Induction of cytotoxicity and production of inflammatory mediators in raw264.7 macrophages by spores grown on six different plasterboards

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (10(4), 10(5), 10(6) spores/10(6) cells), and evaluated 24 h after exposure for the ability to cause cytotoxicity and to stimulate production of nitric oxide (NO), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6). The data indicate clear differences between spores of different microbes in their ability to induce the production of these inflammatory mediators and to cause cell death in macrophages. Also, for each microbe, the induction ability specifically depended on the brand of plasterboard. The spores of Streptomyces californicus collected from all plasterboards were the most potent at inducing NO and cytokine production. Cytotoxicity caused by P. spinulosum and Streptomyces californicus spores was consistent with NO, IL-1beta and IL-6 production induced by those microbes. However, the production of these inflammatory mediators by the spores of Stachybotrys chartarum was not parallel to their ability to cause cell death. The low productions of NO and cytokines were associated with high cytotoxicity caused by the spores of the A. versicolor. These data suggest that growth condition of microbes on different plasterboards affect the ability of microbial spores to induce inflammatory responses and cytotoxicity in macrophages.     

COMPARISON OF MYCOBACTERIA-INDUCED CYTOTOXICITY AND INFLAMMATORY RESPONSES IN HUMAN AND MOUSE CELL LINES

Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factorα, TNF-α; interleukin 6, IL-6; and interleukinβ, IL-1β) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-α induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-α after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1β. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.     

FUNGAL SPORES AS SUCH DO NOT CAUSE NASAL INFLAMMATION IN MOLD EXPOSURE

The increased frequency of respiratory symptoms and diseases among sawmill workers has been linked to occupational exposure to airborne contaminants, especially to different fungi. Similar adverse health effects, together with elevated levels of inflammatory mediators in the nasal lavage (NAL) fluid, have been detected in people working in mold-damaged buildings. However, the indoor fungal spore concentrations in moldy houses are much lower than those in sawmills. To study the effect of fungal spores as such on the inflammatory markers in the NAL fluid, we conducted a similar study in sawmill workers who had an intense exposure to fungal spores. NAL was performed in 11 sawmill workers both during high occupational microbial exposure and during their vacation. Concentrations of nitric oxide (NO), tumor necrosis factor-α (TNFα), interleukin (IL)-4, IL-5, and IL-6 in NAL fluid were analyzed, and occupational exposure to inhalable dust, fungal spores, endotoxins and terpenes was confirmed by personal monitoring during the day of sampling. In addition, health data were collected with a questionnaire. Personal exposures to fungal spores varied from 2 × 10 5 spores/m 3 to 1.5 × 10 6 spores/m 3 (mean 7 × 10 5 spores/m 3) . Concentrations of inhalable dust, endotoxins, and terpenes were relatively low. There were no differences between work and vacation in the concentrations of NO and proinflammatory cytokines in NAL fluid or in the reporting of symptoms, and the levels were lower than in workers at a moldy school building. However, the concentration of TNFα in the NAL fluid was positively correlated with the concentration of terpenes in the working environment (r = .768; p = .006). These results show that microbial exposure as such does not invariably result in inflammatory changes detectable with the NAL method. We suggest that the type of microbial flora or microbial products in the occupational environment determines the proinflammatory potency of microbial exposure.     

Inflammatory responses in RAW264.7 macrophages caused by mycobacteria isolated from moldy houses

Mycobacterial strains (nonpathogenic Mycobacterium terrae, potentially pathogenic Mycobacterium avium-complex and Mycobacterium scrofulaceum), isolated from a moldy building, were studied with respect to their ability to stimulate macrophages (RAW264.7) to produce inflammatory mediators, and to cause cytotoxicity. Reactive oxygen species (ROS) were measured by chemiluminescence, cytokines (TNF-, IL-6, IL-1, IL-10) immunochemically, nitric oxide (NO) by Griess-method, expression of inducible NO-synthase (iNOS) with Western Blot analysis and cytotoxicity with MTT-test. All the strains induced dose- and time-dependent production of NO, IL-6 and TNF- in macrophages, whereas IL-1 or IL-10 production was not detected. The production of ROS and cytotoxicity was increased with the highest doses. Interestingly, different strains had significant differences in their ability to induce these responses, M. terrae being the most potent and M. avium-complex the weakest one. These results indicate that both non- and potentially pathogenic strains of mycobacteria present in moldy buildings are capable of activating inflammatory mechanisms in macrophages.     

Effect of growth medium on potential of Streptomyces Anulatus spores to induce inflammatory responses and cytotoxicity in RAW264.7 macrophages

Epidemiological studies have shown an association between microbial growth in buildings and increased risk of respiratory symptoms and disease related to inflammatory reactions in the inhabitants96. The current study examined the affects of growth conditions of Streptomyces anulatus, isolated from indoor air of a moldy building, on the inflammatory potential of spores of this microbe. Spores were harvested from 15 growth media formulations, applied to RAW264.7 macrophages (10(5), 10(6), or 10(7) spores/million cells), and evaluated for the ability to stimulate production of inflammatory mediators and cytotoxicity in these cells 24 h after exposure. Streptomyces anulatus spores induced dose-dependent production of nitric oxide (NO) in macrophages, reaching a level from 4.2 microM to 39.2 microM depending on the composition of the growth medium of the microbe. Expression of inducible NO synthase (iNOS) was detected in macrophages after exposure to spores collected from all growth media. Production of reactive oxygen species (ROS) was significantly increased only by the highest dose of S. anulatus spores grown on glycerol-arginine agar. Furthermore production of cytokines was affected by growth medium; the highest dose-dependent levels of interleukin 6 (IL-6) ranged from 900 to 7800 pg/ml, and the levels of tumor necrosis factor alpha (TNFalpha) varied from 490 to 3200 pg/ml. The amount of dead macrophages after the exposure varied from 11% to 96%, depending also on the growth media of the microbe. Altogether, our results suggest that the growth medium of S. anulatus has a fundamental role in the ability of the spores to induce inflammatory responses and cytotoxicity in mammalian cells.     

EFFECT OF LINER AND CORE MATERIALS OF PLASTERBOARD ON MICROBIAL GROWTH,SPORE-INDUCED INFLAMMATORY RESPONSES,AND CYTOTOXICITY IN MACROPHAGES

Microorganisms, when grown on wetted plasterboards, can produce bioactive compounds capable of inducing inflammatory and toxic reactions in mammalian cells. The paper liner of plasterboard is commonly regarded as the major substrate for microbial growth. In this study, we cultured Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus on liners and cores of plasterboards in order to examine the role of these main plasterboard components on microbial growth and the resulting bioactivity, which was assessed as the ability of microbial spores to induce inflammatory responses and to evoke cytotoxicity in mouse macrophages. The microbes, isolated from mold problem buildings, were grown under saturated humidity conditions on wetted liners and cores of six different plasterboards. The spores were collected, applied to RAW264.7 macrophages at different doses, and evaluated 24 h after exposure for their ability to evoke cytotoxicity and to stimulate production of nitric oxide (NO), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6). In general, microbial growth was better on the cores than on the liners. All of the studied microbes collected from cores induced a dose-dependent production of TNFα in macrophages. The TNFα production stimulated by spores of Stachybotrys, Aspergillus, and Streptomyces paralleled their cytotoxicity. Spores of Streptomyces and Aspergillus collected from liners were among the most potent inducers of NO and IL-6. Good growth of Stachybotrys on cores was associated with high cytotoxicity. Penicillium grew only on cores, but it did not induce major inflammatory mediator productions, nor was it significantly cytotoxic. These results indicate that previously reported microbial growth on plasterboards and spore-induced production of important inflammatory mediators and cell death in macrophages is not only due to the paper liner of plasterboard, but the core material also has a crucial role.     

Activation of PPAR�� Attenuates IFN�� and IL-1��-induced Cell Proliferation in Astrocytes: Involvement of IL-6 Independent Pathway

The present study demonstrates the effect of fibrates, agonists of PPARα on cytokines-induced proliferation in primary cultured astrocytes. Alone or combination treatment with cytokines, such as IL-1β (10 ng/ml), IFNγ (10 ng/ml), and TNF-α (10 ng/ml) cause a significant increase of cell proliferation in a time-dependent manner. Treatment of astrocytes with bezafibrate and fenofibrate (0, 5, and 10 µM) reduced the IFNγ and IL-1β-induced cell proliferation in a dose-dependent manner. To address the involvement of IL-6 on the IFNγ and IL-1β-induced cell proliferation, released IL-6 level was measured. IFNγ and IL-1β cause an increase of released IL-6 protein level in a time-dependent manner. Furthermore, pretreatment with IL-6 antibody (0, 0.1, 1, 2.5, and 5 ng/ml) dose-dependently inhibited the IFNγ and IL-1β-induced cell proliferation. However, bezafibrate and fenofibrate did not affect increased mRNA and protein levels of IL-6 in IFNγ and IL-1β-stimulated astrocytes. Taken together, these results clearly suggest that activation of PPARα attenuates the IFNγ and IL-1β-induced cell proliferation through IL-6 independent pathway.     

Diesel particles induce nitric oxide production in murine alveolar macrophages and rat airways

The acute adverse health effects among respiratory and cardiovascular patients have been associated with particulate air pollution, containing diesel particles (DP). The mechanisms of these effects are unknown, but they may involve inflammation. We investigated the effects of DP (30-3000 μg/10(6) cells) on cell viability and production of nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in murine RAW 264.7 macrophage cultures in vitro. DP caused a dose- and time-dependent NO-production and was cytotoxic in murine RAW 264.7 macrophages. Cytotoxicity preceded the increases in NO production. DP had minimal effects on cytokine production. A single intratracheal instillation of DP 1 and 5 mg/rat increased NO production and protein concentration in bronchoalveolar lavage fluid, and caused pulmonary edema and hemorrhage. The present results indicate that DP can induce both NO production and cytotoxicity in the lower respiratory tract, which may contribute to the short-term adverse respiratory effects of these particles.     

Fungal spores as such do not cause nasal inflammation in mold exposure

The increased frequency of respiratory symptoms and diseases among sawmill workers has been linked to occupational exposure to airborne contaminants, especially to different fungi. Similar adverse health effects, together with elevated levels of inflammatory mediators in the nasal lavage (NAL) fluid, have been detected in people working in mold-damaged buildings. However, the indoor fungal spore concentrations in moldy houses are much lower than those in sawmills. To study the effect of fungal spores as such on the inflammatory markers in the NAL fluid, we conducted a similar study in sawmill workers who had an intense exposure to fungal spores. NAL was performed in 11 sawmill workers both during high occupational microbial exposure and during their vacation. Concentrations of nitric oxide (NO), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-4, IL-5, and IL-6 in NAL fluid were analyzed, and occupational exposure to inhalable dust, fungal spores, endotoxins and terpenes was confirmed by personal monitoring during the day of sampling. In addition, health data were collected with a questionnaire. Personal exposures to fungal spores varied from 2 x 10(5) spores/m(3) to 1.5 x 10(6) spores/m(3) (mean 7 x 10(5) spores/m(3)). Concentrations of inhalable dust, endotoxins, and terpenes were relatively low. There were no differences between work and vacation in the concentrations of NO and proinflammatory cytokines in NAL fluid or in the reporting of symptoms, and the levels were lower than in workers at a moldy school building. However, the concentration of TNFalpha in the NAL fluid was positively correlated with the concentration of terpenes in the working environment (r =.768; p =.006). These results show that microbial exposure as such does not invariably result in inflammatory changes detectable with the NAL method. We suggest that the type of microbial flora or microbial products in the occupational environment determines the proinflammatory potency of microbial exposure.     

Streptomyces spores from mouldy houses induce nitric oxide, TNFα and IL-6 secretion from RAW264.7 macrophage cell line without causing subsequent cell death

The current view is that only bacterial lipopolysaccharide (LPS) and gamma interferon (IFNγ) are able to alone activate macrophages to secrete nitric oxide (NO), probably a causative agent of cell death. Moreover, some cytokines and gram positive pathogens together with IFNγ induce NO-production. Surprisingly, spores of Streptomyces sp., which are mesophilic gram-positive bacteria found in mouldy houses, stimulated RAW264.7 macrophages to produce pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6), and induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production. However, the Streptomyces spores did not kill NO-producing macrophages, as did both LPS and gram negative bacteria Pseudomonas fluorescens, strong inducers of cytokine- and NO-production. These results imply that Streptomyces sp., induced cytokine and NO-secretion, may play a role in the responses evoked by exposure to these microbes. Moreover, factors other than, or in addition to NO, are necessary for cytotoxicity in murine macrophages.     

Interactions between Streptomyces californicus and Stachybotrys chartarum can induce apoptosis and cell cycle arrest in mouse RAW264.7 macrophages

Exposure to complex mixtures of bacteria and fungi in moisture-damaged buildings is a potential cause of inflammatory related symptoms among occupants. The present study assessed interactions between two characteristic moldy house microbes Streptomyces californicus and Stachybotrys chartarum. Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone. The RAW264.7 cells were exposed to six doses (1 x 10(4) to 3 x 10(6) spores/ml) for 24 h, and the time course of the induced responses was evaluated after 4, 8, 16, and 24 h of exposure (1 x 10(6) spores/ml). The cytotoxic potential of the spores was characterized by the MTT test, DNA content analysis, and enzyme assay for caspase-3 activity. The production of cytokines (IL-1beta, IL-6, IL-10, TNFalpha, and MIP2) was measured immunochemically and nitric oxide by the Griess method. Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G2/M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells. In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells.     

石菖蒲抑制脂多糖诱导的神经胶质细胞炎性反应及其机制

目的 观察石菖蒲对脂多糖(lipopolysaccharide,LPS)诱导的大鼠原代神经胶质细胞炎性反应的抑制作用并探讨其可能机制.方法 分离提取大鼠原代神经胶质细胞,用LPS刺激2h后分别加入不同浓度的石菖蒲含药血清,采用硝酸还原酶法检测石菖蒲对一氧化氮的影响,运用酶联免疫吸附法(ELISA)测定培养上清液中IL-1 β、IL-8、TNF-α水平,通过实时逆转录聚合酶链反应(RT-PCR)测定细胞中诱导型一氧化氮合酶(iNOS) mRNA表达.结果 LPS能够激活神经胶质细胞,不同浓度的石菖蒲含药大鼠血清在不影响细胞存活率的情况下,可以显著降低细胞培养上清液中NO、IL-1β、IL-8、TNF-α水平,抑制细胞内iNOS mRNA表达(P＜0.05).结论 石菖蒲的神经保护机制可能与抑制胶质细胞炎症反应有关.     

苦参碱对细菌脂多糖诱导大鼠枯否细胞释放肿瘤坏死因子及白细胞介素-6的影响

目的是研究苦参碱对细菌脂多糖(lipopolysachrides,LPS)诱导经卡西霉素(calcimycin,Cal)预激活的大鼠枯否细胞分泌肿瘤坏死因子(tumor necrosis factor,TNF)、白细胞介素-6(interleukin-6,IL-6)的影响以及对小鼠体内产生TNF和IL-6的影响。结果,苦参碱125,250及500mg·L^-1剂量依赖性抑制大鼠枯否细胞分泌TNF和IL-6;苦参碱50及100mg·kg^-1降低小鼠体内TNF和IL-6的水平。提示苦参碱的抗炎作用可能与其抑制TNF及IL-6的产生有关。     

Lipopolysaccharide-induced down-regulation of organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is independent of tumor necrosis factor-alpha, Interleukin-1beta, interleukin-6, or inducible nitric oxide synthase.

Organic anion transporting polypeptide 4 (Oatp4; Slc21a10) is expressed almost exclusively in liver, where it mediates uptake of a variety of compounds, including bile acids, as well as other endo- and xenobiotics, across hepatic sinusoidal membranes in a Na+-independent manner. Lipopolysaccharide (LPS) has been shown to decrease Oatp4 mRNA levels in a dose- and time-dependent manner in Toll-like receptor 4 (TLR4)-normal (C3H/OuJ) mice, but not in TLR4-mutant (C3H/HeJ) mice. Moreover, after LPS administration, serum concentrations of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) are markedly lower in TLR4-mutant mice than in TLR4-normal mice. Thus, TLR4 is considered an upstream mediator of LPS-induced decrease in mouse Oatp4 mRNA. LPS is thought to alter liver gene expression through LPS-induced cytokines or nitric oxide (NO). TNF receptor p55 (TNFRp55) and type I IL-1 receptor (IL-1RI) mediate the biological functions of TNF-alpha and IL-1beta, respectively. Therefore, to determine whether endogenous cytokines or NO are mediators of LPS-induced down-regulation of Oatp4, Oatp4 mRNA levels were determined in mice deficient in the TNFRp55, IL-1RI, IL-6, or inducible nitric oxide synthase (iNOS) after LPS administration. Mice homozygous for a targeted deletion of genes for TNFRp55, IL-1RI, IL-6, or iNOS exhibited similar decreases in Oatp4 mRNA levels as wild-type mice after LPS administration. Moreover, in mouse hepatoma cells, treatment with TNF-alpha, IL-1beta, or IL-6 individually or in combination did not suppress activity of mouse Oatp4 promoter (-4.8 kb to +30). Therefore, LPS-induced down-regulation of Oatp4 appears to be independent of TNF-alpha, IL-1beta, IL-6, or iNOS.     

Endotoxin induction of hepatic metallothionein is mediated through cytokines

Metallothionein (MT), a low molecular-weight, cysteine-rich, metal-binding protein, is induced by many environmental factors and a variety of stimuli. Bacterial endotoxin (lipopolysaccharide, LPS) injection is experimentally used to produce acute stress and is an effective inducer of hepatic MT. However, the mechanism of LPS induction of MT is not known. In the present studies, we used two substrains of mice, differing in their production of cytokines after LPS administration, to test the hypothesis that MT induction by LPS is mediated through cytokines. Normal (C3Heb/FeJ) and low cytokine-producing (C3H/HeJ) mice were given various doses of LPS, interleukin-1 (IL-1), interleukin-6 (IL-6), or tumor necrosis factor (TNF), and hepatic MT was determined 24 hr later by the Cd/hemoglobin assay. The low-cytokine-producing mice were much less responsive to the induction of MT by LPS (50 vs 150 micrograms MT/g liver after 1.0 mg LPS/kg, ip) than the normal mice, but were equally responsive to the induction of MT by IL-1 (0.03-1.0 microgram/mouse). IL-6 (0.5-5.0 micrograms/mouse), and TNF (0.005-0.5 microgram/mouse). All the cytokines produced a dose-dependent increase of hepatic MT levels in these two murine substrains (up to five- to sevenfold over controls). In conclusion, these data suggest that LPS induction of MT may be mediated through cytokines.     

Interleukin-6 can prime THP-1 macrophages for enhanced production of tumor necrosis factor-alpha in response to LPS.

Although interferon-gamma has been shown to effectively prime macrophages for enhanced production of tumor necrosis factor-alpha (TNF alpha), it is reasonable to assume that other cytokines present in the extracellular environment may likewise facilitate cytokine biosynthesis. For example, interleukin-6 (IL-6) is synthesized by synovial lining macrophages and fibroblasts, and has been detected (along with TNF alpha) in rheumatoid synovial effusions. Therefore, the purpose of the present study was to determine whether IL-6 influences the production of IL-1 beta and/or TNF alpha by THP-1 macrophages. Although IL-6 treatment alone resulted in only a slight increase in TNF alpha levels, administration of IL-6 followed by Sal. minnesota LPS resulted in a synergistic potentiation of TNF alpha production by THP-1 macrophages. The priming effect of IL-6 could be reversed by boiling, or by the addition of a neutralizing polyclonal antibody against IL-6. Notably, IL-6 only weakly enhanced interleukin-1 beta production. In summary, the ability of IL-6 to potentiate TNF alpha production by THP-1 macrophages may provide insight into the regulation of the cytokine network in inflammatory diseases, such as rheumatoid arthritis.     

Effects of thalidomide on neutrophil respiratory burst, chemotaxis, and transmigration of cytokine- and endotoxin-activated endothelium

Vascular endothelium activated by endotoxin and cytokines plays an important role in organ inflammation and blood leukocyte recruitment. Neutrophils, which are a homogeneous population of effector cells, are rapidly attracted in large numbers to sites of inflammation where they form an early response to infection or injury. Excessive production of various interleukins, TNF, arachidonic acid metabolites, and other substances by neutrophils and macrophages results in systemic endothelial cell injury, a fundamental problem. In the present study, we investigated in vitro the effects of thalidomide (THD) on activation of endothelial cells for enhanced transmigration of neutrophils by lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF), and interleukin-1 (IL-1). Modulation of endotoxin- and cytokine-induced neutrophil chemotaxis and respiratory burst by THD were also studied. Treatment of HUVEC with THD in combination with LPS, TNF, and IL-1, respectively, antagonized LPS-activated transmigration of neutrophils but stimulated the effects of TNF and IL-1. All of the agents used – THD, LPS, TNF, and IL-1 – inhibited neutrophil chemotaxis. Addition of THD to the neutrophils had no effect on LPS-inhibited chemotaxis whereas the TNF- and IL-1-induced chemotaxis was modulated in a bimodal manner. However, THD failed to influence neutrophil respiratory burst activity. Results demonstrate that THD differentially affects mediator-induced activation of HUVEC and neutrophils. Received: 1 April 1997 / Accepted: 4 July 1997     

雷公藤氯内酯醇对小鼠肺泡巨噬细胞体外炎性反应的影响(英文)

目的:探讨雷公藤单体雷公藤氯内酯醇(tripcholorolide,T4)对肺泡巨噬细胞(AM)炎症反应的影响及机制.方法:小鼠AM受脂多糖(LPS)10mg/L刺激的同时,加入T4 500 μg/L或地塞米松 100μmol/L;ELISA法测定上清液中TNFα、IL-1β、IL-6及IL-10浓度:RT-PCR检测上述因子及iNOS基因mRNA的表达.结果:AM受10 mg/L LPS刺激24小时后,上清液中TNFα、IL-1β、IL-6、IL-10及NO释放均明显增加.T4 500 μg/L及地塞米松100μmol/L对上述介质均有不同程度的抑制作用.LPS刺激5小时后,AM中TNFα、IL-6、IL-10和iNOS的mRNA表达均明显增加.T4和地塞米松对上述介质的mRNA表达均有明显抑制作用.另外,T4对TNFα、IL-6、IL-10 mRNA的稳定性无明显影响.结论:T4具有抑制AM中促炎介质和抗炎介质表达的作用.     

Modulation of lipopolysaccharide-induced proinflammatory cytokine production by satratoxins and other macrocyclic trichothecenes in the murine macrophage

The satratoxins and other macrocyclic trichothecene mycotoxins are produced by Stachybotrys, a mold that is often found in water-damaged dwellings and office buildings. To test the potential immunomodulatory effects of these mycotoxins, RAW 264.7 murine macrophage cells were treated with various concentrations of satratoxin G (SG), isosatratoxin F (iSF), satratoxin H (SH), roridin A (RA), and verrucarin A (VA) for 48 h in the presence or absence of suboptimal concentra-tion of lipopolysaccharide (LPS, 50 ng/ml), and tumor necrosis factor-alpha (TNF-alpha ) and interleukin-6 (IL-6) production were assayed by enzyme-linked immunosorbent assay (ELISA). In LPS-stimulated cultures, TNF-alpha supernatant concentrations were significantly increased in the presence of 2.5, 2.5, and 1 ng/ml of SG, SH, and RA, respectively, whereas IL-6 concentrations were not affected by the same concentrations these macrocyclic trichothecenes. When cells that were treated with LPS and SG (2.5 ng/ml) were evaluated by real-time polymerase chain reaction (PCR),TNF-alpha mRNA was found to increase at 24, 36, and 48 h compared to control cells. At higher concentrations, cytokine production and cell viability were markedly impaired in LPS-stimulated cells. Without LPS stimulation, neither TNF-alpha, nor IL-6 was induced. These results indicate that low concentrations of macrocyclic trichothecenes superinduce expression of TNF-alpha, whereas higher concentrations of these toxins are cytotoxic and concurrently reduce cytokine production. The capacity of satratoxins and other macrocyclic trichothecenes to alter cytokine production may play an etiologic role in outbreaks of Stachybotrys-associated human illnesses.     

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.