Homocysteine stimulates inducible nitric oxide synthase expression in macrophages: Antagonizing effect of ginkgolides and bilobalide

Hyperhomocysteinemia is an independent risk factor for atherosclerotic diseases. Inducible nitric oxide synthase (iNOS) is mainly expressed in macrophages upon stimulation. Overproduction of nitric oxide (NO) by iNOS can exacerbate the development of atherosclerosis. Our previous studies demonstrated that the extract of ginkgo biloba leaves (EGb) inhibited the iNOS-mediated NO production in monocyte-derived macrophage. We also reported that homocysteine could stimulate monocyte chemoattractant protein-1 (MCP-1) expression in vascular cells causing enhanced monocyte chemotaxis. The objective of the present study was to investigate the effect of homocysteine on iNOS-mediated NO production in macrophages and the antagonizing effect of EGb. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine for various time periods. Homocysteine at concentrations of 0.05–0.1 mM significantly stimulated NO production and iNOS activity in macrophages via increased expression of iNOS mRNA and protein. The increased iNOS expression was associated with activation of nuclear factor-kappa B (NF-B) arising from reduced expression of inhibitor protein (IB) mRNA as well as increased phosphorylation of IB protein in homocysteine-treated cells. EGb and its terpenoids (ginkgolide A, ginkgolide B and bilobalide) could antagonize the homocysteine effect on iNOS expression in macrophages via their antioxidant effect resulting in attenuation of NF-B activation. Taken together, our results have demonstrated that homocysteine, at pathophysiological concentrations, stimulates iNOS-mediated NO production in macrophages. EGb and its terpenoids can antagonize such stimulatory effect via antioxidation and attenuation of NF-B activation.     

Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei.

Penicillium marneffei is an important opportunistic fungal pathogen. The mechanisms of host defense against P. marneffei are not fully understood. In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P. marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages. P. marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not. These results suggested that O2- played some role in the fungicidal activity of macrophages. However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P. marneffei yeast cells. This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system. On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase. Our results suggested that O2- does not function as the killing mediator of macrophages against P. marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.     

Induction of inducible nitric oxide (NO) synthase mRNA and NO production in macrophages infected with influenza A/PR/8 virus and stimulated with its ether-split product

We investigated the inductive activity of infective influenza A/PR/8/34 (PR8) virus and its ether-split product (ESP) on the expression of inducible nitric oxide (NO) synthase (iNOS) and NO production in RAW264.7 (RAW) cells, a murine macrophage (M psi) cell line, and thioglycolate-elicited peritoneal M psi (TPM). In both cells, PR8 virus infection induced iNOS mRNA between 4 hr and 24 hr, attaining a peak value at 12 hr. In correlation with induction of iNOS mRNA, NO amounts increased significantly from 12 to 24 hr. Moreover, this study demonstrated that ESP with the same hemagglutination titer as PR8 virus could induce iNOS mRNA and NO production, although the inductive activity of ESP was weaker than that of PR8 virus. Considering the dual role (beneficial and detrimental roles) of NO on certain inflammatory disorders and virus infections, the inductive activity of influenza virus on the iNOS-mediated NO production independent of its infectivity might contribute to a modification of influenza virus infection.     

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.     

Combined effect of testosterone and apocynin on nitric oxide and superoxide production in PMA-differentiated THP-1 cells

Human inducible nitric oxide synthase (iNOS) is most readily observed in macrophages from patients with inflammatory diseases like atherosclerosis. The aim of the present study was to find out the combined effect of male sex hormone; testosterone and apocynin (NADPH oxidase inhibitor) on cytokine-induced iNOS production. THP-1 cells were differentiated into macrophages by phorbol myristate acetate (PMA). Expression of iNOS was induced by the addition of cytokine mixture? Testosterone was added at different concentrations (10(-6)-10(-12) M) with apocynin (1 mM). Testosterone (10(-8), 10(-10) M) inhibited NOx production in cytokine-added THP-1 cells which was further confirmed by quantikine assay of iNOS protein and RT-PCR analysis. Testosterone treatment decreased 40% of superoxide anion production. Testosterone showed inhibition of NADPH oxidase, especially expression of p67phox and p47phox (cytosol subunits). Addition of testosterone with apocynin further decreased the expression of p67phox and p47phox subunits of NADPH oxidase. The findings of the present study suggest that, testosterone; the male androgen plays an important role in the prevention of atherogenesis. Even though apocynin does not have any role on NO production, addition of apocynin together with testosterone is effective in suppressing iNOS activity.     

Concomitant production of nitric oxide and superoxide in human macrophages

Many harmful effects of nitric oxide are caused by the reaction of NO with superoxide anion. The present study was carried out to find out the concomitant production of superoxide and to investigate a suitable inhibitor of NO, which is produced by iNOS. THP-1 cells were differentiated into macrophages by PMA and cytokine. Addition of L-NAME showed decrement in superoxide production. Addition of apocynin, aminoguanidine or ONO 1714 brought about a significant reduction in superoxide production. The expressions of p67 and p47(phox) were reduced by the addition of apocynin, aminoguanidine or ONO 1714 whereas xanthine oxidase and cyclooxygenase did not have a major role in superoxide production. The results of the present study show that iNOS and NADPH oxidase play an important role in superoxide release. It suggests that addition of iNOS inhibitor together with apocynin may be more effective in case of therapeutic application in disease conditions like atherosclerosis.     

Cobalt-60 gamma radiation increased the nitric oxide generation in cultured rat vascular smooth muscle cells

Radiation is a promising and new treatment for restenosis following angioplasty. Nitric oxide has been proposed as a potential "anti-restenotic" molecule. We radiated the cultured rat vascular smooth muscle cells with Cobalt-60 gamma radiation at doses of 14 and 25Gy and observed nitrite production, cGMP content, L-arginine uptake, inducible nitric oxide synthase (iNOS) activity, and the gene expression of iNOS. Results showed that radiation at doses of 14 and 25Gy increased cGMP content by 92.4% and 86.4%, respectively. Radiation at the dose of 25Gy increased the iNOS activity and nitrite content, but radiation at the dose of 14Gy had no significant effect on iNOS activity and NO production. Both doses of radiation significantly decreased the L-arginine transport. Radiation at the doses of 14 and 25Gy increased iNOS gene expression significantly, which was consistent with the effect of radiation on iNOS activity. In conclusion, radiation induces the NO generation by up-regulating the iNOS activity.     

Nitric oxide down-regulates caveolin-1 expression in rat brains during focal cerebral ischemia and reperfusion injury

As a signalling molecule of the integral membrane protein family, caveolin participates in cellular signal transduction via interaction with other signalling molecules. The nature of interaction between nitric oxide (NO) and caveolin in the brain, however, remains largely unknown. In this study we investigated the role(s) of NO in regulating caveolin-1 expression in rat ischemic brains with middle cerebral artery occlusion (MCAO). Exposure to 1 h ischemia induced the increases in neuronal nitric oxide synthase (nNOS) and NO concentration with concurrent down-regulation of caveolin-1 expression in the ischemic core of rat brains. Subsequent 24 h or more reperfusion time led to an increase in inducible NOS (iNOS) expression and NO production, as well as a decline of caveolin-1 protein at the core and penumbra of the ischemic brain. Afterwards, NOS inhibitors and an NO donor were utilized to clarify the link between NO production and caveolin-1 expression in the rats with 1 h ischemia plus 24 h reperfusion. N(G)-nitro-l-arginine methyl ester (L-NAME, a non-selective NOS inhibitor), N(6)-(1-iminoethyl)-lysine (NIL, an iNOS inhibitor), and 7-nitroindazole (7-NI, a nNOS inhibitor) prevented the loss of caveolin-1 in the core and penumbra of the ischemic brain, whereas l-N(5)-(1-iminoethyl)-ornithine (L-NIO, an endothelial NOS inhibitor) showed less effect than the other NOS inhibitors. S-Nitroso-N-acetylpenicillamine (SNAP, a NO donor) down-regulated the expression of caveolin-1 protein in normal and ischemic brains. These results, when taken together, suggest that NO modulates the expression of caveolin-1 in the brain and that the loss of caveolin-1 is associated with NO production in the ischemic brain.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

Trichosanthin inhibits antigen-specific T cell expansion through nitric oxide-mediated apoptosis pathway

Trichosanthin (TCS) has been found to exhibit inflammation-suppressing effect but the underlying mechanisms are not clear. In this study, we found that TCS inhibited OVA-specific T cell proliferation in a dose-dependent manner. Such inhibition was correlated with enhanced cell death. At the same time, inducible nitric oxide synthase (iNOS) mRNA expression and protein levels were found increased in cells treated with TCS, and nitric oxide (NO) production by cells was elevated in the presence of TCS. When L-NIL, the specific inhibitor of iNOS, was added to suppress NO production induced by TCS, OVA-specific cell death was significantly inhibited, meanwhile, thymidine incorporation of cells was rescued towards normal levels. These results indicate that TCS could inhibit antigen-specific T cell activation via NO-mediated apoptosis pathway.     

Induction of nitric oxide synthase expression by Vibrio vulnificus cytolysin.

The pore-forming cytolysin of Vibrio vulnificus (VVC) causes severe hypotension and vasodilatation in vivo. Under the condition of bacterial sepsis, large amounts of nitric oxide (NO) produced by inducible NO synthase (iNOS) can contribute to host-induced tissue damage causing hypotension and septic shock. In this study, we investigated the effect of purified VVC on NO production in mouse peritoneal macrophages. VVC induced NO production in the presence of interferon-gamma. Increased NO production was not affected by polymyxin B, and heat inactivation of cytolysin abolished the NO-inducing capability. NO production was induced at the same concentration range of cytolysin for pore formation, as evidenced by the release of preloaded 2-deoxy-d-[(3)H]glucose. At the higher concentrations of cytolysin causing the depletion of cellular ATP, no NO production was observed. Increased expression of iNOS and activation of NFkappaB by VVC were confirmed by Western blotting and gel shift assay, respectively. These results suggest the role of cytolysin as an inducer of iNOS and NO production in macrophage and as a possible virulence determinant in V. vulnificus infection.     

Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism

BACKGROUND: Using specific antibodies against bovine Cu/Zn-superoxide dismutase (EC 1.15.1.1, SOD1) we demonstrated that anti-SOD antibodies (IgG1) are able to promote the intracellular translocation of the antioxidant enzyme. The transduction signalling mediated by IgG1 immune complexes are known to promote a concomitant production of superoxide and nitric oxide leading to the production of peroxynitrites and cell death by apoptosis. The Fc-mediated intracellular delivery of SOD1 thus limited the endogenous production of superoxide. It was thus of interest to confirm that in the absence of superoxide anion, the production of nitric oxide protected cells against apoptosis. Study in greater detail clearly stated that under superoxide anion-free conditions, nitric oxide promoted the cell antioxidant armature and thus protected cells against redox-induced apoptosis. MATERIALS AND METHODS: The murine macrophage cell-lines J774 A1 were preactivated or not with interferon-gamma and were then stimulated by IgG1 immune complexes (IC), free SOD1 or SOD1 IC and superoxide anion, nitric oxide, peroxynitrite, and tumor necrosis factor-alpha (TNF-alpha) production was evaluated. The redox consequences of these activation processes were also evaluated on mitochondrial respiration and apoptosis as well as on the controlled expression of the cellular antioxidant armature. RESULTS: We demonstrated that SOD1 IC induced a Fcgamma receptor (FcgammaR)-dependent intracellular delivery of the antioxidant enzyme in IFN-gamma activated murine macrophages (the J774 AI cell line). The concomitant stimulation of the FcyR and the translocation of the SOD1 in the cytoplasm of IFN-gamma-activated macrophages not only reduced the production of superoxide anion but also induced the expression of the inducible form of nitric oxide synthase (iNOS) and the related NO production. This inducing effect in the absence of superoxide anion production reduced mitochondrial damages and cell death by apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism.     

Alachlor and carbaryl suppress lipopolysaccharide-induced iNOS expression by differentially inhibiting NF-kappaB activation

Nitric oxide (NO) produced by macrophages plays an important role in host defense and inflammation. We found that two agrochemicals, alachlor and carbaryl, inhibit lipopolysaccharide (LPS)-induced NO production by macrophages. In the present study, we investigated this inhibitory mechanism in RAW 264 cells. Both chemicals inhibited LPS-induced iNOS protein and mRNA expression as well as murine iNOS promoter activity. When treating these chemicals with reducing agents, the inhibition by carbaryl was reversed, but not the inhibition by alachlor. These chemicals also inhibited LPS-induced interferon-beta (IFN-beta) expression, an indispensable factor for LPS-induced iNOS expression. The inhibited iNOS expression, however, was not restored by exogenous IFN-beta supplementation. LPS-induced nuclear translocation of NF-kappaB, which is necessary for the expression of IFN-beta and iNOS, was inhibited by these chemicals: however, the LPS-induced degradation of IkappaB-alpha and IkappaB-beta was inhibited only by alachlor. These results indicate that alachlor and carbaryl differentially impair the LPS-induced NF-kappaB activation, leading to the inhibition of NO production.     

诱导型一氧化氮合酶在17β-雌二醇诱导的血管平滑肌细胞周期阻滞中的作用

利用大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMC)作为模型,观察17β-雌二醇(17β-estradiol,E2)对VSMC诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)活性和蛋白表达的影响,并探讨其在内皮素-1(endothlin 1,ET-1)刺激的VSMC周期循环中的作用。检测指标包括同位素法测定iNOS的活性,免疫印迹法(western blot)检测iNOS蛋白表达,流式细胞仪检测细胞周期,观察一氧化氮合酶(nitric oxide synthase,NOS)抑制剂N^G-硝基左旋精氨酸甲酯(N^G-nitro—L—arginine methylester,L—NAME)对E2抑制VSMC细胞周期的影响。结果显示,E2明显增加iNOS的活性和蛋白表达,在30min和12h时能诱导VSMC的iNOS活性明显增加,而60min和24h时VSMC的iNOS活性与对照组无显著差异,不呈明显浓度依赖性,雌激素受体(estrogen receptor,ER)拮抗剂Tamoxifen和L—NAME能明显抑制E2诱导的VSMC iNOS活性增加;E2增加VSMC的iNOS蛋白表达的作用在3h时起效,12h达高峰,以后逐渐下降,呈浓度依赖性,Tamoxifen能明显抑制马诱导的VSMC iNOS蛋白表达;E2明显抑制ET-1诱导的S期细胞百分比和G2 S／G1增加,使VSMC在G1期发生细胞周期阻滞,这些作用可被预先给予L—NAME所明显减轻。上述结果提示,E2使ET—l刺激的VSMC细胞周期循环在G1期发生阻滞与增加VSMC iNOS活性有关,该作用至少部分通过ER介导。     

Absence of involvement of nitric oxide in LP-BM5-induced immunodeficiency syndrome

Abstract To examine the role of nitric oxide (NO) in murine AIDS (MAIDS) pathogenesis, we determined NO production and inducible NOS (iNOS) mRNA expression in the macrophages of LP-BM5-infected mice, together with the in vivo effects of l -NAME, a competitive inhibitor of NO synthase. LP-BM5 infection induced neither spontaneous nitrite production nor iNOS mRNA expression. No differences in IFNγ + LPS-induced nitrite production or iNOS mRNA expression were observed in macrophages from non-infected or infected mice. Spleen weight, ecotropic MuLV replication, the blood lymphocyte phenotype and proliferative response of splenocytes were not modified by l -NAME. LP-BM5 infection did not increase macrophage NO production and NO production did not appear to protect against LP-BM5-induced immunodeficiency.     

Protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in rat cortical neuron-glia cultures

We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron-glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-gamma-induced neurotoxicity in rat cortical neuron-glia cultures with an EC(50) value of 0.09 microM. Radicicol inhibited the LPS/IFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-gamma-induced release of tumor necrosis factor-alpha (TNF-alpha) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-alpha was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-gamma-induced neuronal cell death in neuron-glia cultures is mediated via the inhibition of TNF-alpha release, as well as the suppression of iNOS expression in microglia.     

Effect of N-acetyl- -cysteine on peroxynitrite and superoxide anion production of lung alveolar macrophages in systemic sclerosis

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.     

Lactoferrin feeding augments peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans

Oral administration of lactoferrin (LF), an innate-defense protein present in exocrine secretions such as milk and in neutrophils, is reported to improve host-protection against infections with microorganisms including pathogenic fungi, possibly due to an immunomodulatory effect. This study aimed to evaluate the effect of bovine LF feeding on peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Time course analysis during the 14 days following Candida-priming revealed that LF administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of superoxide anion (O2(-)) and nitric oxide (NO) by peritoneal macrophages at day 7. LF administration facilitated NO production and Candida hyphal-growth inhibition by macrophages derived from Candida-primed mice but not non-primed mice, suggesting that the action of LF is dependent on the immune status of the host. LF administration altered the kinetics of cytokines in the peritoneal lavage fluid of Candida-primed mice. Enhancement of cytokine levels by LF was observed for IL-12 at day 5 and IFN-gamma at day 9, but not for TNF-alpha or IL-10. In conclusion, LF feeding augmented the activities of macrophages in a manner dependent on Candida-priming and these effects may be related to enhanced cytokine levels.