Differential upregulation of TNF-alpha, IL-6, and IL-8 production by deoxynivalenol (vomitoxin) and other 8-ketotrichothecenes in a human macrophage model.

The effects of deoxynivalenol (DON or vomitoxin) and four closely related 8-ketotrichothecenes on proinflammatory cytokine and chemokine production were evaluated in a clonal human macrophage model. U-937 cells, which represent a human monocytelike histocytic lymphoma, were differentiated into macrophages by preincubation with phorbol 12-myristate 13-acetate (PMA). Differentiated macrophages were incubated with DON in the absence or presence of lipopolysaccharide (LPS), and supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), and for the chemokine interleukin-8 (IL-8). In the absence of LPS, DON at 500 or 1,000 ng/ml upregulated TNF-alpha production as early as 3 h and up to 6 h, whereas 100 to 1,000 ng/ml of DON significantly increased production of IL-6 from 3 to 24 h and IL-8 from 6 to 48 h. In cells costimulated with 0.2 microg/ml LPS, DON at 500 or 1000 ng/ml markedly superinduced TNF-alpha and IL-8 production. Although 100 ng/ml of DON also potentiated LPS-induced IL-6 production, 500 or 1,000 ng/ ml of the toxin suppressed the LPS-induced IL-6 response. Four other 8-ketotrichothecenes, fusarenon X, nivalenol, 3-acetyl DON, and 15-acetyl DON, were also capable of upregulating or suppressing TNF-alpha, IL-6, and IL-8 production at concentrations similar to that of DON. In total, the results suggest that DON and other 8-ketotrichothecenes have the potential to both directly induce and superinduce proinflammatory cytokine and chemokine expression in human macrophages, even at toxin concentrations that are cytotoxic.     

Induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan

The immunomodulatory effect of ginsan based on the production of cytokines and the activation of macrophage was studied. Murine peritoneal macrophages (PM) on in vitro treatment with ginsan isolated from Panax ginseng induced mRNA of cytokines such as tumor necrosis factor (TNF)-alpha, interleukin-1 (IL-1)beta, interleukin-6 (IL-6) and interleukin-12 (IL-12); TNF-alpha mRNA induction was maximum within 3 h, IL-6 mRNA was gradually induced up to 24 h, and IL-1beta and IL-12 mRNA were highly induced at 24 h. IL-1beta and IL-6 protein levels also increased within 24 h in a dose-dependent manner and reached a maximum with 100 microg/ml ginsan. IL-12 was induced after 3 days and a high level of induction was detected after 4 days post treatment. Ginsan enhanced the lytic death of L929 cells through TNF-alpha activation. The mRNA expression of nitric oxide synthase (iNOS) was highly induced after 24 h treatment of ginsan, and then NO production was maximum after 48-h treatment with a low dose of 1 microg/ml. The level of iNOS mRNA induction by ginsan was slightly less than that of macrophages activating agents such as LPS plus IFN-gamma. The tumoricidal activity of macrophage cultured with ginsan on Yac-1 cells was enhanced in a dose-dependent manner; growth inhibition increased 1.6-fold with 100 microg/ml ginsan. These results suggest that ginsan exerts as an effective immunomodulator and enhances antitumor activity of macrophages.     

Influence of deoxynivalenol on NF-kappaB activation and IL-8 secretion in human intestinal Caco-2 cells

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. In human intestinal Caco-2 cells, DON activates the mitogen-activated protein kinases (MAPKs). We hypothesized a link between DON ingestion and intestinal inflammation, and used Caco-2 cells to assess the effects of DON, at plausible intestinal concentrations (250-10,000 ng/ml), on inflammatory mediators acting downstream the MAPKs cascade i.e. activation of nuclear factor-kappaB (NF-kappaB) and interleukin-8 (IL-8) secretion. In addition, Caco-2 cells were co-exposed to pro-inflammatory stimuli in order to mimic an inflamed intestinal epithelium. Dose-dependent increases in NF-kappaB activity and IL-8 secretion were observed, reaching 1.4- and 7.6-fold, respectively using DON at 10 microg/ml. Phosphorylation of inhibitor-kappaB (IkappaB) increased (1.6-fold) at DON levels <0.5 microg/ml. Exposure of Caco-2 cells to pro-inflammatory agents, i.e. 25 ng/ml interleukin-1beta, 100 ng/ml tumor necrosis factor-alpha or 10 microg/ml lipopolysaccharides, activated NF-kappaB and increased IL-8 secretion. Synergistic interactions between these stimuli and DON were observed. These data show that DON induces NF-kappaB activation and IL-8 secretion dose-dependently in Caco-2 cells, and this effect was accentuated upon pro-inflammatory stimulation, suggesting DON exposure could cause or exacerbate intestinal inflammation.     

银杏叶提取物对U937泡沫细胞IL-1β、TNF-α及IL-10表达的影响

本研究考察了氧化低密度脂蛋白(ox-LDL)刺激的U937细胞致炎细胞因子白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)、抗炎细胞因子白细胞介素10(IL-10)及其受体(IL-10R)的蛋白及mRNA的表达，同时观察银杏叶提取物(GbE)对它们的作用。U937细胞用100 mg·L^-1 ox-LDL刺激24 h形成泡沫细胞，同时分别加入不同浓度的GbE(0.1, 1及10 μg·L^-1)共孵育，采用酶联免疫吸附试验(ELISA)及逆转录聚合酶链式反应(RT-PCR)方法检测IL-1β、TNF-α、IL-10和IL-10R的蛋白或mRNA表达。U937泡沫细胞组IL-1β、TNF-α、IL-10和IL-10R的蛋白或mRNA的表达较对照组显著增加(P<0.01)。GbE组IL-1β及TNF-α的蛋白和mRNA的表达水平明显降低，IL-10的蛋白、IL-10和IL-10R的mRNA表达水平明显提高，与U937泡沫细胞组相比差异显著(P<0.05, P<0.01)。GbE对U937泡沫细胞致炎细胞因子IL-1β及TNF-α表达的显著抑制作用，对抗炎细胞因子IL-10及其受体IL-10R表达的显著上调作用可能是其抗atherosclerosis(AS)的机制之一。     

Effect of pro-inflammatory cytokines on the toxicity of the arylhydroxylamine metabolites of sulphamethoxazole and dapsone in normal human keratinocytes

Sulphonamides, such as sulphamethoxazole (SMX) and the related sulphone dapsone (DDS), show a higher incidence of cutaneous drug reactions (CDRs) in patients with the acquired immunodeficiency syndrome (AIDS) compared with human immunodeficiency virus (HIV) negative patients. During HIV infection, pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are increased. We hypothesized that this increase in pro-inflammatory cytokines may increase the toxicity of the arylhydroxylamine metabolites of SMX (S-NOH) and DDS (D-NOH) in keratinocytes through a reduction in glutathione (GSH) content. We evaluated the effect of TNF-alpha on GSH levels in normal human epidermal keratinocytes (NHEK) and found a significant decrease in GSH after 24h. Pre-treatment with TNF-alpha also resulted in an increase in the recovery of D-NOH, but failed to alter drug-protein covalent adduct formation in NHEK. We also evaluated the effect of TNF-alpha, IL-1 beta, interferon-gamma (IFN-gamma), lipopolysaccharide (LPS) and conditioned media (obtained from monocytes stimulated with LPS) on the cytotoxicity of pre-formed arylhydroxylamine metabolites in NHEK. Priming cells with cytokines did not significantly alter the cytotoxicity of the metabolites. The effect of pre-treatment with TNF-alpha on reactive oxygen species (ROS) generation in NHEK was also determined. While ROS formation in NHEK was increased in the presence of D-NOH, TNF-alpha did not alter the level of ROS generation. Our data suggest that the level of GSH reduction induced by pro-inflammatory cytokines does not predispose NHEK to cellular toxicity from either S-NOH or D-NOH.     

Effects of diesel exhaust particles on cytokine production by splenocytes stimulated with lipopolysaccharide

It was previously shown that pulmonary exposure of mice to diesel exhaust particles (DEP) enhances inflammatory conditions induced by allergens or bacterial endotoxin (lipopolysaccharide: LPS) via enhanced local expression of cytokines. However, resolution of the underlying mechanisms, in which DEP exaggerate inflammation, remains uncompleted. Investigation of the actions of DEP on mouse-derived mononuclear cells may provide a clue to the mechanisms, because mononuclear cells produce and release several types of cytokines. The present study elucidated the effects of DEP on mononuclear cell reactions stimulated with LPS in vitro. ICR mouse-derived mononuclear cells, isolated from splenocytes, one of the secondary lymphoid tissues, were co-cultured with LPS (1 microg ml(-1)) and DEP (1, 10 or 100 microg ml(-1)). The protein levels of interferon (IFN)-gamma, interleukin (IL)-2, IL-10, and IL-13 in the culture supernatants were measured 72 h after the co-culture. LPS significantly increased the protein levels of IFN-gamma, IL-2 and IL-10. In the presence of LPS, DEP decreased the protein levels in a concentration-dependent manner with an overall trend, whereas DEP (1, 10 microg ml(-1)) moderately elevated the IL-13 level. These results suggest that DEP suppress cytokine production from mononuclear cells stimulated with LPS and provide a possible hint for DEP facilitation on inflammatory conditions, especially related to Th2 response, in vivo.     

Effects of diesel exhaust particles (DEP), carbon black, and silica on macrophage responses to lipopolysaccharide: evidence of DEP suppression of macrophage activity

The effects of diesel exhaust particle (DEP) exposure on alveolar macrophage (AM) response to ex vivo and in vivo lipopolysaccharide (LPS) challenge were determined by monitoring LPS-stimulated production of interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha). The roles of the insoluble particulate and the organic compounds of DEP in altering pulmonary responses were evaluated by comparing the DEP-induced pulmonary responses to those of carbon black (CB), a carbonaceous particle with few adsorbed organic compounds, or to silica, a known pneumotoxic dust. Male Sprague-Dawley rats were exposed to a single intratracheal dose (5 or 35 mg/kg body weight) of DEP, CB, or silica, or to saline vehicle. Rats were sacrificed 1, 3, or 7 d postexposure. To study the responsiveness to the bacterial product LPS, AM isolated from particle-exposed rats were challenged ex vivo with LPS (0.1 microg/10(6) AM) and LPS-stimulated cytokine release was monitored. In addition, rats were exposed intratracheally to a single dose of DEP (5 mg/kg) and 3 d later exposed in vivo to 1 mg/kg LPS for 3 h prior to measurement of cytokine production by AM. DEP exposure resulted in neutrophil infiltration and elevated levels of albumin and lactate dehydrogenase (LDH) activity in the bronchoalveolar lavage fluid; these responses were not substantially different from those elicited by CB or silica exposure. AM from DEP-exposed rats showed increased spontaneous production of IL-1, but not TNF-alpha, while the opposite was true for CB or silica. Upon ex vivo challenge with LPS, AM from DEP-exposed rats showed a significant decrease in the secretion of TNF-alpha and, to a lesser extent, IL-1, compared to the sum of the DEP and LPS effects. In contrast, AM from CB- or silica-exposed rats did not show this decreased responsiveness to subsequent LPS challenge. This inhibitory action of DEP on LPS-stimulated AM production of IL-1 and TNF-alpha was further confirmed by the results obtained from rats exposed to both DEP and LPS in vivo. In summary, these results indicate that while DEP, CB, and silica all induce pulmonary inflammatory responses due to particle stimulation, only DEP suppress AM cytokine release in response to LPS stimulation. The contrasting cellular response with respect to DEP and CB exposures may be due to the presence of adsorbed organic compounds on DEP, which may contribute to the increased susceptibility of hosts to pulmonary infections after DEP exposure.     

Inhibition of COX-2 activity and proinflammatory cytokines (TNF-α and IL-1β) production by water-soluble sub-fractionated parts from bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) venom

Bee venom is used as a traditional medicine for treatment of arthritis. The anti-inflammatory activity of the n-hexane, ethyl acetate, and aqueous partitions from bee venom (Apis mellifera) was studied using cyclooxygenase (COX) activity and pro-inflammatory cytokines (TNF-alpha and IL-1beta) production, in vitro. COX-2 is involved in the production of prostaglandins that mediate pain and support the inflammatory process. The aqueous partition of bee venom showed strong dose-dependent inhibitory effects on COX-2 activity (IC50 = 13.1 microg/mL), but did not inhibit COX-1 activity. The aqueous partition was subfractionated into three parts by molecular weight differences, namely, B-F1 (above 20 KDa), B-F2 (between 10 KDa and 20 KDa) and B-F3 (below 10 KDa). B-F2 and B-F3 strongly inhibited COX-2 activity and COX-2 mRNA expression in a dose-dependent manner, without revealing cytotoxic effects. TNF-alpha and IL-1beta, are potent pro-inflammatory cytokines and are early indicators of the inflammatory process. We also investigated the effects of three subfractions on TNF-alpha and IL-1beta production using ELISA method. All three subfractions, B-F1, B-F2 and B-F3, inhibited TNF-alpha and IL-1beta production. These results suggest the pharmacological activities of bee venom on anti-inflammatory process include the inhibition of COX-2 expression and the blocking of pro-inflammatory cytokines (TNF-alpha, and IL-1beta) production.     

Sustained effect of inhaled diesel exhaust particles on T-lymphocyte-mediated immune responses against Listeria monocytogenes.

Studies have shown that exposure to diesel exhaust particles (DEP) suppresses pulmonary host defense against bacterial infection. The present study was carried out to characterize whether DEP exposure exerts a sustained effect in which inhaled DEP increase the susceptibility of the lung to bacterial infection occurring at a later time. Brown Norway rats were exposed to filtered air or DEP by inhalation at a dose of 21.2 +/- 2.3 mg/m3, 4 h/day for 5 days, and intratracheally instilled with saline or 100,000 Listeria monocytogenes (Listeria) 7 days after the final DEP exposure. Bacterial growth and cellular responses to DEP and Listeria exposures were examined at 3 and 7 days post-infection. The results showed that inhaled DEP prolonged the growth of bacteria, administered 7 days post DEP exposure, in the lung as compared to the air-exposed controls. Pulmonary responses to Listeria infection were characterized by increased production of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-12, and IL-10 by alveolar macrophages (AM) and increased presence of T lymphocytes and their CD4+ and CD8+ subsets in lung draining lymph nodes that secreted elevated levels of IL-2, IL-6, IL-10, and interferon (IFN)-gamma. Diesel exhaust particles were found to inhibit Listeria-induced production of IL-1beta and TNF-alpha, which are responsible for the innate immunity, and IL-12, which initiates the development of T helper (Th)1 responses, but enhance Listeria-induced AM production of IL-10, which prolongs Listeria survival in these phagocytes. The dual action of DEP on AM production of IL-12 and IL-10 correlated with an inhibition of the development of bacteria-specific T lymphocytes by DEP. Cytokine production by lymphocytes from DEP- and Listeria-exposed rats showed a marked decrease in the production of IL-2, IL-10, and IFN-gamma compared to Listeria infection alone, suggesting either that DEP inhibit the production of cytokines by lymphocytes or that these lymphocytes contained T-cell subsets that are different from those of Listeria infection alone and less effective in mediating Th1 immune responses. This study demonstrates that inhaled DEP, after a 7-day resting period, increase the susceptibility of the lung to bacterial infection occurring at a later time by inhibiting macrophage immune function and suppressing the development of T-cell-mediated immune responses. The results support the epidemiological observations that exposure to DEP may be responsible for the pulmonary health effects on humans.     

Curcumin inhibits the increase of glutamate, hydroxyl radicals and PGE2 in the hypothalamus and reduces fever during LPS-induced systemic inflammation in rabbits

Evidence has accumulated to suggest that systemic administration of lipopolysaccharide (LPS), in addition to elevating tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) as well as fever, induces overproduction of glutamate, hydroxyl radicals and prostaglandin E(2) (PGE(2)) in the rabbit's hypothalamus. Current study was attempted to assess whether Curcumin exerts its antipyresis by reducing circulating pro-inflammatory cytokines and hypothalamic glutamate, hydroxyl radicals and PGE(2) in rabbits. The microdialysis probes were stereotaxically and chronically implanted into the preoptic anterior hypothalamus of rabbit brain for determination of glutamate, hydroxyl radicals, and PGE(2) in situ. It was found that systemic administration of LPS (2 microg/kg) induced increased levels of both core temperature and hypothalamic levels of both glutamate and hydroxyl radicals accompanied by increased plasma levels of TNF-alpha, IL-1beta, and IL-6. The rise in both the core temperature and hypothalamic glutamate and hydroxyl radicals could also be induced by direct injection of TNF-alpha, IL-1beta, or IL-6 into the lateral ventricle of rabbit brain. Pretreatment with Curcumin (5-40 mg/kg, i.p.) 1 h before an i.v. dose of LPS significantly reduced the LPS-induced overproduction of circulating TNF-alpha, IL-1beta, and IL-6, and brain glutamate, PGE(2), and hydroxyl radicals. Both the febrile response and overproduction of both glutamate and hydroxyl radicals in the hypothalamus caused by central administration of TNF-alpha, IL-1beta, or IL-6 could be suppressed by Curcumin. These results indicate that systemic injection of Curcumin may exert its antipyresis by inhibiting the glutamate-hydroxyl radicals-PGE(2) pathways in the hypothalamus and circulating TNF-alpha, IL-1beta, and IL-6 accumulation during LPS fever.     

Differential Modulatory Effects of Cholera Toxin and Pertussis Toxin on Pain Behavior Induced by TNF-a, lnterleukin-1β and Interferon- Injected Intrathecally

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in pro-inflammatory cytokine induced pain behaviors. Intrathecal injection of tumor necrosis factor-alpha (TNF-alpha; 100 pg), interleukin-1beta (IL-1beta; 100 pg) and interferon-gamma (INF-gamma; 100 pg) showed pain behavior. Intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 mg) attenuated pain behavior induced by TNF-alpha and INF-gamma administered intrathecally. But intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 microg) did not attenuate pain behavior induced by IL-1beta. On the other hand, intrathecal pretreatment with PTX further increased the pain behavior induced by TNF-alpha and IL-1beta administered intrathecally, especially at the dose of 0.5 microg. But intrathecal pretreatment with PTX did not affect pain behavior induced by INF-gamma. Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play important roles in modulating pain behavior induced by pro-inflammatory cytokines administered spinally. Furthermore, TNF-alpha, IL-1beta and INF-gamma administered spinally appear to produce pain behavior by different mechanisms.     

An Anacardiaceae preparation reduces the expression of inflammation-related genes in murine macrophages

This study investigated the effects of an aqueous extract of the stem bark of Mangifera indica L. (Anacardiaceae; Vimang), which contains a defined mixture of components including polyphenols (principally mangiferin, MA), triterpenes, phytosteroids, fatty acids and microelements, on expression of inflammation mediators in inflammatory murine macrophages after stimulation in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In vitro treatment with Vimang at 4 microg/ml reduced levels of NOS-2 mRNA and NOS-2, while treatment at 40 microg/ml also reduced levels of COX-2 mRNA, COX-2, and prostaglandin E2 (PGE2). Results suggested that MA is involved in these effects. In vitro treatment with Vimang at 40 microg/ml also inhibited mRNA levels of the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha) and colony-stimulating factor (GM-CSF), but did not affect mRNA levels of IL-6 or tumor growth factor-beta (TGF-beta). Extracellular release of TNF-alpha by inflammatory macrophages was inhibited by in vitro treatment with Vimang at the same concentrations that showed inhibition of TNF-alpha mRNA levels. The inhibition of TNF-alpha production appears to be at least partially attributable to MA. Vimang at 4 microg/ml decreased mRNA levels of nuclear factor-kappaB (NF-kappaB) but did not affect expression of the NF-kappaB inhibitor (IkappaB). These data indicate that the potent anti-inflammatory effects of Vimang are due to selective modulation of the expression of inflammation-related genes, leading to attenuation of macrophage activation.     

An inhibitor of p38 MAP kinase downregulates cytokine release induced by sulfur mustard exposure in human epidermal keratinocytes.

Sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) is a potent alkylating agent that induces skin vessication after cutaneous exposure. Previous work has revealed that SM induces the production of inflammatory cytokines, including IL-8, IL-6, TNF-alpha, and IL-1beta, in keratinocytes. The p38 MAP kinase (MAPK14) signaling pathway is activated via phosphorylation in response to cellular stress and has been implicated in the upregulation of cytokines in response to stress. We investigated the role of p38 MAP kinase in inflammatory cytokine upregulation following SM exposure. A dose response study in cultured human epidermal keratinocytes (HEK) revealed increasing phosphorylation of p38 MAP kinase in response to increasing concentrations of SM. A time course at the 200 microM exposure revealed that p38 MAP kinase phosphorylation is induced by 15 min post-exposure, peaks at 30 min and is sustained at peak levels until 8 h post-exposure. Phosphorylation of the upstream kinase MKK3/6 was also detected. Assay of the SM-exposed HEK culture media for cytokines revealed that exposure to 200 microM SM increased IL-8, IL-6, TNF-alpha, and IL-1beta. When cells exposed to 200 microM SM were treated with the p38 MAP kinase inhibitor SB203580, the levels of IL-8, IL-6, and TNF-alpha and IL-1beta were significantly decreased when compared with cells that were untreated. These results show that p38 MAP kinase plays a role in SM-induced cytokine production in HEK and suggest that inhibiting this pathway may alleviate the profound inflammatory response elicited by cutaneous SM exposure.     

Pro-inflammatory potential of ultrafine particles in mono- and co-cultures of primary cardiac cells

Inhalation of particulate air pollution has been associated with increased risks for cardiovascular mortality and morbidity, but the underlying mechanisms are still under discussion. One possible pathway may be that inhaled particles cross the air-blood barrier and interact directly with cardiac tissue. The aim of the present study was to examine the pro-inflammatory potential of particles in cardiac cells. Mono- and co-cultures of primary adult male Wistar (Han) rat cardiomyocytes (CMs) and cardiofibroblasts (CFs) were exposed to increasing concentrations of ultrafine (<100nm) carbon black particles (Printex 90). Expression and release of cytokines (IL-6, IL-1beta and TNF-alpha) were measured by using quantitative real-time PCR and ELISA, respectively. Cytotoxicity was estimated by measuring cellular release of lactate dehydrogenase (LDH). A particle concentration-dependent increase in IL-6 release was observed in both CM mono- and co-cultures (EC(50) approximately 57microg/ml). Furthermore, IL-6 levels detected in both control and particle-exposed co-cultures were synergistically increased compared to mono-cultures (10-19-fold, dependent on the exposure). Experiments with contact and non-contact co-cultures indicate that direct cellular contact is of key importance for the enhanced release of IL-6 in co-cultures. An apparent particle-induced release of IL-1beta was only detected in co-cultures. The release of TNF-alpha was low and did not seem notably influenced by particle exposure. Treatment with an IL-1 receptor antagonist apparently eliminated the particle-induced release of IL-6. In conclusion, ultrafine particles have a pro-inflammatory potential in primary cardiac cells. Furthermore, IL-1 seems critical in triggering particle-induced release of IL-6. These pro-inflammatory responses may be elicited when particles are translocated into the pulmonary circulation upon inhalation or administered intravascularly during medical procedures.     

Immunomodulatory activity of dioscorin, the storage protein of yam (Dioscorea alata cv. Tainong No. 1) tuber.

The purified dioscorin from yam (Dioscorea alata L. cv. Tainong 1) tuber was previously reported (Hsu et al., 2002. J. Agric. Food Chem., 50, 6109-6113). In this report, we evaluated its immunomodulatory ability in vitro in the presence of polymyxin B (50 microg/ml) to eliminate lipopolysaccharide (LPS) contamination. Dioscorin (5-100 microg/ml) was able to stimulate nitric oxide production (expressed as nitrite concentrations) in RAW264.7 cells. The stimulation index on the phagocytosis of RAW264.7 cells against E. coli and the oxidative burst (determined by the intensity of rhodamine fluorescence) of RAW264.7 cells were both enhanced by different concentrations of dioscorin (5-100 microg/ml). The cytokine production, including IL-6, TNF-alpha, and IL-1beta in dioscorin-treated RAW264.7 cells or human monocytes, was measured in the cultured medium. Dioscorin (5-100 microg/ml) was found able to induce IL-6, TNF-alpha, and IL-1beta production in RAW264.7 cells and human monocytes. To evaluate the effects of dioscorin on the proliferation of spleen cells from BALB/c mice, phytohemagglutinin (PHA, 2 microg/ml) alone or PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml) was used to treat spleen cells for 24h. The stimulated proliferation index of splenic cells ranged from 1.38- to 1.48-fold of PHA alone for PHA mixed with different concentrations of dioscorin (10, 25, and 50 microg/ml). We suggest that the tuber storage protein of yam dioscorin functions as an immunomodulatory substance.     

Immunomodulatory activity of betulinic acid by producing pro-inflammatory cytokines and activation of macrophages

Betulinic acid (BA), a pentacyclic triterpene isolated from Lycopus lucidus, has been reported to be a selective inducer of apoptosis in various human cancer and shown anti-inflammatory and immunomodulatory properties. We postulated that BA modulates the immunomodulatory properties at least two groups of protein mediators of inflammation, interleukin-1beta (IL-1beta) and the tumor necrosis factor-alpha (TNF-alpha) on the basis of the critical role of the monocytes and tissue macrophages in inflammatory and immune responses. TNF-alpha and IL-1beta were produced by BA in a dose dependent manner at concentration of 0.625 and 10 microg/mL. The production of NO associated with iNOS was inhibited when treated with LPS at the concentration of 2.5 to 20 microg/mL of BA whereas COX-2 expression was decreased at 2.5 to 20 microg/mL. These modulations of inflammatory mediators were examined in LPS-stimulated RAW 264.7 cells and peritoneal macrophages. The morphology of macrophage was also examined and enhanced surface CD 40 molecule was expressed when treated BA at 0.625 to approximately 5 microg/mL with or without LPS. Furthermore, BA (20 microg/mL) enhanced apoptosis by producing DNA ladder in the RAW 264.7 cells. Our results indicated that BA induced activation of macrophage and pro-inflammatory cytokines. This may provide a molecular basis for the ability of BA to mediate macrophage, suppress inflammation, and modulate the immune response.     

Curcumin inhibition of inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages.

Curcumin, a dietary pigment responsible for the yellow colour of curry, has been used for the treatment of inflammatory diseases and exhibits a variety of pharmacological effects such as anti-inflammatory activity. The mechanism in anti-inflammatory activity of curcumin has been investigated; however, little is known about the effect of curcumin on cytokine production by human peripheral blood monocytes and alveolar macrophages. In the present study, we shed light on the effect of curcumin on inflammatory cytokine production by human peripheral blood monocytes and alveolar macrophages. To this end, we determined the concentrations of interleukin-8 (IL-8), monocyte inflammatory protein-1 (MIP-1alpha), monocyte chemotactic protein-1 (MCP-1), interleukin-1beta (IL-1beta), and tumour necrosis factor-alpha (TNF-alpha) in the culture supernatants from phorbor ester, 4beta phorbor 12beta-myristate-13alpha acetate (PMA)- or lipo-polysaccharide (LPS)-stimulated monocytes and alveolar macrophages in the presence or absence of curcumin. Curcumin inhibited the production of IL-8, MIP-1alpha, MCP-1, IL-1beta, and TNF-alpha by PMA- or LPS-stimulated monocytes and alveolar macrophages in a concentration- and a time-dependent manner. These results show that curcumin exhibits an inhibitory effect on the production of IL-8, MIP-1alpha, MCP-1, IL-1beta, and TNF-alpha by PMA- or LPS-stimulated monocytes and alveolar macrophages.     

Facilitating action of asiaticoside at low doses on burn wound repair and its mechanism

Some reports published from 1967 to 1999 describe the use of ointments containing high doses (0.1 to 0.2%, w/w) C. asiataica herb extracts to enhance wound repair. Lower doses at which burn wound repair is enhanced by such topical applications have not been established yet. We found that the application of asiaticoside at low doses of 10(-8) to 10(-12)% (w/w) facilitated burn wound repair. To clarify the accelerating mechanisms of asiaticoside on burn wound repair, we examined the effects of asiaticoside on the levels of various cytokines produced at the site of the burn wound. The topical application of a low dose (10 pg, 1 ng, or 100 ng/wound area) of asiaticoside increased monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and interleukin (IL)-1beta levels in burn wound exudates. Asiaticoside (10 pg to 100 ng/ml) enhanced MCP-1 production in HaCaT cells, but it had no direct effect on VEGF production. Furthermore, asiaticoside (10 pg to 100 ng/ml) increased the IL-1beta production in THP-1 macrophages with MCP-1, but it had no effect on IL-1beta production without MCP-1 or with lipopolysaccharide (LPS). These findings suggest that the enhancement of burn wound healing by asiaticoside might be due to the promotion of angiogenesis during skin wound repair as a result of the stimulation of VEGF production caused by the increase in MCP-1 expression in keratinocytes and the increase in IL-1beta expression in macrophages induced cooperatively by asiaticoside plus MCP-1.