Effects of oat ß-glucan on the macrophage cytokine response to herpes simplex virus 1 infection in vitro.

Oat ß-glucan can counteract the increased risk for Herpes Simplex Virus 1 (HSV-1) infection in mice, the effects of which have, at least in part, been attributed to macrophages. However, the specific responses of macrophages to oat ß-glucan treatment in this model have yet to be elucidated. We examined the effects of varying doses of oat ß-glucan on the pro-inflammatory cytokine response in both peritoneal and lung macrophages with and without exposure to HSV-1 infection in vitro. Peritoneal and lung macrophages were obtained from mice and cultured with varying concentrations of oat ß-glucan (0 (control), 10, 100, and 1,000 µg) for 24 h and supernatants were collected. A standardized dose of HSV-1 was added for a second 24 h incubation period after which supernatants were again collected. Samples were analyzed for interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α) using enzyme linked immunosorbent assay (ELISA). In most cases, oat ß-glucan resulted in a dose-dependent increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) in lung and peritoneal macrophages with and without exposure to HSV-1 infection. When comparing across macrophage source, this response was greater for IL-1ß and IL-6 in peritoneal macrophages and for TNF-α in lung macrophages. This may be a mechanism for the decreased risk for HSV-1 infection following oat ß-glucan feedings in mice.

Anti-inflammatory activity of select sorghum (Sorghum bicolor) brans.

The bran fractions of certain varieties of sorghum (Sorghum bicolor) grain are rich sources of phytochemicals and antioxidants. In this article, the anti-inflammatory actions of extracts of select sorghum brans were evaluated in two experimental inflammatory systems: (1) the release of cytokines by lipopolysaccharide-activated peripheral blood mononuclear cells and (2) 12-O-tetradecanoylphorbol acetate (TPA)-induced ear edema in mice. A 1:200 dilution of a 10% (wt/vol) ethanol extract of black sorghum bran significantly inhibited the secretion of the pro-inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha. Ethanolic extracts of both black and sumac varieties of sorghum bran significantly reduced edema in inflamed ears as measured by ear thickness and ear punch weight 6 hours following TPA application. The degree of inhibition was similar to that observed with indomethacin. Black sorghum bran significantly diminished the increase in myeloperoxidase activity 24 hours following the application of TPA. No anti-inflammatory activity was observed with white and Mycogen sorghum bran varieties or with oat, wheat, or rice brans in the mouse ear model. The anti-inflammatory activity observed with these brans correlated with their phenolic content and antioxidant activity. These results demonstrate that select sorghum bran varieties possess significant anti-inflammatory activity.

Benefits of oat beta-glucan and sucrose feedings on infection and macrophage antiviral resistance following exercise stress.

Oat beta-glucan can counteract the exercise-induced increased risk for upper respiratory tract infection (URTI) in mice, which is at least partly mediated by its effects on lung macrophages. Substantial evidence in humans indicates that carbohydrate-containing sports drinks can offset the decreased immune function associated with stressful exercise. However, no studies in animals or humans have directly examined their effects on URTI using a controlled virus-challenge model. We examined the effects of sucrose feedings alone and in combination with oat beta-glucan on susceptibility to infection and on macrophage antiviral resistance in mice following stressful exercise. These effects were also examined in rested, nonimmunocompromised control mice. Mice were assigned to one of four groups: H(2)O (water), sucrose (S), oat beta-glucan (ObetaG), and sucrose + oat beta-glucan (S+ObetaG). ObetaG and S treatments consisted of a solution of 50% ObetaG and 6% sucrose, respectively, and were administered in drinking water for 10 consecutive days. Exercise consisted of a treadmill run to fatigue performed on three consecutive days. Mice were then intranasally inoculated with a standardized dose of herpes simplex virus 1 (HSV-1) and monitored for morbidity and mortality for 21 days. Additional mice were used to determine macrophage antiviral resistance. In the exercise experiment, S, ObetaG, and S+ObetaG all reduced morbidity (P < 0.05), while only S+ObetaG reduced mortality (P < 0.05). Macrophage antiviral resistance was also increased in S, ObetaG, and S+ObetaG treatments (P < 0.05). In resting controls, S and S+ObetaG reduced morbidity and mortality (P < 0.05) and showed a trend toward increased macrophage antiviral resistance. There was no significant additive effect of S and ObetaG in either control or exercised animals. These data extend our previous work on the benefits of oat beta-glucan to show that sucrose feedings have similar effects on susceptibility to respiratory infection and macrophage antiviral resistance in both resting controls and following exercise stress.

Effect of IL-6 deficiency on susceptibility to HSV-1 respiratory infection and intrinsic macrophage antiviral resistance.

Cytokines play important roles in the mechanisms of disease development. Interleukin-6 (IL-6) is associated with clearance of herpes simplex virus (HSV) infections and in virus-induced immunopathology. However, the importance of IL-6 in host defense against HSV-1 respiratory infection is unknown. This study tested the effect of knockout mice deficient for IL-6 on susceptibility to HSV-1 respiratory infection and on intrinsic macrophage antiviral resistance to HSV-1. Control C57BL/6 IL-6+/+ mice and IL-6 knockout mice (IL-6-/-) were intranasally inoculated with 50 microL of a standardized dose (3.2 x 10(5)) of HSV-1. Morbidity, mortality, and symptom severity were monitored for 21 days. A subset of mice was sacrificed at 48-h postinfection and lungs were analyzed for viral titers. Peritoneal macrophages were obtained from a third set of mice and assayed for antiviral resistance to HSV-1. IL-6-/- increased morbidity by 84%, mortality by 84%, and symptom severity score on days 7.5 through 11 (p < 0.05). IL-6-/- increased virus titers in the lung 4-fold (p < 0.01) and resulted in a decrease in macrophage antiviral resistance (p < 0.001). Results indicate that IL-6 plays an important role in susceptibility to respiratory infection in mice, which may be mediated at least in part by its effect on macrophage antiviral resistance.

Galectin-3 gene inactivation reduces atherosclerotic lesions and adventitial inflammation in ApoE-deficient mice.

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.

Quercetin reduces illness but not immune perturbations after intensive exercise.

PURPOSE: To investigate the effects of quercetin supplementation on incidence of upper respiratory tract infections (URTI) and exercise-induced changes in immune function. METHODS: Trained male cyclists (N=40) were randomized to quercetin (N=20) or placebo (N=20) groups and, under double-blind procedures, received 3 wk quercetin (1000 mg.d(-1)) or placebo before, during, and for 2 wk after a 3-d period in which subjects cycled for 3 h.d(-1) at approximately 57% Wmax. Blood and saliva samples were collected before and after each of the three exercise sessions and assayed for natural killer cell activity (NKCA), PHA-stimulated lymphocyte proliferation (PHA-LP), polymorphonuclear oxidative-burst activity (POBA), and salivary IgA output (sIgA). RESULTS: Pre- to postexercise changes in NKCA, PHA-LP, POBA, and sIgA did not differ significantly between quercetin and placebo groups. URTI incidence during the 2-wk postexercise period differed significantly between groups (quercetin=1/20 vs placebo=9/20, Kaplan-Meier analysis statistic=8.31, P=0.004). CONCLUSION: Quercetin versus placebo ingestion did not alter exercise-induced changes in several measures of immune function, but it significantly reduced URTI incidence in cyclists during the 2-wk period after intensified exercise.

Quercetin's influence on exercise-induced changes in plasma cytokines and muscle and leukocyte cytokine mRNA.

Trained male cyclists (n = 40) ingested quercetin (Q; n = 20) (1,000 mg/day) or placebo (P; n = 20) supplements under randomized, double-blinded methods for 3 wk before and during a 3-day period in which subjects cycled for 3 h/day at approximately 57% maximal work rate. Blood samples were collected before and after each exercise session and assayed for plasma IL-6, IL-10, IL-1ra, IL-8, TNF-alpha, and monocyte chemoattractant protein 1, and leukocyte IL-10, IL-8, and IL-1ra mRNA. Muscle biopsies were obtained before and after the first and third exercise sessions and assayed for NF-kappaB and cyclooxygenase-2 (COX-2), IL-6, IL-8, IL-1beta, and TNF-alpha mRNA. Postexercise increases in plasma cytokines did not differ between groups, but the pattern of change over the 3-day exercise period tended to be lower in Q vs. P for IL-8 and TNF-alpha (P = 0.094 for both). mRNA increased significantly postexercise for each cytokine measured in blood leukocyte and muscle samples. Leukocyte IL-8 and IL-10 mRNA were significantly reduced in Q vs. P (interaction effects, P = 0.019 and 0.012, respectively) with no other leukocyte or muscle mRNA group differences. Muscle NF-kappaB did not increase postexercise and did not differ between Q and P. Muscle COX-2 mRNA increased significantly postexercise but did not differ between Q and P. In summary, 1 g/day quercetin supplementation by trained cyclists over a 24-day period diminished postexercise expression of leukocyte IL-8 and IL-10 mRNA, indicating that elevated plasma quercetin levels exerted some effects within the blood compartment. Quercetin did not, however, influence any of the muscle measures, including NF-kappaB content, cytokine mRNA, or COX-2 mRNA expression across a 3-day intensified exercise period.

Oat beta-glucan effects on neutrophil respiratory burst activity following exercise.

UNLABELLED: Fatiguing exercise has been associated with a decrease in certain functions of neutrophils, whereas moderate exercise has generally been associated with an increase. Consumption of oat beta-glucan (ObetaG), a soluble fiber and mild immune system enhancer, may offset the immunosuppression associated with intense training and perhaps further enhance the benefits of moderate exercise. PURPOSE: To test the effects of ObetaG consumption on neutrophil function and number after both moderate and fatiguing exercise. METHODS: Male mice were assigned to one of six treatment groups. Fatiguing exercise mice (Ftg-ObetaG and Ftg-H2O) ran to volitional fatigue on a treadmill for three consecutive days, and moderate exercise mice (Mod-ObetaG and Mod-H2O) ran for six consecutive days for 1 h. Control mice (Con-ObetaG and Con-H2O) were exposed to the treadmill environment but did not run. ObetaG was consumed in the drinking water (approximately 0.6 mL x d(-1)) for 10 consecutive days. After rest or exercise on the last day of training, mice were given a 1-mL i.p. injection of thioglycollate. Mice were sacrificed 3 h later; neutrophils were harvested from the peritoneal cavity and counted, and their respiratory burst activity was measured using flow cytometry. RESULTS: Both moderate exercise and ObetaG increased neutrophil burst activity, whereas fatiguing exercise had no effect. Neutrophil number was increased by fatiguing exercise and ObetaG, but not moderate exercise. There were no additive effects of exercise and ObetaG on either of these variables. CONCLUSION: These data suggest that although not additive in their effects, both ObetaG and exercise can alter overall neutrophil respiratory burst activity (number and/or function), but only ObetaG increased both number and function, which may have important ramifications for defense against infection.

Curcumin effects on inflammation and performance recovery following eccentric exercise-induced muscle damage.

Downhill running is associated with fiber damage, inflammation, delayed-onset muscle soreness, and various functional deficits. Curcumin, a constituent of the Indian spice turmeric has been investigated for its anti-inflammatory activity and may offset some of the damage and functional deficits associated with downhill running. This study examined the effects of curcumin on inflammation and recovery of running performance following downhill running in mice. Male mice were assigned to downhill placebo (Down-Plac), downhill curcumin (Down-Cur), uphill placebo (Up-Plac), or uphill curcumin (Up-Cur) groups and run on a treadmill at 22 m/min at -14% or +14% grade, for 150 min. At 48 h or 72 h after the up/downhill run, mice (experiment 1) underwent a treadmill performance run to fatigue. Another subset of mice was placed in voluntary activity wheel cages following the up/downhill run (experiment 2) and their voluntary activity (distance, time and peak speed) was recorded. Additional mice (experiment 3) were killed at 24 h and 48 h following the up/downhill run, and the soleus muscle was harvested for analysis of inflammatory cytokines (IL-1beta, IL-6, and TNF-alpha), and plasma was collected for creatine kinase analysis. Downhill running decreased both treadmill run time to fatigue (48 h and 72 h) and voluntary activity (24 h) (P < 0.05), and curcumin feedings offset these effects on running performance. Downhill running was also associated with an increase in inflammatory cytokines (24 h and 48 h) and creatine kinase (24 h) (P < 0.05) that were blunted by curcumin feedings. These results support the hypothesis that curcumin can reduce inflammation and offset some of the performance deficits associated with eccentric exercise-induced muscle damage.

Quercetin ingestion does not alter cytokine changes in athletes competing in the Western States Endurance Run.

The purpose of this study was to measure the influence of quercetin on plasma cytokines, leukocyte cytokine mRNA, and related variables in ultramarathoners competing in the 160-km Western States Endurance Run (WSER). Sixty-three runners were randomized to quercetin and placebo groups and under double-blinded methods ingested 1000 mg/day quercetin for 3 weeks before the WSER. Thirty-nine of the 63 subjects (n = 18 for quercetin, n = 21 for placebo) finished the race and provided blood samples the morning before the race and 15-30 min postrace. Significant prerace to postrace WSER increases were measured for nine proinflammatory and anti-inflammatory plasma cytokines, cortisol (quercetin = 94%, placebo = 96%), serum C-reactive protein (CRP) (mean +/- SE absolute increase, quercetin = 31.8 +/- 4.2, placebo = 38.2 +/- 5.0 mg/L), and creatine kinase (CK) (quercetin = 21,575 +/- 3,977, placebo = 19,455 +/- 3,969 U/L), with no significant group differences. Interleukin-6 (IL-6) mRNA did not change post-WSER, with a significant decrease measured for leukocyte IL-8 mRNA (0.21 +/- 0.03-fold and 0.25 +/- 0.04-fold change from rest, quercetin and placebo, respectively) and significant increases for IL-1Ra mRNA (1.43 +/- 0.18-fold and 1.40 +/- 0.16-fold change, quercetin and placebo, respectively) and IL-10 mRNA (12.9 +/- 3.9-fold and 17.2 +/- 6.1-fold change, quercetin and placebo, respectively), with no significant differences between groups. In conclusion, quercetin ingestion (1 g/day) by ultramarathon athletes for 3 weeks before a competitive 160-km race significantly increased plasma quercetin levels but failed to attenuate muscle damage, inflammation, increases in plasma cytokine and hormone levels, and alterations in leukocyte cytokine mRNA expression.

Influence of carbohydrate on immune function following 2 h cycling.

The influence of carbohydrate compared with placebo ingestion on changes in immune cell counts and functions following 2 h intensive cycling was studied in 12 trained cyclists who functioned as their own controls. The subjects performed two tests 2 weeks apart where they cycled for 2 h at approximately 64% Watts(max) while receiving 4 mL x kg(-1) x 15 min(-1) carbohydrate (6%) (Cho) or placebo (Pla) beverages. Blood samples were collected 30 min preexercise, and immediately and 1 h postexercise. The samples were assayed for plasma cortisol and epinephrine, blood leukocyte subset counts, PHA-induced lymphocyte proliferation, and natural killer cell activity (NKCA). Compared with Pla ingestion, Cho attenuated exercise-induced changes in plasma cortisol, blood neutrophil, and monocyte counts, but not in total blood lymphocyte, T cell, and NK cell counts, PHA-induced lymphocyte proliferation, and NKCA. Thus despite a strong attenuating influence of carbohydrate ingestion on exercise-induced changes in plasma cortisol and blood neutrophil and monocyte counts, other immune measures related to lymphocyte subset counts, and function were unaffected.

Role of brain IL-1beta on fatigue after exercise-induced muscle damage.

Brain cytokines, induced by various inflammatory challenges, have been linked to sickness behaviors, including fatigue. However, the relationship between brain cytokines and fatigue after exercise is not well understood. Delayed recovery of running performance after muscle-damaging downhill running is associated with increased brain IL-1beta concentration compared with uphill running. However, there has been no systematic evaluation of the direct effect of brain IL-1beta on running performance after exercise-induced muscle damage. This study examined the specific role of brain IL-1beta on running performance (either treadmill or wheel running) after uphill and downhill running by manipulating brain IL-1beta activity via intracerebroventricular injection of either IL-1 receptor antagonist (ra; downhill runners) or IL-1beta (uphill runners). Male C57BL/6 mice were assigned to the following groups: uphill-saline, uphill-IL-1beta, downhill-saline, or downhill-IL-1ra. Mice initially ran on a motor-driven treadmill at 22 m/min and -14% or +14% grade for 150 min. After the run, at 8 h (wheel cage) or 22 h (treadmill), uphill mice received intracerebroventricular injections of IL-1beta (900 pg in 2 microl saline) or saline (2 microl), whereas downhill runners received IL-1ra (1.8 microg in 2 microl saline) or saline (2 microl). Later (2 h), running performance was measured (wheel running activity and treadmill run to fatigue). Injection of IL-1beta significantly decreased wheel running activity in uphill runners (P<0.01), whereas IL-1ra improved wheel running in downhill runners (P<0.05). Similarly, IL-1beta decreased and Il-1ra increased run time to fatigue in the uphill and downhill runners, respectively (P<0.01). These results support the hypothesis that increased brain IL-1beta plays an important role in fatigue after muscle-damaging exercise.

Gender differences in macrophage antiviral function following exercise stress.

INTRODUCTION: In male mice, exhaustive exercise increases susceptibility to respiratory infection following intranasal inoculation with herpes simplex virus-1 (HSV-1), whereas moderate exercise decreases the risk of infection. These responses have been linked with altered macrophage antiviral resistance, among other immune mechanisms. Female mice appear to be better protected from death than male mice following HSV-1 infection, although their response to exercise stress is similar. The possible immune mechanisms, however, have not been explored. PURPOSE: This study was conducted to examine gender differences in macrophage antiviral resistance following repeated moderate and exhaustive treadmill exercise. METHODS: Male (M, N = 36) and female (F, N = 36) CD-1 mice were randomly assigned to moderate exercise (Mod), exhaustive exercise (Exh), or control (C) groups. Exercise was done daily for 3 d; moderate exercise consisted of treadmill running for 90 min, whereas exhaustive exercise consisted of running to volitional fatigue (approximately 50 min). RESULTS: Females had greater macrophage antiviral resistance to HSV-1 than males in C and Mod (P < 0.05), but not Exh; Mod increased resistance, whereas Exh decreased resistance similarly in both genders (P < 0.001). CONCLUSIONS: These data suggest that altered macrophage antiviral resistance to HSV-1 may contribute to gender differences in in vivo resistance to HSV-1 respiratory infection at rest, as well as following moderate and exhaustive exercise.

Antiinflammatory properties of the muscadine grape (Vitis rotundifolia).

The muscadine grape possesses one of the highest antioxidant levels among fruits; yet, the effect of this fruit on mammalian metabolic systems has not received significant attention. To examine the antiinflammatory properties of the muscadine, grape skins were dried, pulverized, and extracted (10% w/v) with 50% ethanol. The extract was then tested in two different assays: the release of superoxide in phorbol myristate acetate-activated neutrophils and the release of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-beta), and interleukin-6 (IL-6)] by lipopolysaccharide-activated peripheral blood mononuclear cells. The release of superoxide and cytokines was inhibited by increasing concentrations of the extract. A 1:100 dilution of the extract inhibited superoxide release by approximately 60% while the release of TNF-alpha and IL-1beta was reduced at a dilution of 1:200 by approximately 15 and 90%, respectively (all P < 0.05). The inhibition pattern on the release of IL-6 was similar to that seen with TNF-alpha. In a related in vivo study, rats were fed a diet containing 5% (wt/wt) dried muscadine grape skins for 14 days and then were injected with carrageenan in the foot pad. After 3 h, paw edema was measured and the rats on the grape skin diet had approximately 50% less paw edema than controls (P < 0.05). These results demonstrate that the muscadine grape skin powder possesses significant in vitro and in vivo antiinflammatory properties.

Gender differences in viral infection after repeated exercise stress.

UNLABELLED: Fatiguing exercise can increase susceptibility to respiratory infection after intranasal inoculation with herpes simplex virus-1 (HSV-1) in male mice. Although gender differences in susceptibility to certain pathogens do exist, it is unknown whether female mice will respond differently than males in response to strenuous exercise and HSV-1 infection. PURPOSE: To test the effects of gender on susceptibility to HSV-1 respiratory infection after repeated exhaustive exercise. METHODS: Male (N = 86) and female (N = 89) CD-1 mice (approximately 60 d old) were randomly assigned to exercise (Ex) or control (C) groups. Exercise consisted of 3 d of treadmill running at 36 m x min(-1) at 8% grade until volitional fatigue (135 +/- 5min). Fifteen minutes after the last bout of exercise, Ex and C mice were inoculated intranasally with a standard dose (LD30) of HSV-1. Mice were monitored for 21 d for morbidity (time to sickness and symptom severity) and mortality. RESULTS: Run time to fatigue was significantly longer in females than males (P = 0.027). Significant gender differences in susceptibility to infection were found after exercise stress. In males, exercise stress resulted in increased morbidity (66%, P < 0.05) and mortality (30%, P < 0.05) whereas in females, exercise stress only resulted in increased morbidity (66%, P < 0.05). CONCLUSION: Results suggest that although males and females have similar morbidity rates after infection and exercise stress, females recover to a greater extent and are ultimately better protected from death.

Effects of oat beta-glucan on innate immunity and infection after exercise stress.

PURPOSE: To test the effects of oat beta-glucan (ObetaG) on respiratory infection, macrophage antiviral resistance, and NK cytotoxicity. METHODS: Mice were randomly assigned to one of four groups: Ex-H2O, Ex-ObetaG, Con-H2O, or Con-ObetaG. ObetaG was fed in the drinking water for 10 d before intranasal inoculation of HSV-1 or sacrifice. Exercise consisted of treadmill running to volitional fatigue (approximately 140 min) for three consecutive days. Fifteen minutes after the last bout of exercise or rest, mice (N = 24) were intranasally inoculated with a standardized dose of HSV-1. Mice were monitored twice daily for morbidity and mortality. Additional mice were sacrificed after exercise, peritoneal macrophages were obtained via i.p. lavage and assayed for antiviral resistance to HSV-1 (N = 18), and spleens were harvested and assayed for NK cell cytotoxicity (N = 12). RESULTS: Exercise stress was associated with a 28% increase in morbidity (P = 0.036) and 18% increase in mortality (P = 0.15). Ingestion of ObetaG before infection prevented this increase in morbidity (P = 0.048) and mortality (P = 0.05). Exercise stress was associated with a decrease in macrophage antiviral resistance (P = 0.007), which was blocked by ingestion of ObetaG (P < 0.001). There were no effects of exercise or ObetaG on NK cytotoxicity. CONCLUSION: These data suggest that daily ingestion of ObetaG may offset the increased risk of URTI associated with exercise stress, which may be mediated, at least in part, by an increase in macrophage antiviral resistance.

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins.

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.