Synthesis of interleukin 1beta and interleukin 6 by stimulated rat peritoneal macrophages: modulation by glutamine

Synthesis and secretion of IL-1beta and IL-6 were compared in LPS-stimulated rat peritoneal macrophages, and the effect of glutamine studied. LPS induced a parallel increase in mRNA and synthesis of IL-1beta and IL-6. IL-1beta accumulated mainly in the cytosol and IL-6 in the culture medium. Glutamine addition increased the synthesis of both cytokines, but the overall production (intra-+extracellular) of IL-1beta increased two-fold, although that of IL-6 increased only 1.3-fold. The influence of glutamine is discussed.     

Determination of quantitative parameters of Escherichia coli phagocytosis by mouse peritoneal macrophages

Quantitative parameters of phagocytosis of fluorescein-labeled Escherichia coli cells by mouse peritoneal macrophages were studied using a fluorimetric method. E. coli cells were conjugated with fluoresceinisothiocyanate (FITC) and then incubated with macrophages. At the end of incubation, phagocytosis was stopped by the addition of a lysing solution (0.5% Triton X-100 in 0.01 M phosphate buffer in 0.15 M saline, pH 7.4). Trypan blue at a concentration of 0.04% was used as a quenching agent to differentiate between attached and ingested E. coli cells. It was shown that phagocytosis of E. coli cells depended on temperature and opsonization of bacteria. The number of E. coli cells ingested by macrophages increased rapidly for the initial 60 min of incubation at 37°C. To achieve optimal uptake of E. coli cells, their opsonization with 5% native serum was needed. The uptake of nonopsonized bacteria by macrophages was significantly lower than that of the opsonized ones (p < 0.05). Sodium azide was shown to produce a dose-dependent suppression of phagocytosis of E. coli cells by mouse peritoneal macrophages.     

5-series peptido-leukotriene synthesis in mouse peritoneal macrophages: modulation by dietary n-3 fatty acids

This study was undertaken to elucidate the metabolic fate of dietary eicosapentaenoic acid (20:5n-3), a major n-3 polyunsaturated fatty acid constituent of fish oil, in the mouse peritoneal macrophage. Mice were fed diets containing menhaden fish oil (FO) or safflower oil (SO). After 3 weeks, resident or responsive peritoneal macrophages were isolated and stimulated with zymosan and A23187, respectively. We demonstrate the novel synthesis of leukotriene C5 (LTC5), derived from 20:5n-3, in addition to LTC4 in both resident and responsive macrophages from FO fed mice. The structures of these peptido-leukotrienes were characterized by their retention times on reverse phase high pressure liquid chromatography, by their u.v. absorbance spectra, and by cross-reactivity with peptido-leukotriene antibody. These results demonstrate for the first time that macrophages are capable of metabolizing dietary 20:5n-3 to LTC5, a pentaene peptido-leukotriene.     

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.     

Biochemical characterization of mouse microsomal prostaglandin E synthase-1 and its colocalization with cyclooxygenase-2 in peritoneal macrophages.

We cloned the cDNA for mouse microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and expressed the recombinant enzyme in Escherichia coli. The membrane fraction containing recombinant mPGES-1 catalyzed the isomerization of PGH2 to PGE2 in the presence of GSH with K(m) values of 130 microM for PGH2 and 37 microM for GSH, a turnover number of 600 min(-1), and a k(cat)/K(m) ratio of 4.6 min(-1) microM(-1). Recombinant mPGES-1 was purified and used to generate a polyclonal antibody highly specific for mPGES-1. The antibody showed a single band on Western blotting of microsomal fractions from lipopolysaccharide-treated mouse peritoneal macrophages. Northern and Western blotting analyses revealed that mPGES-1 was induced together with cyclooxygenase-2 in mouse macrophages after treatment of the cells with lipopolysaccharide. Confocal immunofluorescence microscopy revealed that both mPGES-1 and cyclooxygenase-2 were colocalized in the lipopolysaccharide-treated macrophages. Taken together, these results demonstrate that mPGES-1 is an efficient downstream enzyme for the production of PGE2 in the activated macrophages treated by lipopolysaccharide.     

大田软海绵酸对FL细胞DNA的损伤及凋亡相关蛋白表达的影响

本文旨在探讨大田软海绵酸对人羊膜细胞DNA的损伤及凋亡相关蛋白表达的影响。实验用0、20、40、608、0、100 nmol/L OA诱导FL细胞4h后,检测DNA损伤程度的彗星实验表明,OA对FL细胞DNA的损伤随染毒浓度的升高而增加。蛋白免疫印迹法显示凋亡相关蛋白Bcl-2、Bax和p53的表达与染毒浓度呈负相关;用100 nmol/L OA分别诱导2h、4h、8h后发现,三种蛋白的表达与染毒时间也呈负相关。由此可知在OA诱导的FL细胞凋亡中,损伤DNA,降低Bcl-2蛋白的表达可能参与了凋亡的部分作用,而Bax和p53蛋白则可能与OA诱导的细胞增殖有关。     

Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages

, , and 1986. Inhibition of lysosomal fusion by Trypanosoma cruzi in peritoneal macrophages. International Journal for Parasitology 16: 629–632. Prelabelling of lysosomes with acridine orange has been performed in order to verify whether metacyclic forms of Trypanosoma cruzi are capable of inhibiting lysosomal fusion during the first moments of interiorization in non-sensitized mouse peritoneal macrophages. Thus, the degree of degranulation (lysosomal fusion) in metacyclic forms is low while epimastigote forms present higher levels. When epimastigote forms are made to interact with the macrophages in the presence of various concentrations of the medium used for transformations of epimastigotes to metacyclic forms or when interaction was performed in the presence of NH₄Cl, the degree of degranulation was similar to that obtained when interaction was carried out with metacyclic forms.

The present results suggest that during the first moments of the interaction of T. cruzi, only the infective forms may increase the cytoplasmic pH value of the host phagocytic cell, avoiding lysosomal fusion and the subsequent destruction of the parasite.     

Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrow-derived macrophages upon coculturing with late apoptotic cells

Recently, we found that resident peritoneal macrophages produce MIP-2, one of the major chemokines for neutrophils, upon coculturing with late apoptotic cells, and that intraperitoneal injection of late apoptotic cells into the peritoneal cavity causes neutrophil infiltration via MIP-2. It is not known, however, whether or not macrophages are heterogeneous in such MIP-2 production. In this study, we examined changes in the surface phenotype during the differentiation of bone marrow cells into macrophages due to M-CSF and GM-CSF, and then examined the production of cytokines, namely IL-12 p40, MIP-2, IL-10, and TGF-β, following phagocytosis of late apoptotic cells with these macrophages or LPS stimulation of these macrophages. GM-CSF and M-CSF induced macrophage populations with distinct but overlapping cell surface phenotype. Although these macrophages phagocytosed late apoptotic cells to a similar extent, they produced either IL-12 p40 or IL-10, whereas they produced MIP-2 to a similar extent after the coculture, raising the possibility that late apoptotic cells may induce neutrophil infiltration in any organs, such as the liver and lungs, where the macrophages are differentiated by either M-CSF or GM-CSF, respectively.     

Delivery to macrophages and toxic action of etoposide carried in mouse red blood cells

Erythrocytes could be used as physiological carriers of active compounds. Several substances can be loaded into erythrocytes by hypotonic dialysis methods. Furthermore, carrier erythrocyte membrane can be chemically modified in order to promote increased arrival of the loaded compound to macrophages. In this work, we have prepared erythrocytes loaded with etoposide. We found conditions to obtain high etoposide encapsulation yields with minor alteration of some cell parameters of these carrier erythrocytes. Etoposide loaded into erythrocytes is mainly localised in the cytoplasmic compartment. Membrane modification of etoposide-loaded erythrocytes with band 3 crosslinkers produces an increased incorporation of the drug into macrophages mainly by phagocytosis process. The toxic effect of etoposide conveyed in these carrier erythrocytes determined as DNA fragmentation in macrophages was higher than that shown by free etoposide added at the same concentration in the culture medium to macrophages. These results seem to indicate the usefulness of this model to deliver this anti-tumour compound to macrophages, which might be useful in therapy.     

Influence of the galactosyl ligand structure on the interaction of galactosylated liposomes with mouse peritoneal macrophages

Liposomes bearing at their surface mono- and triantennary galactosyl ligands were prepared and their interaction with the galactose receptor of mouse peritoneal macrophages studied. Triantennary structures were synthesized by coupling derivatives of 1-thio--d-galactose to the amino groups of lysyl-lysine dipeptide. Galactosylated liposomes were obtained either by synthesis of neo-galactolipids followed by their incorporation into the vesicles or by neo-galactosylation of preformed liposomes by reaction between thiol-functionalized galactosyl ligands and vesicles bearing maleimido groups. The interaction of the galactosylated liposomes with the macrophage lectin was remarkably sensitive to the topology of the ligands, i.e., a spacer-arm length about 3 nm was necessary and, in contrast to results obtained with the galactose receptor of other cells, the triantennary structure did not provide additional binding. Related to the strategy of drug delivery with targeted liposomes, these results indicate that lectins from different cells might possibly be distinguished by using multiantennary ligands having optimal geometries.Abbreviations Gal d-galactose - GalNAc 2-acetamido-2-deoxy-d-galactose - PC l--phosphatidylcholine - PE l--phosphatidylethanolamine - DPPE dipalmitoyl-l--phosphatidylethanolamine - PG l--phosphatidylglycerol - SPDP N-succinimidyl-3-(2-pyridyldithio)propionate - SMPB succinimidyl-4-(p-maleimidophenyl)butyrate - MPB-PE 4-(p-maleimidophenyl)butyryl-PE - Succ-DPPE N-succinyl-DPPE - NHS N-hydroxysuccinimide - DCC N,N-dicyclohexylcarbodiimide - EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - NHS-Succ-DPPE NHS ester ofN-succinyl-DPPE - REV vesicles obtained by reversed phase evaporation - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - DMEM Dulbecco's modified Eagle's medium.     

Mycobacteria exploit nitric oxide‐induced transformation of macrophages into permissive giant cells

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so‐called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double‐edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO‐induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria‐infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.     

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2ú-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2ú- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.     

Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

Expression and distribution of very late antigen-5 in mouse peritoneal macrophages upon ingestion of fibronectin-bound Staphylococcus aureus

Many pathogens colonize host tissues by binding to the extracellular matrix via their cell surface adhesion molecules, which are called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). Staphylococcus aureus expresses several of these adhesion molecules, some of which bind to fibronectin. Of these adhesion molecules, fibronectin-binding proteins play a role in the pathogenicity of S. aureus, although it is not yet clear whether they enhance its virulence. We have previously shown that fibronectin-bound S. aureus is efficiently phagocytosed by thioglycolate-induced mouse peritoneal macrophages. Bacterial ingestion is mediated by Very Late Antigen-5 (VLA-5; alpha5beta1 integrin) and is accompanied by the formation of adhesion complexes. Here we show that the expression of VLA-5 is restricted to thioglycolate-induced inflammatory macrophages and is not found in the resident macrophages. When cells were in suspension, alpha5 integrin was not expressed on the surface of either resident or inflammatory macrophages, whereas in adherent cells, this integrin was distributed on the surface of inflammatory but not resident macrophages. A high level of this integrin was present in the cytoplasmic region only in inflammatory macrophages. In agreement with this, fibronectin-mediated phagocytosis of S. aureus was observed only in the inflammatory macrophages. In inflammatory macrophages ingesting fibronectin-bound S. aureus, alpha5 integrin was concentrated close to the phagocytosed bacteria. This change in distribution was not found in macrophages ingesting untreated bacteria. Together with our previous work, these results indicate that, upon ingestion of fibronectin-bound S. aureus, VLA-5 accumulates in the area of phagocytosis in inflammatory macrophages, where it forms adhesion complexes.     

Analysis of the p53 gene alterations in mouse embryo fibroblast cell line Balb 3T12 and its derivative 312

We have analysed the status of the p53 gene in the mouse embryo fibroblast cell line Balb 3T12 (TD₅₀=10⁶) and its transformed clonal derivative 312 (TD₅₀=10⁴) with an aim to determine whether there exists a correlation between increased tumorigenicity and clonal expansion of cells bearing a mutation in the p53 gene. While Southern hybridizations did not show any obvious changes in the p53 gene organization in 3T12 and 312 cells, sequencing the p53 cDNA revealed that 3T12 is mutated at the amino acid residue 233 (Tyr→ Asp) whereas 312 is mutated at the residue 132 (Cys→Trp). Exploiting the altered RFLP pattern due to mutations, we identified that 3T12 contains p53 alleles that are different from the already identified mutant p53. On the basis of these observations, we conclude that 3T12 and 312 have evolved independently.     

The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons

DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.     

Different effects of p58PITSLRE on the apoptosis induced by etoposide,cycloheximide and serum-withdrawal in human hepatocarcinoma cells

Minimal overexpression of the p58^PITSLRE protein kinase in Chinese hamster ovary cells induces telephase delay, abnormal cytokinesis, retarded cell growth and apoptosis. Fas mediated T cell death is correlated with p58^PITSLRE proteolysis and an increase in its histone H1 kinase activity. In this study, it was found that p58^PITSLRE had different effects on the apoptosis induced by etoposide, cycloheximide and serum-withdrawal in human hepatocarcinoma cells. The ectopic expression of p58^PITSLRE in human hepatocarcinoma cells suppressed apoptosis induced by etoposide, while enhancing the apoptosis induced by cycloheximide and serum-withdrawal respectively. Elevated expression of p58^PITSLRE was found during the apoptosis induced by etoposide, whereas most of p58^PITSLRE was proteolytically processed during apoptosis induced by cycloheximide and serum-withdrawal. Furthermore, transient transfection of p50^PITSLRE resembling the proteolytic form of p58^PITSLRE enhanced the 7721 cells susceptibility to apoptosis induced by all the three stimuli. These findings suggest that the full-length p58^PITSLRE might protect the cells from the apoptosis induced by etoposide and its proteolysis might contribute to and enhance the apoptosis induced by cycloheximide and serum-withdrawal respectively.