. Soc. 57:503-509.[Medline]
2. Kehn, P. & Fernandes, G. (2001) The importance of omega-3 fatty acids in the attenuation of immune-mediated diseases. J. Clin. Immunol. 21:99-101.[Medline]
3. Calder, P. C. (1998) Immunoregulatory and anti-inflammatory effects of n-3 polyunsaturated fatty acids. Braz. J. Med. Biol. Res. 31:467-490.[Medline]
4. Kromhout, D., Bosschieter, E. B. & de Lezenne, C. C. (1985) The inverse relation between fish consumption and 20-year mortality from coronary heart disease. N. Engl. J. Med. 312:1205-1209.[Abstract]
5. Cathcart, E. S. & Gonnerman, W. A. (1991) Fish oil fatty acids and experimental arthritis. Rheum. Dis. Clin. North Am. 17:235-242.[Medline]
6. Robinson, D. R., Prickett, J. D., Makoul, G. T., Steinberg, A. D. & Colvin, R. B. (1986) Dietary fish oil reduces progression of established renal disease in (NZB x NZW)F1 mice and delays renal disease in BXSB and MRL/1 strains. Arthritis Rheum 29:539-546.[Medline]
7. Robinson, D. R., Xu, L. L., Tateno, S., Guo, M. & Colvin, R. B. (1993) Suppression of autoimmune disease by dietary n-3 fatty acids. J. Lipid Res. 34:1435-1444.[Abstract]
8. Empey, L. R., Jewell, L. D., Garg, M. L., Thomson, A. B., Clandinin, M. T. & Fedorak, R. N. (1991) Fish oil-enriched diet is mucosal protective against acetic acid-induced colitis in rats. Can. J. Physiol Pharmacol. 69:480-487.[Medline]
9. Belch, J.J.F. (1990) Essential fatty acids and rheumatoid arthritis. Horrobin, D. F. eds. Omega-6 Essential Fatty Acids: Pathophysiology and Roles in Clinical Medicine 1990:223-237 Wiley-Liss New York, NY. .
10. Grimminger, F. & Mayser, P. (1995) Lipid mediators, free fatty acids and psoriasis. Prostaglandins Leukot. Essent. Fatty Acids 52:1-15.[Medline]
11. Burton, J. L. (1990) Esential fatty acids in atopic eczema: clinical studies. Horrobin, D. F. eds. Omega-6 Essential Fatty Acids: Pathophysiology and Roles in Clinical Medicine 1990:67-73 Wiley-Liss New York, NY. .
12.
Walton, A. J., Snaith, M. L., Locniskar, M., Cumberland, A. G., Morrow, W. J. & Isenberg, D. A. (1991) Dietary fish oil and the severity of symptoms in patients with systemic lupus erythematosus. Ann. Rheum. Dis. 50:463-466.
13.
D’Amico, G. (1987) The commonest glomerulonephritis in the world: IgA nephropathy. Q. J. Med. 64:709-727.
14. Hellegers, D. M. (1993) IgA nephropathy: questions and answers (a guide for adult patients). IgA Nephropathy Support Network 1993 Granby MA (abs.).
15.
Donadio, J. V., Jr, Bergstralh, E. J., Offord, K. P., Spencer, D. C. & Holley, K. E. (1994) A controlled trial of fish oil in IgA nephropathy. Mayo Nephrology Collaborative Group [see comments]. N. Engl. J. Med. 331:1194-1199.
16.
Harper, S. J., Allen, A. C., Pringle, J. H. & Feehally, J. (1996) Increased dimeric IgA producing B cells in the bone marrow in IgA nephropathy determined by in situ hybridisation for J chain mRNA. J. Clin. Pathol. 49:38-42.
17.
Donadio, J. V., Jr, Grande, J. P., Bergstralh, E. J., Dart, R. A., Larson, T. S. & Spencer, D. C. (1999) The long-term outcome of patients with IgA nephropathy treated with fish oil in a controlled trial. Mayo Nephrology Collaborative Group [In Process Citation]. J. Am. Soc. Nephrol. 10:1772-1777.
18. Hunley, T. E. & Kon, V. (1999) IgA nephropathy. Curr. Opin. Pediatr. 11:152-157.[Medline]
19. Emancipator, S. N. & Lamm, M. E. (1989) IgA nephropathy: pathogenesis of the most common form of glomerulonephritis. Lab. Investig. 60:168-183.[Medline]
20. Endo, Y. & Kanbayashi, H. (1994) Etiology of IgA nephropathy syndrome. Pathol. Int. 44:1-13.[Medline]
21. Schena, F. P., D’Altri, C., Cerullo, G., Manno, C. & Gesualdo, L. (2001) ACE gene polymorphism and IgA nephropathy: an ethnically homogeneous study and a meta-analysis. Kidney Int 60:732-740.[Medline]
22. Coppo, R., Basolo, B., Rollino, C., Roccatello, D., Martina, G., Amore, A. & Piccoli, G. (1986) Dietary gluten and primary IgA nephropathy [letter]. N. Engl. J. Med. 315:1167-1168.[Medline]
23. Coppo, R. (1988) The pathogenetic potential of environmental antigens in IgA nephropathy. Am. J. Kidney Dis. 12:420-424.[Medline]
24. Hinoshita, F., Suzuki, Y., Yokoyama, K., Hara, S., Yamada, A., Ogura, Y., Hashimoto, H., Tomura, S., Marumo, F. & Ueno, Y. (1997) Experimental IgA nephropathy induced by a low-dose environmental mycotoxin, nivalenol. Nephron 75:469-478.[Medline]
25. Harper, L. & Savage, C. O. (1999) Treatment of IgA nephropathy [comment]. Lancet 353:860-862.[Medline]
26. Donadio, J. V., Jr (1991) Omega-3 polyunsaturated fatty acids: a potential new treatment of immune renal disease. Mayo Clin. Proc. 66:1018-1028.[Medline]
27. Holman, R. T., Johnson, S. B., Bibus, D., Spencer, D. C. & Donadio, J. V., Jr (1994) Essential fatty acid deficiency profiles in idiopathic immunoglobulin A nephropathy. Am. J. Kidney Dis. 23:648-654.[Medline]
28. Wakai, K., Kawamura, T., Matsuo, S., Hotta, N. & Ohno, Y. (1999) Risk factors for IgA nephropathy: a case-control study in Japan. Am. J. Kidney Dis. 33:738-745.[Medline]
29. Hamazaki, T., Tateno, S. & Shishido, H. (1984) Eicosapentaenoic acid and IgA nephropathy. Lancet 1:1017-1018.
30.
Donadio, J. V., Jr, Larson, T. S., Bergstralh, E. J. & Grande, J. P. (2001) A randomized trial of high-dose compared with low-dose omega-3 fatty acids in severe IgA nephropathy. J. Am. Soc. Nephrol. 12:791-799.
31. Bennett, W. M., Walker, R. G. & Kincaid-Smith, P. (1989) Treatment of IgA nephropathy with eicosapentanoic acid (EPA): a two-year prospective trial. Clin. Nephrol. 31:128-131.[Medline]
32. Pettersson, E. E., Rekola, S., Berglund, L., Sundqvist, K. G., Angelin, B., Diczfalusy, U., Bjorkhem, I. & Bergstrom, J. (1994) Treatment of IgA nephropathy with omega-3-polyunsaturated fatty acids: a prospective, double-blind, randomized study. Clin. Nephrol. 41:183-190.[Medline]
33. Montinaro, V., Gesualdo, L. & Schena, F. P. (1999) The relevance of experimental models in the pathogenetic investigation of primary IgA nephropathy. Ann. Med. Interne (Paris) 150:99-107.[Medline]
34. Pestka, J. J., Moorman, M. A. & Warner, R. L. (1989) Dysregulation of IgA production and IgA nephropathy induced by the trichothecene vomitoxin. Food Chem. Toxicol. 27:361-368.[Medline]
35. Dong, W., Sell, J. E. & Pestka, J. J. (1991) Quantitative assessment of mesangial immunoglobulin A (IgA) accumulation, elevated circulating IgA immune complexes, and hematuria during vomitoxin-induced IgA nephropathy. Fundam. Appl. Toxicol. 17:197-207.[Medline]
36. Dong, W. & Pestka, J. J. (1993) Persistent dysregulation of IgA production and IgA nephropathy in the B6C3F1 mouse following withdrawal of dietary vomitoxin (deoxynivalenol). Fundam. Appl. Toxicol. 20:38-47.[Medline]
37. Pestka, J. J., Dong, W., Warner, R. L., Rasooly, L., Bondy, G. S. & Brooks, K. H. (1990) Elevated membrane IgA+ and CD4+ (T helper) populations in murine Peyer’s patch and splenic lymphocytes during dietary administration of the trichothecene vomitoxin (deoxynivalenol). Food Chem. Toxicol. 28:409-420.[Medline]
38. Pestka, J. J., Dong, W., Warner, R. L., Rasooly, L. & Bondy, G. S. (1990) Effect of dietary administration of the trichothecene vomitoxin (deoxynivalenol) on IgA and IgG secretion by Peyer’s patch and splenic lymphocytes. Food Chem. Toxicol. 28:693-699.[Medline]
39. Bondy, G. S. & Pestka, J. J. (1991) Dietary exposure to the trichothecene vomitoxin (deoxynivalenol) stimulates terminal differentiation of Peyer’s patch B cells to IgA secreting plasma cells. Toxicol. Appl. Pharmacol. 108:520-530.[Medline]
40. Bondy, G. S. & Pestka, J. J. (2000) Immunomodulation by fungal toxins. J. Toxicol. Environ. Health B Crit. Rev. 3:109-143.[Medline]
41. Witt, M. F., Hart, L. P. & Pestka, J. J. (1985) Purification of deoxynivalenol (vomitoxin) by water-saturated silica gel chromatography. J. Agric. Food Chem. 33:745-748.
42. Thompson, W. L. & Wannemacher, R. W., Jr (1986) Structure-function relationships of 12,13-epoxytrichothecene mycotoxins in cell culture: comparison to whole animal lethality. Toxicon 24:985-994.[Medline]
43. American Institute of Nutrition (1980) Second report of the ad hoc committee on standards for nutritional studies. J. Nutr. 110:1726.
44. Reeves, P. G., Nielsen, F. H. & Fahey-G, C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
45. Inai, H., Chen, A., Wyatt, R. J. & Rifai, A. (1987) Composition of IgA immune complexes precipitation with polyethylene glycol. A model for isolation and analysis of immune complexes. J. Immunol. Methods 103:239-245.
46. Valenzuela, R. & Deodhar, S. D. (1980) Interpretation of immunofluorescent patterns in renal diseases. Pathobiol. Ann. 10:183-221.[Medline]
47. Rotter, B. A., Prelusky, D. B. & Pestka, J. J. (1996) Toxicology of deoxynivalenol (vomitoxin). J. Toxicol. Environ. Health 48:1-34.[Medline]
48. Greene, D. M., Bondy, G. S., Azcona Olivera, J. I. & Pestka, J. J. (1994) Role of gender and strain in vomitoxin-induced dysregulation of IgA production and IgA nephropathy in the mouse. J. Toxicol. Environ. Health 43:37-50.[Medline]
49. Rasooly, L. & Pestka, J. J. (1994) Polyclonal autoreactive IgA increase and mesangial deposition during vomitoxin-induced IgA nephropahty in the BALB/c mouse. Food Chem. Toxicol. 32:329-336.[Medline]
50. Peterson, L. D., Thies, F., Sanderson, P., Newsholme, E. A. & Calder, P. C. (1998) Low levels of eicosapentaenoic and docosahexaenoic acids mimic the effects of fish oil upon rat lymphocytes. Life Sci 62:2209-2217.[Medline]
51. Peterson, L. D., Jeffery, N. M., Thies, F., Sanderson, P., Newsholme, E. A. & Calder, P. C. (1998) Eicosapentaenoic and docosahexaenoic acids alter rat spleen leukocyte fatty acid composition and prostaglandin E2 production but have different effects on lymphocyte functions and cell-mediated immunity. Lipids 33:171-180.[Medline]
52. Sadeghi, S., Wallace, F. A. & Calder, P. C. (1999) Dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice. Immunology 96:404-410.[Medline]
53. Yaqoob, P. & Calder, P. (1995) Effects of dietary lipid manipulation upon inflammatory mediator production by murine macrophages. Cell Immunol 163:120-128.[Medline]
54. Greene, D. M., Azcona Olivera, J. I. & Pestka, J. J. (1994) Vomitoxin (deoxynivalenol)-induced IgA nephropathy in the B6C3F1 mouse: dose response and male predilection. Toxicology 92:245-260.[Medline]
55. Weimann, B. J. & Hermann, D. (1999) Inhibition of autoimmune deterioration in MRL/lpr mice by vitamin E. Int. J. Vitam. Nutr. Res. 69:255-261.[Medline]
56. Trachtman, H., Chan, J. C., Chan, W., Valderrama, E., Brandt, R., Wakely, P., Futterweit, S., Maesaka, J. & Ma, C. (1996) Vitamin E ameliorates renal injury in an experimental model of immunoglobulin A nephropathy. Pediatr. Res. 40:620-626.[Medline]
57. Kuemmerle, N. B., Chan, W., Krieg, R. J., Jr, Norkus, E. P., Trachtman, H. & Chan, J. C. (1998) Effects of fish oil and alpha-tocopherol in immunoglobulin A nephropathy in the rat. Pediatr. Res. 43:791-797.[Medline]
58. Scolari, F. (1999) Familial IgA nephropathy. J. Nephrol. 12:213-219.[Medline]
59. VanCott, J. L., Staats, H. F., Pascual, D. W., Roberts, M., Chatfield, S. N., Yamamoto, M., Coste, M., Carter, P. B., Kiyono, H. & McGhee, J. R. (1996) Regulation of mucosal and systemic antibody responses by T helper cell subsets, macrophages, and derived cytokines following oral immunization with live recombinant Salmonella. J. Immunol. 156:1504-1514.[Abstract]
60.
Ramsay, A. J., Husband, A. J., Ramshaw, I. A., Bao, S., Matthaei, K. I., Koehler, G. & Kopf, M. (1994) The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science (Washington, DC) 264:561-563.
61. Baba, Y., Akagi, H., Fukushima, K., Kosaka, M., Hattori, K., Nishizaki, K., Ogawa, T., Masuda, Y. & Shikata, K. (1999) Quantitative analysis of interleukin 6 (IL-6) in patients with IgA nephropathy after tonsillectomy. Auris Nasus Larynx 26:177-182.[Medline]
62. Ranieri, E., Gesualdo, L., Petrarulo, F. & Schena, F. P. (1996) Urinary IL-6/EGF ratio: a useful prognostic marker for the progression of renal damage in IgA nephropathy. Kidney Int 50:1990-2001.[Medline]
63. Nakamura, T., Ebihara, I., Takahashi, T., Yamamoto, M., Tomino, Y. & Koide, H. (1993) Increased interleukin 6 mRNA expression by peripheral blood T cells from patients with IgA nephropathy. Autoimmunity 15:171-179.[Medline]
64. Yan, D., Zhou, H. R., Brooks, K. H. & Pestka, J. J. (1997) Potential role for IL-5 and IL-6 in enhanced IgA secretion by Peyer’s patch cells isolated from mice acutely exposed to vomitoxin. Toxicology 122:145-158.[Medline]
65. Yan, D., Zhou, H. R., Brooks, K. H. & Pestka, J. J. (1998) Role of macrophages in elevated IgA and IL-6 production by Peyer’s patch cultures following acute oral vomitoxin exposure. Toxicol. Appl. Pharmacol. 148:261-273.[Medline]
66. Rasooly, L., Abouzied, M. M., Brooks, K. H. & Pestka, J. J. (1994) Polyspecific and autoreactive IgA secreted by hybridomas derived from Peyer’s patches of vomitoxin-fed mice: characterization and possible pathogenic role in IgA nephropathy. Food Chem. Toxicol. 32:337-348.[Medline]
67. Matsiota, P., Dosquet, P., Louzir, H., Druet, E., Druet, P. & Avrameas, S. (1990) IgA polyspecific autoantibodies in IgA nephropathy. Clin. Exp. Immunol. 79:361-366.[Medline]
68. Ouyang, Y. L., Li, S. & Pestka, J. J. (1996) Effects of vomitoxin (deoxynivalenol) on transcription factor NF-kappa B/Rel binding activity in murine EL-4 thymoma and primary CD4+ T cells. Toxicol. Appl. Pharmacol. 140:328-336.[Medline]
69. Li, S., Ouyang, Y., Yang, G. H. & Pestka, J. J. (2000) Modulation of transcription factor AP-1 activity in murine EL-4 thymoma cells by vomitoxin (deoxynivalenol). Toxicol. Appl. Pharmacol. 163:17-25.[Medline]
70. Wong, S., Schwartz, R. C. & Pestka, J. J. (2001) Superinduction of TNF-alpha and IL-6 in macrophages by vomitoxin (deoxynivalenol) modulated by mRNA stabilization. Toxicology 161:139-149.[Medline]
71. Zhou, H. R., Yan, D. & Pestka, J. J. (1997) Differential cytokine mRNA expression in mice after oral exposure to the trichothecene vomitoxin (deoxynivalenol): dose response and time course. Toxicol. Appl. Pharmacol. 144:294-305.[Medline]
72. Yan, D., Zhou, H. R., Brooks, K. H. & Pestka, J. J. (1997) Potential role for IL-5 and IL-6 in enhanced IgA secretion by Peyer’s patch cells isolated from mice acutely exposed to vomitoxin. Toxicology 122:145-158.
73. Pestka, J. J. & Zhou, H. R. (2000) Interleukin-6-deficient mice refractory to IgA dysregulation but not anorexia induction by vomitoxin (deoxynivalenol) ingestion. Food Chem. Toxicol. 38:565-575.[Medline]
74. Chandrasekar, B. & Fernandes, G. (1994) Decreased pro-inflammatory cytokines and increased antioxidant enzyme gene expression by omega-3 lipids in murine lupus nephritis. Biochem. Biophys. Res. Commun. 200:893-898.[Medline]
75. Meydani, S. N., Endres, S., Woods, M. M., Goldin, B. R., Soo, C., Morrill-Labrode, A., Dinarello, C. A. & Gorbach, S. L. (1991) Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte proliferation: comparison between young and older women. J. Nutr. 121:547-555.
This article has been cited by other articles:
|
|
 |
|
  E. Beli, M. Li, C. Cuff, and J. J. Pestka Docosahexaenoic Acid-Enriched Fish Oil Consumption Modulates Immunoglobulin Responses to and Clearance of Enteric Reovirus Infection in Mice J. Nutr., April 1, 2008; 138(4): 813 - 819. [Abstract] [Full Text] [PDF]
|
|
