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Dietary PUFAs differentially modify canine neutrophil composition and function
Poster Presentations Wednesday 23 July
EFA & Eicosanoids 1997- Edinburgh
P121
P122
Dietary fish oil decreases the production of pro-inflammatory mediators by murine macrophages. Fiona Wallace, Sarah Neely,
Dietary fish oil increases mRNA levels for Serum Amyloid A in Syrian Hamsters.
Elizabeth Miles and Philip Calder, School of Biological Sciences, University of Southampton, Southampton, U.K. Despite a number of studies, the effects of dietary fish oil upon the production of pro-inflammatory cytokines remain controversial with m a n y contradictory reports in the literature. To investigate the effect of fish oil and other fipids further, we fed male C57B16 mice for 12 weeks upon a low fat (LF) diet (25 g/kg corn oil) or on diets containing 200 g/kg coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO). Peritoneal macrophages were elicited with thioglycollate and were cultured for 24 hours at a density of 1 x 106 cells/ml in the presence of 10 ~tg/ml lipopolysaccharide and 5% v/v fetal calf serum. The concentrations of PGE2, IL-I[~, IL-6 and TNF-c~ (ng/ml) in the m e d i u m were measured by ELISA and the concentration of nitrite (an indicator of nitric oxide production) (~tM) was measured by a colourimetric assay. Data are m e a n + SEM for 4 to 6 animals fed on each diet. Values not sharing a c o m m o n superscript letter are significantly different (ANOVA). Diet LF CO OO SO FO
Nitrite 24.45:2.3 11.6± 3.0 20.5+3.4 19.85:6.2 24.6_+3.0
Concentration in medium PGE 2 IL-113 IL-6 TNF-tx 74.7-+11.4 a 0.15 +0.03 a 0.85+0.0119 3.1-+0.6 a 57.8-+13.6a 0.13-+0.01a 0.86 + 0.02b 2.0 + 0.3ab 50.8-+3.7 b 0.11 +0.01 a 0.88 +0.03 b 1.3 +0.4 bc 50.4-+4.1 b 0.11 ±0.02 a 0.95 + 0.05a 0.70+0.07 cd 13.4_+6.2c 0.057-+0.004 b 0.83+0.05 b 0.12+0.02 d
Diet did not affect nitrite production, although there was a trend towards lower production by cells from animals fed coconut oil. As expected, fish oil feeding caused a significant decrease in PGE2 production. The production of two pro-inflammatory cytokines (IL113 and TNF-c0 was markedly reduced if the mice were fed fish oil. IL-6 production was enhanced by safflower oil feeding. Thus, this study demonstrates that fish oil feeding greatly diminishes the ability of m a c r o p h a g e s to produce a range of i n f l a m m a t o r y mediators.
243
Hardard6ttir, I., Feingold, K.R., Sipe, J., and Griinfeld, C. Science Institute, University of Iceland, R e y k j a v ~ , Iceland Dietary fish oils can attenuate inflammation and prevent autoimmunity. However, their effects on infection vary. The present study explored the effects of dietary fish oils on m R N A levels for serum amyloid A (SAA) in several tissues in the hamster after endotoxin (LPS) administration. Male Syrian hamsters were fed diets supplemented with 1.5% of ethyl esters of olive oil, safflower oil or fish oil. The hamsters were injected with LPS and 16 hours later tissues were removed. Poly A R N A was isolated from livers, spleens, hearts and duodenum and analysed by Northern blotting. The m R N A levels for SAA increased, following LPS treatment, by 60% in the liver and 50% in the heart of hamsters fed the fish oil enriched diets when compared to that in hamsters fed the olive oil enriched diet. m R N A levels for S A A in livers and hearts of hamsters fed the safflower oil enriched diet were in between the m R N A levels for SAA in hamsters fed the fish oil and the olive oil enriched diets. In spleen the trend was the same as that in livers and hearts although the increase in m R N A levels for S A A in hamsters fed the fish oil enriched diet was not statistically different when compared to that in hamsters fed the olive oil enriched diet. There was no difference in the m R N A levels for SAA in the duodenum of hamsters fed any of the three diets. These data demonstrate that dietary fish oil increases m R N A levels for S A A in several tissues in the hamsters after stimulation in vivo with endotoxin. SAA is one of the major acute phase proteins and is believed to play a beneficial role during infection. Hence the increase in m R N A levels for S A A in the hamsters fed the fish oil enriched diet may indicate a beneficial effect of fish oils during infection.
P123
P124
Dietary PUFAs Differentially Modify Canine Neutrophil Composition and Function. M.K. Waldron ~, J.E. Bauer l, W.J.
Mononuclear Cell Functions in Healthy Volunteers Fed Borage Oil (Borago
Burkholder l, S.S. Hannah 2. tTexas A & M University, College Station, TX, USA, 2 Ralston Purina Co., St. Louis, MO, U S A Effects of dietary 18 and 20 carbon polyunsaturated fatty acids on neutrophil phospholipid membrane composition and function were investigated in dogs. We hypothesized that 18:3n-3 is chain elongated and desaturated to 20:5n-3 and 22:6n-3 and that membrane modification alters neutrophil function. 27 adult mixed breed dogs were randomized into three groups and acclimated to a basal diet for 14 days. The basal diet was then supplemented with sunflower (SFO), linseed oil (LSO), or menhaden oil (MHO) for 42 days (day 56). Cholesterol content of the supplements were equilibrated to MHO levels. % Keats from n-3 fatty acids was 15.9, 6.7, and 0.3 (LSO, MHO, SFO), with n-6/n-3 ratios of 0.5, 0.6, and 78.6 (LSO, MHO, SFO). Phagocytosis was quantified using flow cytometry. No significant differences between the groups in serum cholesterol or lipoprotein subfractions were observed. Respiratory burst was significantly reduced in the MHO group. The MHO group had higher relative amounts of 20:5n-3, 22:5n-3, and 22:6n-3 than the LSO and SFO groups. No enrichment of 22:6n-3 was observed in the LSO group, but this group had higher relative amounts of 18:3n-3, 20:5n-3, and 22:5n-3 vs. the SFO group and higher 18:3n-3 vs. the MHO group were observed. The MHO group also had lower 20:4n-6 than LSO and SFO groups. Repeated measures ANOVA revealed no significant differences in phagocytic function between groups. A time effect was seen with a significant decrease in function between day 14 and day 0 and day 56 (p< 0.05) when all three groups were combined. Fatty acid analysis of neutrophil membrane phospholipid revealed increased P/S ratio between day 14 and day 56 (0.27 vs. 0.50, p < 0.05). Our data shows that active incorporation of PUFAs into the phospholipid membrane occurs and that a low fat/high saturated fatty acid diet when compared to a high fat/high PUFA diet decreases phagocytic function. Taken together the data indicate that 18 and 20 carbon PUFAs are uniquely incorporated into the membrane and modify neutrophil function even at similar n.-6/n-3 ratios.
officinalis).*#Harbigc LS, *Fisher B and *Fadlon EJ. #School of Chemical and Life Sciences. University of Greenwich, London SE 18 6PF UK *Department of Immunology. UMDS. St Thomas' Hospital. London SEI 7EH UK.
Aim: We have investigated the effects of dietary supplementation with borage oil ill healthy volunteers on peripheral blood mononuclear cells (PBMC). Methods: Eight healthy subjects orally ingested 15ml of borage oil (23 % "tlinolenic acid and 37 % linoleic acid) daily for 12 weeks. Peripheral blood was collected by venepunclure before and at weeks 4, 8, and 12 of supplementation. Tile proportions of T cell phenotypes in whole blood were assessed using direclly conjugated antibodies and flow cytometric analysis, PBMC separated by density gradient (Lymphoprep) were either labelled with SlCr for adherence assays to activated (10 lU/ml TNF-ct) and non-activated HUVEC or cultured with PHA (5lsg/ml) for 24 hr. Tile 24 hr supernatants werq collected for cytokine and eicosanoid measurements by ELISA (R&D and Cayman). The T cell proliferative response to PHA (5p.g/ml) was measured by 3[H] Ihymidioe uplake over tile final seven hours of a 96 well plate three day culture. Results: Table 1. Effect of Borage Oil Supplementation on T cell Phenotype and Cvtokine Production (Mean ± SD). Weeks Supplementation 0 4 8 12 CD4+CD45RA+ 28±17 22±17" 23±19 37±17' CD4+CD45RO+ 43±10 46±9.7 45±14 515:8.4 TGF[31 (pg/ml) 309±216 471+994.* 615±351"* 9495:614' IL-4 (pg/ml) 67±51 44:1:38 295:17" 30±15' Sign!ficance of difference from baseline value **P<0. 001, * P
EFA & Eicosanoids 1997- Edinburgh
P121
P122
Dietary fish oil decreases the production of pro-inflammatory mediators by murine macrophages. Fiona Wallace, Sarah Neely,
Dietary fish oil increases mRNA levels for Serum Amyloid A in Syrian Hamsters.
Elizabeth Miles and Philip Calder, School of Biological Sciences, University of Southampton, Southampton, U.K. Despite a number of studies, the effects of dietary fish oil upon the production of pro-inflammatory cytokines remain controversial with m a n y contradictory reports in the literature. To investigate the effect of fish oil and other fipids further, we fed male C57B16 mice for 12 weeks upon a low fat (LF) diet (25 g/kg corn oil) or on diets containing 200 g/kg coconut oil (CO), olive oil (OO), safflower oil (SO) or fish oil (FO). Peritoneal macrophages were elicited with thioglycollate and were cultured for 24 hours at a density of 1 x 106 cells/ml in the presence of 10 ~tg/ml lipopolysaccharide and 5% v/v fetal calf serum. The concentrations of PGE2, IL-I[~, IL-6 and TNF-c~ (ng/ml) in the m e d i u m were measured by ELISA and the concentration of nitrite (an indicator of nitric oxide production) (~tM) was measured by a colourimetric assay. Data are m e a n + SEM for 4 to 6 animals fed on each diet. Values not sharing a c o m m o n superscript letter are significantly different (ANOVA). Diet LF CO OO SO FO
Nitrite 24.45:2.3 11.6± 3.0 20.5+3.4 19.85:6.2 24.6_+3.0
Concentration in medium PGE 2 IL-113 IL-6 TNF-tx 74.7-+11.4 a 0.15 +0.03 a 0.85+0.0119 3.1-+0.6 a 57.8-+13.6a 0.13-+0.01a 0.86 + 0.02b 2.0 + 0.3ab 50.8-+3.7 b 0.11 +0.01 a 0.88 +0.03 b 1.3 +0.4 bc 50.4-+4.1 b 0.11 ±0.02 a 0.95 + 0.05a 0.70+0.07 cd 13.4_+6.2c 0.057-+0.004 b 0.83+0.05 b 0.12+0.02 d
Diet did not affect nitrite production, although there was a trend towards lower production by cells from animals fed coconut oil. As expected, fish oil feeding caused a significant decrease in PGE2 production. The production of two pro-inflammatory cytokines (IL113 and TNF-c0 was markedly reduced if the mice were fed fish oil. IL-6 production was enhanced by safflower oil feeding. Thus, this study demonstrates that fish oil feeding greatly diminishes the ability of m a c r o p h a g e s to produce a range of i n f l a m m a t o r y mediators.
243
Hardard6ttir, I., Feingold, K.R., Sipe, J., and Griinfeld, C. Science Institute, University of Iceland, R e y k j a v ~ , Iceland Dietary fish oils can attenuate inflammation and prevent autoimmunity. However, their effects on infection vary. The present study explored the effects of dietary fish oils on m R N A levels for serum amyloid A (SAA) in several tissues in the hamster after endotoxin (LPS) administration. Male Syrian hamsters were fed diets supplemented with 1.5% of ethyl esters of olive oil, safflower oil or fish oil. The hamsters were injected with LPS and 16 hours later tissues were removed. Poly A R N A was isolated from livers, spleens, hearts and duodenum and analysed by Northern blotting. The m R N A levels for SAA increased, following LPS treatment, by 60% in the liver and 50% in the heart of hamsters fed the fish oil enriched diets when compared to that in hamsters fed the olive oil enriched diet. m R N A levels for S A A in livers and hearts of hamsters fed the safflower oil enriched diet were in between the m R N A levels for SAA in hamsters fed the fish oil and the olive oil enriched diets. In spleen the trend was the same as that in livers and hearts although the increase in m R N A levels for S A A in hamsters fed the fish oil enriched diet was not statistically different when compared to that in hamsters fed the olive oil enriched diet. There was no difference in the m R N A levels for SAA in the duodenum of hamsters fed any of the three diets. These data demonstrate that dietary fish oil increases m R N A levels for S A A in several tissues in the hamsters after stimulation in vivo with endotoxin. SAA is one of the major acute phase proteins and is believed to play a beneficial role during infection. Hence the increase in m R N A levels for S A A in the hamsters fed the fish oil enriched diet may indicate a beneficial effect of fish oils during infection.
P123
P124
Dietary PUFAs Differentially Modify Canine Neutrophil Composition and Function. M.K. Waldron ~, J.E. Bauer l, W.J.
Mononuclear Cell Functions in Healthy Volunteers Fed Borage Oil (Borago
Burkholder l, S.S. Hannah 2. tTexas A & M University, College Station, TX, USA, 2 Ralston Purina Co., St. Louis, MO, U S A Effects of dietary 18 and 20 carbon polyunsaturated fatty acids on neutrophil phospholipid membrane composition and function were investigated in dogs. We hypothesized that 18:3n-3 is chain elongated and desaturated to 20:5n-3 and 22:6n-3 and that membrane modification alters neutrophil function. 27 adult mixed breed dogs were randomized into three groups and acclimated to a basal diet for 14 days. The basal diet was then supplemented with sunflower (SFO), linseed oil (LSO), or menhaden oil (MHO) for 42 days (day 56). Cholesterol content of the supplements were equilibrated to MHO levels. % Keats from n-3 fatty acids was 15.9, 6.7, and 0.3 (LSO, MHO, SFO), with n-6/n-3 ratios of 0.5, 0.6, and 78.6 (LSO, MHO, SFO). Phagocytosis was quantified using flow cytometry. No significant differences between the groups in serum cholesterol or lipoprotein subfractions were observed. Respiratory burst was significantly reduced in the MHO group. The MHO group had higher relative amounts of 20:5n-3, 22:5n-3, and 22:6n-3 than the LSO and SFO groups. No enrichment of 22:6n-3 was observed in the LSO group, but this group had higher relative amounts of 18:3n-3, 20:5n-3, and 22:5n-3 vs. the SFO group and higher 18:3n-3 vs. the MHO group were observed. The MHO group also had lower 20:4n-6 than LSO and SFO groups. Repeated measures ANOVA revealed no significant differences in phagocytic function between groups. A time effect was seen with a significant decrease in function between day 14 and day 0 and day 56 (p< 0.05) when all three groups were combined. Fatty acid analysis of neutrophil membrane phospholipid revealed increased P/S ratio between day 14 and day 56 (0.27 vs. 0.50, p < 0.05). Our data shows that active incorporation of PUFAs into the phospholipid membrane occurs and that a low fat/high saturated fatty acid diet when compared to a high fat/high PUFA diet decreases phagocytic function. Taken together the data indicate that 18 and 20 carbon PUFAs are uniquely incorporated into the membrane and modify neutrophil function even at similar n.-6/n-3 ratios.
officinalis).*#Harbigc LS, *Fisher B and *Fadlon EJ. #School of Chemical and Life Sciences. University of Greenwich, London SE 18 6PF UK *Department of Immunology. UMDS. St Thomas' Hospital. London SEI 7EH UK.
Aim: We have investigated the effects of dietary supplementation with borage oil ill healthy volunteers on peripheral blood mononuclear cells (PBMC). Methods: Eight healthy subjects orally ingested 15ml of borage oil (23 % "tlinolenic acid and 37 % linoleic acid) daily for 12 weeks. Peripheral blood was collected by venepunclure before and at weeks 4, 8, and 12 of supplementation. Tile proportions of T cell phenotypes in whole blood were assessed using direclly conjugated antibodies and flow cytometric analysis, PBMC separated by density gradient (Lymphoprep) were either labelled with SlCr for adherence assays to activated (10 lU/ml TNF-ct) and non-activated HUVEC or cultured with PHA (5lsg/ml) for 24 hr. Tile 24 hr supernatants werq collected for cytokine and eicosanoid measurements by ELISA (R&D and Cayman). The T cell proliferative response to PHA (5p.g/ml) was measured by 3[H] Ihymidioe uplake over tile final seven hours of a 96 well plate three day culture. Results: Table 1. Effect of Borage Oil Supplementation on T cell Phenotype and Cvtokine Production (Mean ± SD). Weeks Supplementation 0 4 8 12 CD4+CD45RA+ 28±17 22±17" 23±19 37±17' CD4+CD45RO+ 43±10 46±9.7 45±14 515:8.4 TGF[31 (pg/ml) 309±216 471+994.* 615±351"* 9495:614' IL-4 (pg/ml) 67±51 44:1:38 295:17" 30±15' Sign!ficance of difference from baseline value **P<0. 001, * P