Paclitaxel (Taxol)-Induced Killing of Leishmania major in Murine Macrophages

The antitumor drug paclitaxel (Taxol) has been demonstrated to be a lipopolysaccharide mimetic in murine macrophages. In this study, the capacity of paclitaxel to activate macrophages to become microbicidal for Leishmania major was examined. Paclitaxel and gamma interferon synergized to kill intracellular L. major in Lpsⁿ, but not Lps^d, macrophages by a nitric oxide (NO·)-dependent mechanism.     

Lipopolysaccharide and Its Analog Antagonists Display Differential Serum Factor Dependencies for Induction of Cytokine Genes in Murine Macrophages

Monocytes/macrophages play a central role in mediating the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria by the production of proinflammatory mediators. Recently, it was shown that the expression of cytokine genes for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interferon-inducible protein-10 (IP-10) by murine macrophages in response to low concentrations of LPS is entirely CD14 dependent. In this report, we show that murine macrophages respond to low concentrations of LPS (≤2 ng/ml) in the complete absence of serum, leading to the induction of TNF-α and IL-1β genes. In contrast to the TNF-α and IL-1β genes, the IP-10 gene is poorly induced in the absence of serum. The addition of recombinant human soluble CD14 (rsCD14) had very little effect on the levels of serum-free, LPS-induced TNF-α, IL-1β, and IP-10 genes. In contrast, the addition of recombinant human LPS-binding protein (rLBP) had opposing effects on the LPS-induced TNF-α or IL-1β and IP-10 genes. rLBP inhibited LPS-induced TNF-α and IL-1β genes, while it reconstituted IP-10 gene expression to levels induced in the presence of serum. These results provide further evidence that the induction of TNF-α or IL-1β genes occurs via a pathway that is distinct from one that leads to the induction of the IP-10 gene and that the pathways diverge at the level of the initial interaction between LPS and cellular CD14. Additionally, the results presented here indicate that LPS structural analog antagonists Rhodobacter sphaeroides diphosphoryl lipid A and SDZ 880.431 are able to inhibit LPS-induced TNF-α and IL-1β in the absence of serum, while a synthetic analog of Rhodobacter capsulatus lipid A (B 975) requires both rsCD14 and rLBP to function as an inhibitor.     

霍乱弧菌LPS诱导小鼠腹腔巨噬细胞产生TNF的研究

本研究采用霍乱弧菌古典生物型５６９Ｂ菌株及ＥＩ-Ｔｏｒ生物型菌株，以Ｗｅｓｔｐｈａｌ的热酚-水法及高速离心等方法提取ＬＰＳ，血凝法测其效价，并比较不同菌株的ＬＰＳ刺激小鼠腹腔巨噬细胞产生ＴＮＦ的情况。实验表明５６９Ｂ菌株ＬＰＳ血凝效价较高，诱导小鼠腹腔巨噬细胞产生ＴＮＦ的能力较强。     

Cytokine Induction in Murine Macrophages Infected with Virulent and Avirulent Rhodococcus equi

To look for a possible correlation between the virulence of Rhodococcus equi and its cytokine-inducing capacity, we evaluated intracellular survival and measured cytokine induction by mouse macrophages infected with a virulent strain containing an 85-kb plasmid and expressing VapA (103⁺), its avirulent plasmid-cured derivative (103⁻), and heat-killed 103⁺ (HK). After incubation with similar numbers of bacteria, macrophages infected with 103⁻ contained significantly more organisms than those infected with 103⁺ or HK. The number of bacteria in the macrophages infected with 103⁻ and HK decreased progressively, whereas the 103⁺ numbers remained constant over 48 h. Interleukin 1β (IL-1β), IL-6, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-α) mRNA induction peaked at 4 h and returned to baseline between 12 and 48 h postinfection. IL-1β, IL-6, IL-10, and TNF-α concentrations assessed by enzyme-linked immunosorbent assay generally agreed well with mRNA expression; IL-12 could, however, not be detected. For all the cytokines detected, mean concentrations in the supernatants were consistently higher in the 103⁻-infected monolayers than in those infected with 103⁺, although, with the exception of IL-1β, the differences were not statistically significant. R. equi HK was a poor inducer of cytokine production. In conclusion, virulent and avirulent R. equi strains induced similar levels of cytokine synthesis. The slightly greater induction of most cytokines observed following infection with 103⁻ is likely secondary to greater uptake by macrophages rather than to a direct role of VapA or another plasmid-encoded product in downregulating cytokine induction.     

Lipopolysaccharide Accelerates Neuropilin-1 Protein Degradation by Activating the Large GTPase Dynamin-1 in Macrophages

Inflammation - Neuropilin-1 (Nrp1) is highly expressed in macrophages and plays a critical role in acute and chronic inflammation-associated diseases, such as sepsis, type II diabetes, and...     

Phlebotomus papatasi Saliva Inhibits Protein Phosphatase Activity and Nitric Oxide Production by Murine Macrophages

Leishmania parasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand fly Phlebotomus papatasi is the vector of Leishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show that P. papatasi saliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate that P. papatasi saliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that the P. papatasi salivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by which P. papatasi saliva exacerbates cutaneous leishmaniasis.     

Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival

Cryptococcus neoformans is a facultative intracellular opportunistic pathogen and the leading cause of fungal meningitis in humans. In the absence of a protective cellular immune response, the inhalation of C. neoformans cells or spores results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan, which constitutes approximately 90% of the capsular material. In the lungs, surfactant protein A (SP-A) and SP-D contribute to immune defense by facilitating the aggregation, uptake, and killing of many microorganisms by phagocytic cells. We hypothesized that SP-D plays a role in C. neoformans pathogenesis by binding to and enhancing the phagocytosis of the yeast. Here, the abilities of SP-D to bind to and facilitate the phagocytosis and survival of the wild-type encapsulated strain H99 and the cap59Δ mutant hypocapsular strain are assessed. SP-D binding to cap59Δ mutant cells was approximately sixfold greater than binding to wild-type cells. SP-D enhanced the phagocytosis of cap59Δ cells by approximately fourfold in vitro. To investigate SP-D binding in vivo, SP-D^−/− mice were intranasally inoculated with Alexa Fluor 488-labeled cap59Δ or H99 cells. By confocal microscopy, a greater number of phagocytosed C. neoformans cells in wild-type mice than in SP-D^−/− mice was observed, consistent with in vitro data. Interestingly, SP-D protected C. neoformans cells against macrophage-mediated defense mechanisms in vitro, as demonstrated by an analysis of fungal viability using a CFU assay. These findings provide evidence that C. neoformans subverts host defense mechanisms involving surfactant, establishing a novel virulence paradigm that may be targeted for therapy.Cryptococcus neoformans is an opportunistic fungal pathogen that usually infects the host via the lungs and then disseminates to the central nervous system, where it can cause life-threatening disease in immunocompromised patients. Immunocompetent individuals generally clear the pulmonary infection, although an outbreak of infections with C. gattii serotype B in apparently immunocompetent individuals on Vancouver Island, Canada, and in the surrounding areas occurred recently (21).Outside the cell wall, which contains chitin and chitosan (5), α-1,3-glucans (48), and β-1,3- and β-1,6-glucans (28), C. neoformans elaborates a polysaccharide capsule composed primarily of the polysaccharide glucuronoxylomannan (GXM). Minor capsular components include galactoxylomannan (GalXM) and mannoprotein 1 (MP1). The capsule is a key virulence factor, with myriad roles including antiphagocytic activities (29) and immunomodulatory functions during adaptive immune responses (15, 46, 61). Interestingly, the capsule has also been shown previously to be important for dissemination from the lungs (9), and the capsular component GalXM has been shown to facilitate dissemination to the central nervous system (44). The capsule also plays a prominent role during interactions of C. neoformans with macrophages (7, 29, 33, 51). Consequently, it is of fundamental interest to understand the role of capsular components in C. neoformans clearance by innate immune cells in the lungs.Surfactant is a lipid-rich material that has been recognized traditionally to be essential for reducing surface tension in the lungs but, more recently, has been appreciated as orchestrating important innate immune functions (60). Surfactant is synthesized primarily by type II cells of the aveoli, although surfactant proteins (SPs) are also produced by cells of the distal airways. In addition to lipids, there are four SPs, designated SP-A, SP-B, SP-C, and SP-D. SP-B and SP-C are hydrophobic and remain closely associated with the lipid component of surfactant, participating in its surface tension-lowering ability and biophysical properties. SP-A and SP-D are more hydrophilic and have important innate immune functions as a result of their abilities to evoke both proinflammatory and anti-inflammatory responses (23). These dual functions of SP-A and SP-D depend on their structures, which include a C-terminal carbohydrate recognition domain (CRD) and an N-terminal collagen-like region. SP-A and SP-D are members of the collectin family of proteins, which bind via their CRDs in a calcium-dependent manner to sugars present on the surfaces of microorganisms, allowing SP-A and SP-D to function as pattern recognition molecules. Via binding to the carbohydrate moieties of many different microorganisms, collectins opsonize and enhance the phagocytosis of microbial pathogens (6, 30, 32, 47).To date, relatively little is known about the role played by SPs during innate immune responses to C. neoformans infection. Both SP-A and SP-D have been observed to bind to acapsular strains, resulting in their aggregation (50). Although SP-A binds to C. neoformans, it does not enhance its uptake (58), and the susceptibility of SP-A null mice to C. neoformans infection is similar to that of wild-type animals, suggesting that SP-A does not play a major role in C. neoformans host defense in vivo (26). SP-D, but not SP-A, binds MP1 and GXM in vitro, and GXM has been shown previously to inhibit the SP-D-dependent aggregation of C. neoformans cells (55). In addition, SP-D interacts with β-1,6-glucan present in the cell walls of Saccharomyces cerevisiae and Aspergillus fumigatus (2). It is of interest that exogenous SP-D administration is protective in a mouse model of invasive aspergillosis (52). Additionally, SP-D is important for mounting an appropriate immune response to a number of different fungal pathogens, including A. fumigatus (40, 41) and Pneumocystis carinii (4), although any physiological role during the initial stages of C. neoformans infection remains unclear. We hypothesized that SP-D plays a role in the early phase of C. neoformans infection by binding to and enhancing the phagocytosis of the yeast.Here, we characterize SP-D binding to the acapsular C. neoformans cap59Δ strain and the isogenic wild-type strain H99 and demonstrate that SP-D enhances C. neoformans phagocytosis in vitro and in vivo. Paradoxically, we show that the pretreatment of C. neoformans with SP-D results in increased fungal growth in the presence of macrophages. The findings presented here expand our understanding of the role of SP-D in initial pulmonary immune responses to the fungus and may lead to novel therapies for patients presenting with pulmonary cryptococcal infections.     

Administration of Silica Sensitizes Lipopolysaccharide Responsiveness of Murine Macrophages but Inhibits T and B Cell Priming by Inhibition of Antigen Presenting Function

Macrophages play a key role in natural host defense against infection by a variety of pathogens. In addition, macrophages initiate the development of acquired immunity via antigen processing and presentation. The role of macrophages in resistance to pathogens, the development of autoimmune diseases and the induction of acquired immunity has been studied by treatment of rodents with reagents which are cytotoxic. We have studied the effects of one such reagent, silica, on the function of spleen macrophages and peritoneal exudate cells (PEC). Intraperitoneal administration of silica caused the accumulation of spleen macrophages and neutrophils, reduction in the number of B cells and had a modest effect on T cell abundance. The percentage of CD11b⁺ PEC was not affected by silica treatment but total PEC recovery was diminished 5-8 fold. Silica treatment did not cause release of TNF-alpha or IL-1-beta but, when stimulated with lipopolysaccharide (LPS) in vitro after silica treatment, PEC or spleen macrophages produced elevated levels of both cytokines compared to controls. In contrast, release of IL-12 from non-LPS treated PEC was stimulated 4-5 fold by silica treatment. In addition, sensitivity to LPS toxicity in vivo was significantly enhanced by silica. The ability of macrophages to present antigen to a T cell clone in vitro was found to be dramatically inhibited by silica treatment, as was the ability to prime antigen-specific T cells and B cells by antigen injection. Collectively these data demonstrate that silica treatment enhances macrophage sensitivity to LPS exposure but inhibits antigen processing and presentation.     

地塞米松对小鼠脾脏巨噬细胞内TLR4和TLR2表达的影响

为观察小鼠脾巨噬细胞内的TLR4和TLR2在脂多糖 (LPS )刺激后 ,地塞米松 (Dx )对其表达量的影响 ,在向BALB/c小鼠腹腔内注射LPS和Dx后采用贴壁法分离小鼠脾巨噬细胞 ,通过半定量RT PCR方法测定了TLR4和TLR2mRNA的表达量。结果显示 :①LPS刺激后 ,小鼠脾巨噬细胞内TLR4mRNA显著上调 (吸光度比为 0 11) ,TLR2上调不明显 (吸光度比为0 0 0 8) ;②预先注射不同剂量Dx ,分别为 0 1mg/kg、 1mg/kg、 10mg/kg ,再用LPS刺激小鼠。注射了Dx后巨噬细胞TLR4mRNA表达量较单纯注射LPS的小鼠显著降低 (吸光度比分别为 0 0 93、 0 0 5 0和 0 0 14 ) ;但TLR2mRNA表达量随着Dx使用剂量增加而增加 (吸光度比分别为 0 0 5 4、 0 0 80和 0 16 1)。该项研究表明TLR4是参与LPS引起炎症反应的主要Toll样受体 ,而TLR2不起主要作用 ;Dx对LPS刺激后TLR 4mRNA的表达有抑制作用 ,而对TLR2mRNA的表达有增强作用 ;Dx抑制LPS引起的炎症反应可能与抑制TLR4的表达有关     

Strain-Dependent Activation of Monocytes and Inflammatory Macrophages by Lipopolysaccharide of Porphyromonas gingivalis

Porphyromonas gingivalis is one of the pathogens associated with periodontal diseases, and its lipopolysaccharide (LPS) has been suggested as a possible virulence factor, acting by stimulation of host cells to secrete proinflammatory mediators. However, recent studies have shown that P. gingivalis LPS inhibited some components of the inflammatory response. The present study was designed to test the hypothesis that there are strain-dependent variations in the ability of P. gingivalis LPS to elicit the host inflammatory response. By using LPS preparations from two strains of P. gingivalis, W50 and A7346, the responses of mouse macrophages and human monocytes were evaluated by measuring the secretion of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Both direct and indirect (priming) effects were investigated. LPS from Salmonella typhosa was used as a reference LPS. P. gingivalis A7436 LPS induced lower secreted levels of NO from the tested cells than S. typhosa LPS but induced similar levels of TNF-α. In contrast, LPS from P. gingivalis W50 did not induce NO or TNF-α secretion. Preincubation of macrophages with LPS from S. typhosa or P. gingivalis A7436 prior to stimulation with S. typhosa LPS upregulated NO secretion and downregulated TNF-α secretion, while preincubation with P. gingivalis W50 LPS enhanced both TNF-α and NO secretory responses. These results demonstrate that LPSs derived from different strains of P. gingivalis vary in their biological activities in vitro. The findings may have an impact on our understanding of the range of P. gingivalis virulence in vivo.     

Interferon Production by Corynebacterium Parvum and BCG-Activated Murine Spleen Macrophages

Macrophages were identified to be a major source of interferon produced in murine spleen cell cultures after intravenous injection of Corynebacterium parvum (C. parvum), strain CN 6134 or Bacille Calmette Guérin (BCG). Another strain of C. parvum, CN 5888, which lacks RES stimulating activity and adjuvant activity in vivo, was not effective when injected intravenously. Protein synthesis was required for interferon activity to be produced and protein synthesis was also required for the antiviral state to be expressed. The antiviral activity was relatively stable to pH 2 and neutralized by an antiserum against virus-induced fibroblast interferon, thus exhibiting some properties of type I interferon. In vitro only CN 6134, the biologically active strain, could induce small amounts of interferon in spleen macrophage cultures. Macrophages from CN 6134 or BCG-infected athymic nu/nu mice produced similar interferon titers as their controls. It is concluded that infection with certain immunomodulators can activate splenic macrophages via a predominantly T-cell independent mechanism. Interferon in turn may operate locally as a mediator of immunoregulation.     

Secretory Leukocyte Protease Inhibitor Interferes with Uptake of Lipopolysaccharide by Macrophages

Macrophages are among the most sensitive targets of bacterial endotoxin (LPS), responding to minute amounts of LPS by releasing a battery of inflammatory mediators. Transfection of macrophages with secretory leukocyte protease inhibitor (SLPI) renders these cells refractory to LPS stimulation. Here we show that uptake of LPS from soluble CD14 (sCD14)-LPS complexes by SLPI-overexpressing cells was only 50% of that seen in control cells. SLPI transfectants and mock transfectants did not differ in the surface expression of CD14 or CD18. We show, in addition, that recombinant human SLPI can bind to purified endotoxin in vitro. SLPI caused a decrease in the binding of LPS to sCD14 as assessed both by fluorescence quenching of labeled LPS and by nondenaturing polyacrylamide gel electrophoresis. These results suggest that the inhibitory effect of SLPI on macrophage responses to LPS may, in part, be due to its blockade of LPS transfer to soluble CD14 and its interference with uptake of LPS from LPS-sCD14 complexes by macrophages.     

Interferon Induction in Mice by Lipopolysaccharide from Brucella abortus

Endotoxin or lipopolysaccharide isolated from Brucella abortus (strain 456) produced a typical endotoxin-type interferon response in mice with peak levels of the inhibitor occurring 2 h after intravenous injection of the stimuli. Brucella lipopolysaccharide was a much less effective interferon stimulus than the lipopolysaccharide isolated from Salmonella typhimurium (strain LT-2).     

Differential Recovery of Macrophages from Endotoxin-Tolerant States Elicited by Lipopolysaccharide and Enzymatic Treatments

Exposure of macrophages to endotoxin (lipopolysaccharide, LPS) leads to a suppression of their capacity to bind LPS and to produce cytokines after reexposure to LPS. This phenomenon is termed endotoxin tolerance, or LPS-induced desensitization. LPS also stimulates the secretion of serine proteases in macrophages, and activates membrane phospholipases. We have investigated the role of trypsin (a serine protease) and of a phosphatidylinositol-specific phospholipase C (PI-PLC, which cleaves GPI-anchored molecules such as CD14), on LPS-induced desensitization. The results obtained by treatment with PI-PLC or in the presence of protease inhibitors, suggested that activation of phospholipases and proteases are not involved in LPS-induced desensitization. However, trypsin treatment of macrophages abolished both LPS binding and cytokine responses. The recovery of macrophages from this trypsin-induced tolerance (restoration of TNF-α synthesis without reexpression of LPS-binding sites) was very different from that following LPS-induced tolerance (reexpression of LPS-binding sites without restoration of TNF-α synthesis). The results are consistent with the hypothesis that signaling LPS-receptors might be synthesized de novo after trypsin degradation, whereas non-signaling LPS-receptors might be internalized and recycled after preexposure to LPS.     

Role of Macrophages in Resistance to Murine Cytomegalovirus

The role of macrophages in protecting mice from murine cytomegalovirus (MCMV) was studied in Swiss, CBA/J, and C57BL/6J mice. CBA/J mice were more resistant to virus than were C57BL/6J mice at all ages tested. Prior treatment of adult Swiss mice with 60 mg of silica, a dose selectively toxic to macrophages, increased mortality due to MCMV infection. Transfer of syngeneic adult macrophages to suckling mice significantly increased their resistance to subsequent MCMV infection. Transfer of syngeneic, nonimmune adult lymphocytes to suckling mice also had a lesser but significant protective effect against subsequent MCMV challenge. In vitro infection of adult CBA/J and C57BL/6J macrophages with virulent and attenuated MCMV resulted in productive infection in only a small percentage of cells and recovery of very little virus from the extracellular fluid. Infection of CBA macrophages was no less productive than C57BL/6J nor was infection with virulent virus more productive than with attenuated virus. Histological examination of the livers of MCMV-infected CBA/J and C57BL/6J mice suggested that divergent cellular immune responses to infection might account for differences in susceptibility. It is postulated that the macrophage may facilitate the inductive phase of cellular immunity, one possible explanation for its demonstrated importance in host defenses against MCMV.     

改良型TNF-α和IFN-γ诱导小鼠腹腔巨噬细胞产生NO和细胞毒作用的影响

应用改良型ＴＮＦ α（Ｉ ＴＮＦ α）和ＩＦＮ γ刺激小鼠腹腔巨噬细胞（ＭΦ）诱生一氧化氮（ＮＯ）,并观察对细胞毒作用的影响。结果：ＩＦＮ γ可激活小鼠腹腔ＭΦ产生小量ＮＯ,并伴随产生低度细胞毒活性,尤其是对经ＩＦＮ γ处理的靶细胞（γ Ｌ１２１０细胞）为显著。单用Ｉ ＴＮＦ α刺激的ＭΦ,诱生ＮＯ不显著或微量,对靶细胞也无明显细胞毒作用。但Ｉ ＴＮＦ α（１００ｎｇ／ｍｌ）和ＩＦＮ γ（５０Ｕ／ｍｌ）共同刺激ＭΦ,可产生有意义量的ＮＯ（Ｐ＜００５）,对γ Ｌ１２１０细胞有显著细胞作用（Ｐ＜００１）,而对未经ＩＦＮ γ处理的靶细胞（Ｌ１２１０ｃｅｌｓ）无明显细胞毒作用。这可能是ＩＦＮ γ促进ＭΦ诱生ＮＯ和靶细胞表面粘附分子的表达,加强效靶识别和结合,提高了对靶细胞的杀伤力。     

Production of Interleukin-12 by Murine Macrophages in Response to Bacterial Peptidoglycan

Peptidoglycan (PG), a component of the bacterial cell wall, has various immunomodulating activities, including the capacity to induce delayed-type hypersensitivity reactions to antigens administered in Freund’s adjuvant. We report that PG induces interleukin-12 (IL-12) mRNA production and IL-12 secretion by mouse macrophages. The capacity of PG to induce IL-12 production, like its previously reported immunomodulating activities, was dependent on the structure of its peptide subunit. PG from Bacillus megaterium and Staphylococcus aureus induced IL-12 production, whereas PG from Micrococcus luteus and Corynebacterium poinsettiae did not. The ability of most bacterial PGs to induce IL-12 production suggests that they play an important role in triggering host defense mechanisms against bacterial infections.     

Altered IL-1β and IL-2 mRNA Expression in Murine Splenic Cells After Concomitant Stimulation by Semliki Forest Virus and Lipopolysaccharide

Co-infection with virus and bacteria happens frequently and often results in an exacerbated clinical course of the disease, possibly due to mechanisms including altered cytokine production. In the present study, the authors investigated the combined effects of avirulent Semliki Forest virus (SFV-A7) and bacterial lipopolysaccharide (LPS) on the interleukin-1β (IL-1β) and IL-2 gene expression in murine splenic cells. The authors found that 10 ng/ml of LPS in the culture medium induced expression of IL-1β but not IL-2, while infection with SFV-A7 did not induce either of these two cytokines. However, when SFV-A7 and LPS were applied together, a synergistic increase of both IL-1β and IL-2 was observed. Further experiments showed that addition of SFV-A7 3 h before LPS enhanced, whereas addition of the virus 3 h after the LPS inhibited, IL-1β gene expression. These results indicate that an interaction of virus and Gram-negative bacteria can result in an altered cytokine gene expression.