Structural significance of the acyl group at the C-10 position and the A ring of the taxane core of paclitaxel for inducing nitric oxide and tumor necrosis factor production by murine macrophages.

The antitumor agent, paclitaxel (Taxol), mimics the actions of lipopolysaccharide (LPS) on murine macrophages (Mphi). Various synthetic analogs of paclitaxel were examined for their potencies to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by murine peritoneal Mphi, and by human peripheral blood cells. The benzoyl group at C-2, the hydroxy group at C-7 and the acetyl group at C-10 were found to be critically important sites to activate murine Mphi. Nor-seco-taxoid analogs lacking the A ring of the taxane core of paclitaxel were inactive, but inhibit paclitaxel- or LPS-induced NO production. All the compounds tested did not induce TNF production by human blood cells.

Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells.

Pentaacyl diphosphoryl lipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-alpha nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPS) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occurred when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of 125I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occurred when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.

The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid A for macrophage binding and cytokine induction.

Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of 125I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells and to induce cytokine-release in J774.1 cells. LPS, synthetic Escherichia coli-type lipid A (compound 506) and tetraacyl precursor Ia (compound 406) inhibited the binding of 125I-LPS to macrophage-like J774.1 cells and induced the release of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6). Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release. Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 (alpha-phosphonooxyethyl analogue of 406) > 406 >> 404 (4'-monophosphoryl partial structure of 406) > 405 (1-monophosphoryl partial structure of 406). In the case of hexaacyl preparations, compounds 506, PE-1 (alpha-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a beta-phosphonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction. The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells. These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e. cytokine induction.

Quinolactacins A, B and C: novel quinolone compounds from Penicillium sp. EPF-6. I. Taxonomy, production, isolation and biological properties.

Quinolactacins A (1), B (2) and C (3), novel quinolone antibiotics have been found from the cultured broth of a fungal strain isolated from the larvae of the mulberry pyralid Margaronia pyloalis Welker). The fungal strain, EPF-6 was identified as Penicillium sp. from its morphological characteristics. Quinolactacins were obtained from the culture medium by solvent extraction and chromatographic purification. Compound 1 showed inhibitory activity against tumor necrosis factor (TNF) production induced by murine peritoneal macrophages and macrophage-like J774.1 cells stimulated with lipopolysaccharide (LPS).

Microtubule-disrupting agents inhibit nitric oxide production in murine peritoneal macrophages stimulated with lipopolysaccharide or paclitaxel (Taxol).

Paclitaxel (Taxol), a yew-derived antimitotic agent which binds to microtubules, can mimic certain effects of lipopolysaccharide (LPS) on macrophages from LPS responder mouse strains. The production of nitric oxide (NO) by the peritoneal macrophages of LPS responder C3H/HeN mice stimulated with taxol or LPS was partially, but not completely, suppressed by microtubule-disrupting agents, such as colchicine, podophyllotoxin, vinblastine, and nocodazole, but not by lumicolchicine, an inactive derivative of colchicine. Inducible NO synthase protein expression induced by taxol and LPS in the macrophages was also suppressed by colchicine, but colchicine did not suppress the transcription of iNOS mRNA in the macrophages after stimulation with taxol or LPS. These findings suggest that microtubules function in the posttranscriptional processes of iNOS protein expression rather than in the transcriptional process of iNOS mRNA and the synthetic process of NO molecules.

Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS.

Some lipopolysaccharide (LPS) preparations from S- or R-form members of the family Enterobacteriaceae and oral black-pigmented bacteria (Porphyromonas gingivalis and Prevotella intermedia) are known to activate LPS-refractory C3H/HeJ macrophages. When contaminating proteins are removed from R-form LPS of Enterobacteriaceae by repurification, however, this ability is lost. In the present study, we investigated the capacity of LPS from P. gingivalis, P. intermedia, Salmonella minnesota, and Salmonella abortusequi to induce production of tumor necrosis factor (TNF) in gamma interferon-primed C3H/HeJ macrophages before and after repurification. P. abortusequi S-LPS was fractionated by centrifugal partition chromatography into two LPS forms: SL-LPS, having homologous long O-polysaccharide chains, and SS-LPS having short oligosaccharide chains. Prior to repurification, all LPS forms except SL-LPS induced TNF production in both C3H/HeJ and C3H/HeN macrophages. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that repurification removed contaminating protein from the preparations, and repurified SS-LPS and S. minnesota Ra-LPS no longer stimulated TNF production in C3H/HeJ macrophages, although C3H/HeN macrophages remained responsive. In contrast, repurified oral bacterial LPS retained the capacity to induce TNF production in C3H/HeJ macrophages. Oral bacterial LPS preparations also were not antagonized by excess inactive, repurified SL-LPS; Ra-LPS; Rhodobacter sphaeroides lipid A, a competitive LPS antagonist, or paclitaxel, an LPS agonist, and they were comparatively resistant to polymyxin B treatment. Nevertheless, oral bacterial LPS was less toxic to D-galactosamine-treated C3H/HeN mice than was LPS from Salmonella. These findings indicate that the active molecule(s) and mode of action of LPS from P. gingivalis and P. intermedia are quite different from those of LPS from Salmonella.

CD14 is not involved in Rhodobacter sphaeroides diphosphoryl lipid A inhibition of tumor necrosis factor alpha and nitric oxide induction by taxol in murine macrophages.

Taxol, a microtubule stabilizer with anticancer activity, mimics the actions of lipopolysaccharide (LPS) on murine macrophages in vitro. Recently, it was shown that taxol-induced macrophage activation was inhibited by the LPS antagonist Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA). To investigate the mechanisms of taxol-induced macrophage activation, the present study focused on the interaction of LPS, RsDPLA, and taxol in the activation of and binding to macrophages. Taxol alone induced murine C3H/He macrophages to secrete tumor necrosis factor alpha (TNF) and to produce nitric oxide (NO) with kinetics similar to that of LPS. Macrophages from LPS-hyporesponsive C3H/HeJ mice, in contrast, did not yield any detectable TNF and NO production in response to LPS or taxol. RsDPLA inhibited taxol-induced TNF and NO production from C3H/He macrophages in a dose-dependent manner. The inhibition by RsDPLA was specific for LPS and taxol in that RsDPLA did not inhibit heat-killed Listeria monocytogenes- or zymosan-induced TNF production. Polymyxin B blocked the inhibitory effect of RsDPLA on taxol-induced TNF production. The inhibitory activity of RsDPLA appeared to be reversible since macrophages still responded to taxol in inducing TNF production after the RsDPLA was washed out with phosphate-buffered saline prior to the addition of taxol. Taxol-induced TNF production was not inhibited by colchicine, vinblastine, or 10-deacetylbaccatine III. A mutant cell line, J7.DEF3, defective in expression of a CD14 antigen, responded equally well to taxol by producing TNF as did the parent J774.1 cells. This suggested that the activation of macrophages by taxol does not require CD14. Taxol-induced TNF production by the mutant cells was also inhibited by RsDPLA. 125I-labeled LPS and 3H-labeled taxol was reported to bind to J774.1 cells predominantly via CD14 and microtubules, respectively. The binding of 125I-labeled LPS to J7.DEF3 cells was about 30 to 40% of that to J774.1 cells. The binding of 125I-LPS to J774.1 cells was inhibited by unlabeled LPS and RsDPLA but not by taxol. On the other hand, 3H-labeled taxol bound to both J774.1 cells and J7.DEF3 cells in similar time- and dose-dependent manners. The binding of [3H]taxol to these cells was inhibited by taxol but not by LPS or RsDPLA. Although the binding studies failed to examine cross competition for binding to macrophages, a possible explanation of these results is that LPS, RsDPLA, and taxol share the same molecule(s) on murine macrophages for their functional receptor(s), which is neither CD14 nor tubulin.

Structural elucidation of a capsular polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn's disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF-COSY, TOCSY, ROESY, HSQC-TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: -->3)beta-D-Glcp(1-->6)[alpha-D-GalpNAc(1-->2)beta-D-Galp(1-->4)]beta-D-GlcpNAc(1-->3)alpha-D-Galp(1-->4)beta-D-Manp(1-->.

Lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) in mice is principally due to maternal cause but not fetal sensitivity to LPS.

The present study deals with whether lipopolysaccharide (LPS)-induced intra-uterine fetal death (IUFD) is related to LPS-susceptibility of either mother or fetus and how LPS or LPS-induced TNF causes IUFD. LPS-susceptible C3H/HeN or -hypo-susceptible C3H/HeJ pregnant mice and the mice mated reciprocally with these mice were used on days 14 to 16 of gestation for experiments. All of fetuses in pregnant C3H/HeN mice mated with either C3H/HeN males [HeN(HeN)] or C3H/HeJ males [HeN(HeJ)] were killed within 24 hr when injected intravenously (i.v.) with 50 or 100 microg of LPS. On the other hand, the majority of fetuses in C3H/HeJ females mated with either C3H/HeJ males [HeJ(HeJ)] or C3H/HeN males [HeJ(HeN)] survived when injected i.v. with even 400 microg of LPS. These findings indicate that LPS-induced IUFD depends on the maternal LPS-responsiveness. LPS injected into mothers could pass through placenta to fetuses, since an injection with 125I-labeled LPS or IgG into pregnant mice resulted in considerable levels of radioactivity in fetuses as well as placenta. Cultured peritoneal macrophages derived from F1 mice of HeJ(HeN) or HeN(HeJ) mice, produced nitric oxide (NO) and tumor necrosis factor (TNF) in response to LPS, although the levels of NO and TNF were lower in comparison with those of C3H/HeN macrophage cultures, suggesting a possibility that the fetus as well as F1 cells might be responsible to LPS. LPS-induced IUFD was not blocked by treatment with anti-TNF antibody which inhibited LPS-induced TNF production in pregnant females, although an injection of recombinant TNFalpha instead of LPS could induce IUFD, suggesting that the cause of IUFD cannot be attributed to mother-derived TNF alone. The roles of LPS passed through placenta and LPS-induced mediators on IUFD were discussed.

Structural significance of the benzoyl group at the C-3'-N position of paclitaxel for nitric oxide and tumor necrosis factor production by murine macrophages.

The antitumor agent paclitaxel (Taxol) mimics the actions of lipopolysaccharide (LPS) on murine macrophages (M phi). Recently, we have shown that the benzoyl group at the C-3' position of paclitaxel is the most important site to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by C3H/HeN M phi (Biochem. Biophys. Res. Commun. 210, 678-686, 1996). In the present study, synthetic analogs of paclitaxel with replacement of the C-3'-N position were examined for their potencies to induce NO and TNF production by peritoneal M phi of LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice, by human blood cells and human M phi. In this structure-activity relationship study, we found that (i) the p-substitution of the benzoyl group definitely affects the activity to activate C3H/HeN M phi, (ii) the analogs having a methyl or chloro group at the p-position exhibit stronger activity than that of paclitaxel, (iii) there is good correlation between NO and TNF production by the M phi in response to compounds, (iv) the compounds tested do not induce either NO or TNF production by C3H/HeJ M phi or TNF production by human cells, (v) a previous treatment of C3H/HeN M phi with the inactive compounds can hardly affect either paclitaxel- or LPS-induced TNF production by the M phi, (vi) paclitaxel and its analogs marginally affect LPS-induced TNF production by human blood cells, and (vii) there is no correlation between the NO/TNF inducibility to C3H/HeN M phi and growth inhibitory activity against M phi-like J774.1 and J7.DEF3 cells.

Isolation of a macrophage-like cell line defective in binding of lipopolysaccharide. Influence of serum and lipopolysaccharide chain length on macrophage activation.

A mutant cell line (J7.DEF.3) derived from murine macrophage-like J774.1 cells, was isolated on the basis of defective specific 125I-labeled LPS-binding in the presence of serum. Although J7.DEF.3 cells still respond to LPS in inducing TNF-alpha release and nitric oxide (NO) formation, these cells nevertheless showed significantly decreased responsiveness to LPS relative to the J774.1 parent. Under serum-free conditions, no differences between J774.1 and J7.DEF.3 cells in response to LPS were observed. The time kinetics of responsiveness to LPS also showed a delay in the onset of TNF-alpha release and NO formation in the mutant cells relative to parent cells. Importantly, this decrease in responsiveness to LPS relative to parental cells was dependent on the length of the polysaccharide portion of LPS. The defect in the mutant cells has been shown to be specific for LPS, in that these cells respond to heat-killed Listeria monocytogenes and to zymosan to a similar extent as do the parental cells. Collectively these results suggest that the defect in the J7.DEF.3 mutant cells may be related to a cellular LPS-binding molecule that, in turn, may depend upon an LPS-binding serum component.

Onapristone (ZK299) blocks the suppressive effect of progesterone, but not that of dexamethasone, on inducible nitric oxide synthase gene expression and nitric oxide production in murine macrophages.

Suppressive effects of progesterone on inducible nitric oxide synthase (iNOS) protein expression and nitric oxide (NO) production in murine peritoneal macrophages in response to bacterial lipopolysaccharide (LPS) and the inhibition of the suppressive activity of progesterone by onapristone (ZK299), a synthetic progesterone inhibitor, were studied. Progesterone suppressed dose-dependently LPS-induced NO production by macrophages, and scarcely detectable expression of iNOS was seen in the macrophages. ZK299 liberated the macrophages from the inhibitory effect of progesterone. Although dexamethasone, a synthetic glucocorticoid, can potently suppress LPS-induced NO production by macrophages, ZK299 did not liberate the suppression by dexamethasone, suggesting that these two corticosteroids induce suppression through independent mechanisms. RT-PCR analysis showed that murine macrophages expressed no progesterone-receptor. These findings indicate that the inhibitory effect of progesterone occurs at least on the level of iNOS protein expression in the signaling pathway after the LPS-stimulus. Furthermore, our present data may suggest the existence of a yet unknown type of progesterone-receptor in murine macrophages, the binding to which is responsible for the inhibitory effect of progesterone, or that progesterone may act non-specifically on the macrophages without involvement of any receptor.

Macrophage activation in response to S-form lipopolysaccharides (LPS) separated by centrifugal partition chromatography from wild-type LPS: effects of the O-polysaccharide portion of LPS.

The S-form lipopolysaccharide (LPS) was effectively separated from a native preparation of smooth-type Salmonella abortus equi LPS by means of the centrifugal partition chromatography (CPC). To clarify the mechanisms by which LPS activates macrophages, CPC-separated S-form LPS was assessed for its ability to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by murine macrophage-like J774.1 cells in comparison with other fractions of LPS which lacks most of O-polysaccharides. LPS dose-response and time-kinetics studies showed that serum factor(s) regulated especially the onset of TNF-alpha secretion in stimulation with S-form LPS. These results strongly suggest that the native (unfractionated) LPS activates macrophages in both O-polysaccharide/serum-dependent and -independent pathways.

Detection of lipopolysaccharide-binding proteins on membranes of murine lymphocyte and macrophage-like cell lines.

Lipopolysaccharide-(LPS) binding proteins present on murine-lymphocyte and macrophage-like cell lines were identified by a ligand-blotting method and subsequent immunological detection of bound LPS. Membrane proteins of the murine-pre-B-cell line 70Z/3 were separated by SDS-PAGE, transferred electrophoretically onto nitrocellulose, and the blot was incubated with LPS of the Salmonella minnesota Re-mutant R595 (mRe-LPS). LPS bound to proteins on nitrocellulose was immunologically detected by anti-mRe-LPS antibodies; LPS was associated with one of the membrane proteins of 70Z/3 cells. This protein was 40 kDa under reducing and 45 kDa under non-reducing conditions, respectively. Treatment of 70Z/3 cells with pronase led to the disappearance of the LPS-binding protein indicating its surface location. Excess free lipid A, which represents the biologically active region of LPS, inhibited the binding of mRe-LPS to the protein. This LPS-binding protein was also identified on the pre-B-cell line CYG8, the B-cell line CYG101 and the murine-T-cell line BW5147. It was, however, not detectable on the B-cell line CYG34 and the myeloma-cell line P3-X63-Ag8.653. No other LPS-binding protein could be detected on these cell lines. In the murine-macrophage-like cell line J774.1, two LPS-binding proteins, one of 40 kDa and one of 80 kDa, were detected. These results indicate that mRe-LPS is specifically bound to a 40-kDa protein of lymphocytes, whereas in the case of macrophages it is associated with two LPS-binding proteins of 40 and 80 kDa.

Participation of CD14 in the phagocytosis of smooth-type Salmonella typhimurium by the macrophage-like cell line, J774.1.

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF.3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248betaH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248betaH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.

Endotoxin contamination in fetal bovine serum and its influence on tumor necrosis factor production by macrophage-like cells J774.1 cultured in the presence of the serum.

Trace amounts of endotoxin (lipopolysaccharide: LPS) are assumed to contaminate commercially available fetal bovine serum (FBS) for tissue or cell culture during the manufacturing process. We examined how cultured cells were affected by the endotoxin and how much endotoxin was in the FBS. Macrophage-like J774.1 cells maintained in RPMI1640 medium supplemented with FBS containing low doses of LPS for 15 or 21 days showed less TNF production in response to LPS than the cells maintained under LPS-free conditions, and the affected responses of the cells were not recovered by an additional 21 day culture in medium with LPS-free FBS. Concentrations of LPS in 40 lots of FBS obtained from 13 international manufacturers were measured by a highly sensitive and LPS-specific chromogenic limulus assay. The median of endotoxin levels in these lots was 46 ng/ml and the maximum was 38.8 ng/ml. Relatively higher concentrations of LPS (> 1 ng/ml) or lower levels (< 10 pg/ml) were found in 9 and 6 lots, respectively. The majority of the FBS lots contained various levels of (1-->3)-beta-D-glucan, and all lots contained high-density lipoprotein (HDL). However, no correlation was found between LPS and (1-->3)-beta-D-glucan or HDL level in the lots. Each FBS was added to macrophage-like J774.1 cells which had been maintained in LPS-free medium. Five lots of FBS induced significant TNF production by the cells without addition of any stimulant. These active 5 FBS contained relatively higher levels of LPS and pretreatment of the FBS with polymyxin B eliminated their ability to induce TNF production. No correlation was found between (1-->3)-beta-D-glucan levels in FBS and the TNF-inducing capability of FBS. These results show that considerable lots of FBS contain significant levels of LPS, which must affect cell culture.