Induction of early gene expression in murine macrophages by synthetic lipid A analogs with differing endotoxic potentials

Numerous lipid A analogs have been synthesized in an attempt to dissociate endotoxic activities from beneficial immunomodulatory activities. In the present study, we have evaluated select lipid A analogs in macrophages for their ability to induce a panel of lipopolysaccharide (LPS)-inducible genes to gain insights into the molecular mechanisms which underlie endotoxicity. We evaluated three monosaccharide lipid A analogs: SDZ MRL 953, an agonist with an improved therapeutic margin over endotoxin; SDZ 281.288, a more toxic analog; and SDZ 880.431, an analog with proven LPS-inhibitory activity. In addition, three disaccharide lipid A analogs (i.e., lipid IVA, SDZ 880.611, and SDZ 880.924) that differ in acylation and phosphorylation patterns were also examined and compared with synthetic lipid A. With the exception of SDZ 880.431, each of these structurally diverse analogs was able to induce the complete panel of LPS-inducible genes, specifically genes which encode tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, 75-kDa type 2 TNF receptor (D7), IP-10, D3, and D8. These results underscore that macrophage stimulation by lipid A analogs is permissive to considerable structural diversity. Structures with favorable therapeutic indices (SDZ MRL 953, SDZ 880.611, and SDZ 880.924) were not different from structures with poor therapeutic indices (lipid A, lipid IVA, and SDZ 281.288) with regard to gene induction. Nonetheless, the nontoxic SDZ MRL 953 was approximately 1,000-fold less potent than synthetic lipid A at inducing TNF-alpha secretion, and perhaps this contributes to the lack of toxicity exhibited by this compound. The ability of compound SDZ 880.431 to inhibit TNF-alpha secretion induced by both SDZ MRL 953 and smooth LPS suggests that the monosaccharide and smooth LPS share a receptor or a portion thereof. A pattern of protein tyrosine phosphorylation similar to that induced by LPS was stimulated by the monosaccharide SDZ MRL 953 and SDZ 281.288 and disaccharides lipid IVA, SDZ 880.924, and SDZ 880.611, providing evidence for a common signalling pathway.     

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-alpha), interleukin-1beta (IL-1beta), 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 (相似文献   

CD14-dependent and CD14-independent signaling pathways in murine macrophages from normal and CD14 knockout mice stimulated with lipopolysaccharide or taxol

The antitumor agent, Taxol, shares with bacterial LPS the ability to activate murine macrophages, and its LPS-mimetic effects are blocked by LPS analogue antagonists. Since CD14 is central to the recognition of LPS by macrophages, we sought to examine a role for CD14 in the response to Taxol vs LPS. A comparison of responses of macrophages from wild-type mice with those from mice lacking CD14 due to a targeted disruption of the CD14 gene (CD14-deficient knockout (CD14KO)) revealed that like LPS, Taxol induces both CD14-dependent and -independent pathways of gene activation, although the CD14 dependency of Taxol stimulation is much less striking than that observed with LPS. The macrophage interaction with low concentrations of LPS (< or = 10 ng/ml) is largely CD14 dependent, as evidenced by the lack of induction of TNF-alpha, IL-1beta, and interferon-inducible protein-10 (IP-10) genes by CD14KO macrophages cultured in the absence of soluble CD14 (i.e., in autologous CD14KO -/- mouse serum). However, at high concentrations of LPS or Taxol, a CD14-independent pathway of activation is observed: this pathway leads to minimal IP-10 gene induction, even though induction of TNF-alpha and IL-1beta occurs. Measurements of TNF secretion followed a similar pattern to that observed at the level of steady state mRNA. These data suggest the existence of two pathways of activation by both LPS and Taxol: one that is CD14 dependent and leads to induction of TNF-alpha, IL-1beta, and IP-10 gene induction, and a CD14-independent pathway that results in the induction of TNF-alpha and IL-1beta, with minimal induction of IP-10.     

Lipopolysaccharide induction of MARCKS-related protein and cytokine secretion are differentially impaired in microglia from LPS-nonresponsive (C3H/HeJ) mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (> or = 100 ng/ml) than in normal (C3H/OuJ) microglia (< or = 10 ng/ml). By contrast, TNF alpha production was not significantly increased in C3H/HeJ microglia even at 1 microgram LPS/ml. Microglia expressed PKC isoforms alpha, beta, delta, and zeta (but not gamma and epsilon); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lpsd mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. European Organization for Research and Treatment of Cancer--Childhood Leukemia Cooperative Group

Osteoclasts (OCL) resorb bone. They are essential for the development of normal bones and the repair of impaired bones. The function of OCL is presumed to be supported by cytokines and other biological mediators, including tumor necrosis factor (TNF)-alpha and nitric oxide (NO). Bacterial lipopolysaccharide (LPS) is a potent inducer of TNF-alpha and inducible nitric oxide synthase (iNOS), which is the specific enzyme for synthesizing NO from L-arginine. To obtain direct evidence on LPS-induced TNF-alpha production and iNOS expression by OCL, OCL-enriched cultures were prepared by 7-day cocultures of bone marrow cells of adult BALB/c mice and osteoblastic cells (OBs) derived from calvaria of newborn BALB/c mice, and the generation of TNF-alpha and iNOS in OCL stimulated with LPS was examined immunocytochemically. When the cultured cells were stimulated with 100 ng/ml of LPS, OCL clearly showed TNF-alpha and iNOS expression. Without LPS-stimulation, no expression was observed. TNF activity in the culture supernatants of the OCL-enriched cultures in the presence of LPS was also detected by cytotoxic assay that used TNF-sensitive L929 cells. The dentin resorption activity of OCL was estimated by area and number of pits formed on dentin slices, which were covered by the OCL fraction and cultured in the presence or absence of LPS, sodium nitroprusside (SNP; a NO generating compound), N(G)-monomethyl L-arginine acetate (L-NMMA; a competitive inhibitor of NO synthase (NOS)), or LPS plus L-NMMA. Pit formation was obviously inhibited in the presence of SNP and slightly inhibited in the presence of L-NMMA, but it was not affected in the presence of LPS or LPS plus L-NMMA. These findings indicate that OCL produces TNF and expresses iNOS in response to LPS, but the LPS-activation of OCL scarcely affects pit formation by them.     

Effect of hepatitis B virus on tumour necrosis factor (TNF alpha) gene expression in human THP-1 monocytic and Namalwa B-cell lines

In response to viruses, monocytes and B cells produce TNF alpha. Therefore, we investigated TNF alpha gene expression and protein secretion in a human monocytic cell line, THP-1, and a Burkitt's lymphoma B-cell line, Namalwa, in response to hepatitis B virus (HBV). Stimulation by phorbol myristate acetate (PMA) (100 ng/ml for 48 h) induced TNF alpha secretion in THP-1 and Namalwa cells (100 to 300 pg/ml). In THP cells, the optimum response (> 2000 pg/ml) was obtained in the presence of a second mitogenic signal such as lipopolysaccharide (LPS) (10 microg/ml for 24 h). In our activation conditions, Northern blot analysis revealed a marked accumulation of TNF alpha mRNA species at 1.7 kb in both cell lines. When PMA- or PMA+LPS-stimulated THP-1 cells were exposed to HBV, TNF alpha protein and mRNA significantly decreased (> 50%). In contrast, HBV exposure of PMA-activated Namalwa cells resulted in strongly increased TNF alpha protein secretion (1 ng/ml). In this case, HBV induced TNF alpha mRNA accumulation that consisted of two types: a regular 1.7 kb and two novel high molecular weight (HMW) species at 3.7 and 4.3 kb. Exposure of stimulated THP-1 and Namalwa cells to HBV resulted in HBs and pre-S1 antigen production in the supernatants. In addition, HMW HBV DNA forms were detected in both cell lines, but with distinct HindIII restriction patterns. These findings indicate that TNF alpha gene expression may be differently regulated by HBV in activated human macrophages and B cells, and thus TNF alpha may be involved in the pathogenesis of HBV.     

The locus of tumor necrosis factor-alpha action in lung inflammation

The pulmonary host response to infection and inflammation appears, at least in part, to be compartmentalized from the systemic host response. Tumor necrosis factor-alpha (TNF-alpha) has been implicated in lung inflammation and injury, but its site(s) of action has not been clearly defined. To investigate this, transgenic mice (surfactant apoprotein C promotor/soluble TNF receptor type II-Fc fusion protein ([SPCTNFRIIFc] mice) were generated in which TNF-alpha was selectively antagonized in the distal lung through tissue-specific expression of sTNFRIIFc, a soluble TNF inhibitor. The lung inflammatory response in these mice to pulmonary challenge with Micropolyspora faeni antigen or lipopolysaccharide (LPS) was compared with the response of wild-type mice, wild-type mice treated with recombinant sTNFRIIFc intravenously, and type I TNF-receptor knockout mice. Recruitment of polymorphonuclear leukocytes (PMN) to the lung after challenge with M. faeni antigen was essentially abolished in the TNFRI knockout mice and markedly reduced in the SPCTNFRIIFc mice. Wild-type mice given sTNFRIIFc intravenously in amounts resulting in lung concentrations similar to those in SPCTNFRIIFc mice also showed significantly reduced lung PMN recruitment, whereas those given doses that achieved such concentrations in the blood but low levels in the lung did not. In contrast, PMN recruitment to the lung following aerosol challenge with LPS was reduced significantly in the TNFRI knockout mice and in mice given high-dose sTNFRIIFc intravenously, but was not reduced significantly in SPCTNFRIIFc mice. Thus, inhibition of PMN recruitment in response to M. faeni antigen correlated largely with the extent of intrapulmonary inhibition of TNF-alpha, whereas the response to LPS correlated best with the extent of extrapulmonary inhibition of TNF-alpha. These studies indicate that TNF-alpha may act at different loci to mediate lung inflammation, with the site of action depending in part on the nature of the inflammatory stimulus, and that SPCTNFRIIFc mice provide a tool by which the locus of TNF action can be addressed.     

Human retinal pigment epithelial cell interleukin-8 and monocyte chemotactic protein-1 modulation by T-lymphocyte products

PURPOSE: The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression. METHODS: HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively. RESULTS: ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results. CONCLUSIONS: T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.     

HindIII liposomes suppress delayed-type hypersensitivity responses in vivo and induce epidermal IL-10 in vitro

The purpose of this study was to evaluate the effects of resident islet macrophage activation on beta cell function. Treatment of freshly isolated rat islets with TNF-alpha and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by beta cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24 degrees C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1alpha and IL-1beta mRNA expression in rat islets. Immunocytochemical colocalization of IL-1beta with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1beta appears to mediate the inhibitory actions of TNF + LPS on beta cell function as TNF + LPS-induced expression of IL-1beta is fourfold higher than IL-1alpha, and Ab neutralization of IL-1beta prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of beta cells during the development of autoimmune diabetes.     

Structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) in a murine macrophage-like cell line, J774.1

The structure-activity relationships of lipopolysaccharide (LPS) in tumor necrosis factor-alpha (TNF-alpha) production and induction of macrophage cell death in the presence of cycloheximide (CHX) were examined in a murine macrophage-like cell line, J774.1. TNF-alpha production is one of the characteristic phenotypes of LPS-activated macrophages, and is observed upon incubation with LPS. On the contrary, macrophage cell death, which had been found in our laboratory (Amano F., Karahashi H., J. Endotoxin Res., 3, 415423 (1996)) and was examined as the release of lactate dehydrogenase (LDH) from cells into the culture supernatant, was observed 2.5 h after the addition of LPS in the presence of CHX. However, both TNF-alpha production and macrophage cell death were similarly dependent on the structures of LPS and lipid A. At more than 10 ng/ml, wild-type LPS from E.coli and S. minnesota exhibited the highest activity, and LPS as well as diphosphoryl lipid A from S. minnesota rough mutants also exhibited activity, but E. coli LPS detoxified by alkaline treatment or monophosphoryl lipid A from S. minnesota exhibited no activity even at 100 ng/ml. These results suggest that LPS-induced macrophage cell death in the presence of CHX shows similar requirements for LPS and/or lipid A structures as for the macrophage activation at higher doses than 10 ng/ml, although the former was not observed at 1 ng/ml LPS, while the latter was. However, TNF-alpha does not seem to be involved in the induction of macrophage cell death, because a neutralizing anti-TNF-alpha antibody failed to block the macrophage cell death and because recombinant TNF-alpha had little effect on the extent of LDH release in the presence or absence of LPS and/or CHX, and also because TNF-alpha production by LPS was abolished in the presence of CHX.     

Peroxynitrite mediates IL-8 gene expression and production in lipopolysaccharide-stimulated human whole blood

Recent evidence indicates that free oxygen radicals, in particular hydroxyl radicals, may act as intracellular second messengers for the induction of IL-8, a potent chemoattractant and activator of neutrophil granulocytes. Here we report that peroxynitrite (ONOO-), formed by a reaction of nitric oxide (NO) with superoxide, mediates IL-8 gene expression and IL-8 production in LPS-stimulated human whole blood. The NO synthase inhibitors aminoguanidine and NG-nitro-L-arginine methyl ester (L-NAME) blocked IL-8 release by approximately 90% in response to LPS (1 microg/ml), but did not affect the production of IL-1beta or TNF-alpha. Both aminoguanidine and L-NAME blocked the induction of IL-8 mRNA by LPS. Authentic ONOO- (2.5-80 microM) augmented IL-8 mRNA expression and stimulated IL-8 release in a concentration-dependent manner, whereas the NO-releasing compounds, S-nitroso-N-acetyl-DL-penicillamine and sodium nitroprusside failed to induce cytokine production. Combination of the NO-generating chemicals with a superoxide-generating system (xanthine/xanthine oxidase) markedly increased IL-8 release. Enhanced ONOO- formation was detected in granulocytes, monocytes, lymphocytes, and plasma after challenge with LPS. Furthermore, pyrrolidine dithiocarbamate, an inhibitor of activation of nuclear factor-gammaB, markedly attenuated the induction of IL-8 mRNA expression and IL-8 release by either LPS or ONOO-. Our study identifies ONOO- as a novel signaling mechanism for IL-8 gene expression and suggests that inhibition of ONOO- formation or scavenging ONOO- may represent a novel therapeutic approach to inhibit IL-8 production that could lead to reduction of neutrophil accumulation and activation.     

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.