Thais Martins de Lima

Ranking the toxicity of fatty acids on Jurkat and Raji cells by flow cytometric analysis

The fatty acids have an important role in the control of leukocyte metabolism and function. Higher concentrations of certain fatty acids, particularly polyunsaturated fatty acids (PUFAs) and volatile fatty acids, can cause cell death via apoptosis or, when concentrations are greater, necrosis. In this study, we determined the highest concentrations of various fatty acids that are non-toxic to two human leukemic cell lines, Jurkat (T-lymphocyte) and Raji (B-lymphocyte). Toxicity was evaluated by either loss of membrane integrity and/or DNA fragmentation using flow cytometric analysis. There were no remarkable differences for the toxicity of the fatty acids between B and T cell lines. The cytotoxicity of the fatty acids was related to the carbon chain length and number of double bonds: docosahexaenoic acid=eicosapentaenoic acid=arachidonic acid=gamma-linolenic acid=stearic acid=palmitic acid > linoleic acid=palmitoleic acid > vacenic acid=lauric acid > oleic acid > elaidic acid > capric acid > butyric acid > caprylic acid=caproic acid=propionic acid. The proportion of cells undergoing apoptosis or necrosis, induced by the fatty acids tested, remains to be investigated.

Comparative toxicity of fatty acids on a macrophage cell line (J774)

In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARgamma (peroxisome-proliferator-activated receptor gamma) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARgamma activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.

Beneficial effect of glutamine on exercise-induced apoptosis of rat neutrophils.

INTRODUCTION/PURPOSE The effect of a single bout of intensive exercise on apoptosis of rat neutrophils and the possible prevention by glutamine administration was examined. The experiments were performed in sexually immature and sexually mature male rats as to examine the possible involvement of sexual maturation in the effect of exercise. METHODS Exercise was carried out on a treadmill for 1 h before rats were killed by decapitation. Aqueous solution of glutamine was given by gavage (1 g.kg-1 body weight), 1 h before exercise. Neutrophils were obtained by intraperitoneal lavage with phosphate-buffered saline (PBS), 4 h after injection of oyster glycogen solution. The cells were then analyzed for apoptosis by flow cytometry and fluorescence microscopy. Pro- and antiapoptotic gene expression was evaluated by reverse transcriptase chain reaction (RT-PCR). RESULTS Neutrophils obtained from immature and mature exercised rats showed an increase in DNA fragmentation, chromatin condensation, and phosphatidylserine externalization. This suggests that all neutrophils suffered apoptosis. To study the possible mechanism involved, the production of reactive oxygen metabolites, expression of genes involved in apoptosis and mitochondrial transmembrane potential were examined. Acute exercise raised reactive oxygen metabolites production by neutrophils. Exercise did not change the expression of antiapoptotic (bcl-xL) and apoptotic (bax and bcl-xS) genes in neutrophils from immature rats but caused a significant increase of bax and bcl-xS expression and provoked a significant decrease of bcl-xL expression in cells from mature rats. Exercise also induced a marked loss of mitochondrial depolarization in neutrophils. Oral glutamine supplementation partially prevented the exercise-induced apoptosis in neutrophils from sexually immature and mature rats. CONCLUSION The protective effect of glutamine on neutrophil apoptosis induced by acute exercise possibly occurs by preservation of mitochondrial function.

Mechanisms by which fatty acids regulate leucocyte function.

Fatty acids (FAs) have been shown to alter leucocyte function and thus to modulate inflammatory and immune responses. In this review, the effects of FAs on several aspects of lymphocyte, neutrophil and macrophage function are discussed. The mechanisms by which FAs modulate the production of lipid mediators, activity of intracellular signalling pathways, activity of lipid-raft-associated proteins, binding to TLRs (Toll-like receptors), control of gene expression, activation of transcription factors, induction of cell death and production of reactive oxygen and nitrogen species are described in this review. The rationale for the use of specific FAs to treat patients with impaired immune function is explained. Substantial improvement in the therapeutic usage of FAs or FA derivatives may be possible based on an improvement in the understanding of the precise molecular mechanisms of action with respect to the different leucocyte types and outcome with respect to the inflammatory responses.

Toxicity of a Soybean Oil Emulsion on Human Lymphocytes and Neutrophils

BACKGROUND The incorporation of lipid emulsions in parenteral diets is a requirement for energy and essential fatty acid supply to critically ill patients. In this study, the toxicity of a lipid emulsion rich (60%) in triacylglycerol of omega-6 polyunsaturated fatty acids on leukocytes from healthy volunteers was investigated. METHODS Eleven volunteers were recruited, and blood samples were collected before infusion of a soybean oil emulsion, immediately afterwards, and 18 hours later. The cells were studied immediately after isolation and again after 24 hours or 48 hours in culture. The following determinations were made: composition and concentration of fatty acids in plasma, lymphocytes and neutrophils, lymphocyte proliferation, levels of cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, reactive oxygen species production, and neutral lipid accumulation. RESULTS Soybean oil emulsion decreased lymphocyte proliferation and provoked neutrophil and lymphocyte apoptosis and necrosis. Evidence is presented herein that soybean oil emulsion is less toxic to neutrophils than to lymphocytes. The mechanism of cell death induced by this oil emulsion was characterized by mitochondrial membrane depolarization and neutral lipid accumulation but did not alter reactive oxygen species production. CONCLUSIONS Soybean oil emulsion given as a single dose of 500 mL promotes lymphocyte and neutrophil death that may enhance the susceptibility of the patients to infections.

Seven DNA polymorphisms at the candidate genes of atherosclerosis in Brazilian women with angiographically documented coronary artery disease.

The possible association of genetic markers at the apolipoprotein E (HhaI polymorphism), apolipoprotein B (XbaI, EcoRI and Ins/Del polymorphisms), and low-density lipoprotein receptor (LDLR) (AvaII, HincII and PvuII polymorphisms) with coronary artery disease (CAD) was evaluated in 50 Brazilian women with CAD diagnosed by angiography and in 100 healthy women (controls). The frequency of E3/E4 genotype for HhaI polymorphism at the Apo E gene was significantly higher in CAD patients than in controls (40% vs. 14%, respectively, P<0.001). Similarly, the X-X- genotype for XbaI polymorphism was more frequent in CAD individuals than controls (42% vs. 12%, P<0.0001). The A+A+ and P1P1 genotypes for AvaII and PvuII polymorphisms at the LDLR locus were also higher in CAD subjects than controls (44% vs. 16%, P<0.001 and 64% vs. 39%, P<0.05, respectively). The estimated relative risks for CAD in women carrying the E3/E4, X-X-, A+A+ and P1P1 genotypes were 4.1 [95% confidence interval (CI), 3.0-5.6], 5.3 (95% CI, 3.8-7.5), 4.1 (95% CI, 3.0-5.5), and 2.8 (95% CI, 2.2-3.6), respectively. This study demonstrates that Apo E, Apo B and LDLR gene polymorphisms are associated with CAD in Brazilian Caucasian women.

Effects of EPA and DHA on proliferation, cytokine production, and gene expression in Raji cells

The effects of EPA and DHA on the function and gene expression of a B-lymphocyte cell line (Raji) were investigated. Proliferation; production of interleukin-10 (IL-10), tumor necrosis factor (TNF)-α, and interferon (INF)-γ; and expression of pleiotropic genes were evaluated. Cell proliferation was increased in the presence of 12.5 μM EPA (approximately twofold) and 12.5 μM DHA (approximately 1.5-fold). EPA and DHA (25 μM) also decreased production of the key immunoregulatory cytokines IL-10, TNF-α, and INF-γ. EPA and DHA changed the expression of specific genes, but this effect was more marked for EPA (25.9% of genes investigated) compared with DHA (8.4% of genes investigated). EPA and DHA affected the expression of genes clustered as: cytokines, signal transduction, transcription, cell cycle, defense and repair, apoptosis, cell adhesion, cytoskeleton, and hormones. The most remarkable changes were observed in the genes of signal transduction and transcription. These results led us to conclude that the mechanism of DHA and EPA effects on B-lymphocyte functions includes regulation of gene expression. Thus, the ingestion of fish oil, a rich source of EPA and DHA, may have a strong effect on B-lymphocyte function in vivo. However, remarkable differences were observed between DHA and EPA, demonstrating that specific effects of these FA may be responsible for the marked differences in edible oil effects on immune function in vivo reported by others.

Fatty acid control of nitric oxide production by macrophages

Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF‐κB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1–10 μM) and inhibited it at high doses (50–200 μM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 μM) was observed. The activity of NF‐κB was time‐dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.

Regulation of interleukin-2 signaling by fatty acids in human lymphocytes

Docosahexaenoic (DHA; C22:6 n-3), eicosapentaenoic (EPA; C20:5 n-3), palmitic (PA; C16:0), and stearic (SA; C18:0) acids decrease lymphocyte proliferation in concentrations of >50 μM, as observed in our previous study. However, oleic acid (OA; C18:1 n-9) and linoleic acid (LA; C18:2 n-6) increase lymphocyte proliferation at 25 μM. In this study, the effect of these FAs on the interleukin-2 (IL-2) signaling pathway in human lymphocytes was investigated. Cells were isolated from heparinized venous blood of healthy human donors by density-gradient sedimentation. Cells were stimulated with 5 μg/ml concanavalin A and treated with FAs in the absence or presence of IL-2 for 1 hour. CD25-α externalization was analyzed by flow cytometry, and Janus kinase 1 (JAK1), JAK3, signal transducer and activator of transcription (STAT) 5, extracellular signal-regulated kinases (ERKs) 1 and 2, Akt, and protein kinase C (PKC)-ζ phosphorylation were analyzed by Western blotting. The expression of CD25-α at the cell surface was increased by DHA, SA, and PA but was unaffected by EPA, OA, and LA. PA, SA, DHA, and EPA decreased JAK1, JAK3, STAT5, and Akt phosphorylation induced by IL-2, but OA and LA did not cause any effect. OA and LA increased ERK1/2 phosphorylation, whereas the other FAs caused a marked decrease. PKC-ζ phosphorylation was decreased by OA and LA and was not altered by the remaining FAs. In conclusion, the inhibitory effect of PA, SA, DHA, and EPA on lymphocyte proliferation observed in our previous study was attributable to a decrease in JAK/STAT, ERK, and Akt pathways activated by IL-2. Probably, OA and LA stimulated lymphocyte proliferation by increasing ERK1/2 phosphorylation through PKC-ζ activation. The inhibition of JAK1, JAK3, STAT5, ERK1/2, and Akt phosphorylation caused by DHA, SA, and PA is associated with an alteration of CD25 expression at the cell surface.

Effect of olive oil-based emulsion on human lymphocyte and neutrophil death.

BACKGROUND The incorporation of lipid emulsions in parenteral diets is a requirement for energy and essential fatty acid supply to critically ill patients. The most frequently used IV lipid emulsions (LE) are composed with long-chain triacylglycerols rich in omega-6 polyunsaturated fatty acids (PUFA) from soybean oil, but these LE promote lymphocyte and neutrophil death. A new emulsion containing 20% soybean oil and 80% olive oil rich in omega-9 monounsaturated fatty acids (MUFA) has been hypothesized not to cause impairment of immune function. In this study, the toxicity of an olive oil-based emulsion (OOE) on lymphocytes and neutrophils from healthy volunteers was investigated. METHODS Twenty volunteers were recruited and blood was collected before a 6-hour infusion of an OOE, immediately after infusion, and again 18 hours postinfusion. Lymphocytes and neutrophils were isolated by gradient density. The cells were studied immediately after isolation and after 24 hours or 48 hours in culture. The following determinations were carried out: triacylglycerol levels and fatty acid composition and levels in plasma, lymphocyte proliferation, production of reactive oxygen species, and parameters of lymphocyte and neutrophil death (viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, and neutral lipid accumulation). RESULTS OOE decreased lymphocyte proliferation, provoked lymphocyte necrosis, and had no effect on the proportion of viable neutrophils. The mechanism of cell death induced by OOE involved neutral lipid accumulation but had no effect on mitochondrial membrane depolarization. CONCLUSIONS The OOE given as a single dose of 500 mL induced low toxicity to lymphocytes from healthy volunteers, probably by necrosis.