Modulation of lipopolysaccharide-induced macrophage gene expression by Rhodobacter sphaeroides lipid A and SDZ 880.431

Rhodobacter sphaeroides lipid A (RsDPLA) and SDZ 880.431 (3-aza-lipid X-4-phosphate) are prototypic lipopolysaccharide (LPS) antagonists. Herein, we examined the ability of these structures to regulate murine macrophage tumor necrosis factor (TNF) secretion and LPS-inducible gene expression (tumor necrosis factor alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], IP-10, type 2 TNF receptor [TNFR-2], D3, and D8 genes). We report that RsDPLA alone (> 1 microgram/ml) induced low levels of TNF-alpha secretion and a selective pattern of gene expression in peritoneal exudate macrophages; SDZ 880.431 alone was completely inactive. When LPS was present at a low concentration (1 ng/ml), RsDPLA and SDZ 880.431 blocked TNF secretion and gene induction in a concentration-dependent fashion. In general, gene induction was measurably reduced by 10 to 30 ng of RsDPLA per ml or 300 ng of SDZ 880.431 per ml, but inhibition could be uniformly overridden by increasing the concentration of LPS. Although induction of all six genes by LPS was suppressed by either inhibitor, effective inhibitor concentrations depended on the gene of interest. Induction of TNFR-2 by LPS was relatively resistant to inhibition by RsDPLA, and induction of TNFR-2 and D3 was relatively resistant to inhibition by SDZ 880.431. When LPS was present at > or = 100 ng/ml, correspondingly high concentrations (> or = 20 micrograms/ml) of either inhibitor influenced gene expression in a bidirectional manner. Under these conditions, LPS-induced expression of IP-10, D3, and D8 was suppressed regardless of the LPS concentration used (concentrations tested up to 50 micrograms/ml), while expression of TNF-alpha mRNA was enhanced about fourfold. In toto, RsDPLA and SDZ 880.431, when present at low concentrations, act in a manner consistent with competitive inhibition of LPS, while at higher concentrations, these structures inhibit certain LPS responses noncompetitively and synergize with LPS for other responses.     

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.     

Establishment of functional human pituitary tumor cell cultures

Five primary human pituitary tumor cell cultures were initiated from adenoma fragments obtained from patients with prolactin-secreting adenomas and acromegaly. Functional cell cultures were maintained and propagated in monolayer or suspension culture for up to 9 months. Optimal cell viability and growth were achieved using Ham's F10 medium enriched with 20% fetal bovine serum, although cells from a patient with acromegaly also grew in serum-free, defined, hormone-containing medium. Bromocriptine (100 ng/ml) did not alter the growth curve of replicating cells derived from a patient with acromegaly. These cells initially secreted 5.5 micrograms human growth hormone/10(6) cells, and hormone production diminished after 6 wk. Prolactin secretion by cells derived from prolactinomas (0.5 to 1.3 micrograms/10(6) cells/24 h) was stimulated by thyrotropin-releasing hormone (10 ng/ml) in two of the cultures. Both dopamine (10 ng/ml) and nickel chloride (1 mM) suppressed PRL secretion. These studies demonstrate that responsive human pituitary tumor cell cultures can be initiated and maintained.     

Intestinal epithelial cells down-regulate macrophage tumor necrosis factor-alpha secretion: a mechanism for immune homeostasis in the gut-associated lymphoid tissue

BACKGROUND: The gut lumen contains more than 10(6) organisms per gram of luminal contents. The mechanisms that limit the response of macrophages in the lamina propria to these microbial antigens are unknown, although an intrinsic defect in this mechanism may contribute to the development of inflammatory bowel disease. Intestinal epithelial cells (IEC) may play an important role in mediating tonic down-regulation of local immune cell activation. The purpose of this study was to discern whether IEC might modulate macrophage activation in response to a variety of microbial stimuli. METHODS: Thioglycollate-elicited murine peritoneal macrophages were activated by endotoxin, zymosan, Escherichia coli, and Candida albicans in the presence or absence of IEC from the rat intestinal epithelial cell line IEC-6. Macrophage tumor necrosis factor-alpha (TNF-alpha) secretion was determined by enzyme-linked immunosorbent assay. RESULTS: Lipopolysaccharide or zymosan-activated macrophages in coculture with IEC secreted significantly less TNF-alpha than macrophages cultured alone. The inhibitory effect of the IEC was dependent on their activation by lipopolysaccharide. Interleukin-1 alpha production was not affected. IEC-mediated suppression of macrophage TNF-alpha secretion was reversed by indomethacin but not by neutralizing antibody to TGF-beta. CONCLUSIONS: Lipopolysaccharide-activated IEC down-regulate macrophage TNF-alpha secretion in response to microbial stimuli through a prostanoid-mediated mechanism. IEC may mediate tonic down-regulation of immune cell activation in the gut-associated lymphoid tissue and may thereby regulate local and systemic inflammatory responses.     

The threshold for immune cell reactivity to milk antigens decreases in cow's milk allergy with intestinal symptoms

BACKGROUND: In cow's milk allergy (CMA) with intestinal symptoms, peripheral blood mononuclear cells (PBMCs) secrete tumor necrosis factor-alpha (TNF-alpha), altering intestinal function. However, the type of cow's milk protein (CMP) that triggers symptoms (intact or intestinally processed) is not known, and neither is the minimal amount required. METHODS: PBMCs were isolated from infants with active CMA or cured infants just before a new challenge and stimulated with intact or intestinally processed CMP. Supernatants were tested for cytokine content and for their ability to perturb intestinal barrier capacity, measured in Using chambers in HT29-19A intestinal cells. RESULTS: PBMCs from infants with active CMA secreted more TNF-alpha, when they were stimulated with intact rather than intestinally processed CMPs, and more TNF-alpha than PBMCs from cured infants. Accordingly, supernatants from PBMCs stimulated with intact but not intestinally processed CMPs significantly increased intestinal permeability. The CMP concentration required to trigger TNF-alpha secretion capable of altering intestinal function was very small in infants with active CMA (approximately 2 micrograms/ml), but about 300 times higher in cured infants. CONCLUSION: Intact rather than intestinally processed proteins stimulate PBMCs to release TNF-alpha and alter intestinal barrier capacity. The threshold for PBMC reactivity to milk antigens drops considerably during active CMA with intestinal symptoms.     

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.     

CD44 occupancy prevents macrophage multinucleation

Cells of the mononuclear phagocyte lineage have the capability to adhere to and fuse with each other and to differentiate into osteoclasts and giant cells. To investigate the macrophage adhesion/fusion mechanism, we focused our attention on CD44, a surface glycoprotein known to play a role in hematopoietic cell-cell adhesion. We report that CD44 expression by macrophages is highly and transiently induced by fusogenic conditions both in vitro and in vivo. We show that CD44 ligands, hyaluronic acid, chondroitin sulfates, and osteopontin prevent macrophage multinucleation. In addition, we report that the recombinant extracellular domain of CD44 binds fusing macrophages and prevents multinucleation in vitro. These data suggest that CD44 may control the mononucleated status of macrophages in tissues by virtue of mediating cell-cell interaction.     

Oxidized lipoproteins including HDL and their lipid peroxidation products inhibit TNF-alpha secretion by THP-1 human macrophages

It has been established that oxidized LDL (ox-LDL) modifies cytokine secretion by macrophages, for example, by reducing tumor necrosis factor alpha (TNF-(alpha) m-RNA. However, little is known about the effects of oxidized high density lipoprotein (ox-HDL). This study reports the effects of ox-HDL subfractions 2 and 3 (ox-HDL2, ox-HDL3) compared with that of ox-LDL and some products of oxidation (hydroperoxides and aldehydes) on the secretion of TNF-alpha from THP-1 human monocytes derived macrophages in vitro. HDL2, HDL3 and LDL were oxidized with 10 microM Cu++ for 12 h and/or 24 h. Native and oxidized HDL and LDL were incubated for 24 h with macrophages with or without LPS (10 ng/ml) after which TNF-alpha secretion was measured in the culture medium. Lipid hydroperoxides and apolar aldehydes were also incubated with the cells for 2 h following which the medium was replaced and TNF-alpha secretion measured after a further 22 h of incubation. An inhibition of TNF-alpha by ox-HDL2 (p < .05), ox-HDL3 (p < .05) and ox-LDL (p < .05) from THP-1 macrophages was observed in the presence and absence of LPS. This inhibition remained the same after incubation with ox-HDL 12 h and 24 h. Hydroperoxides of linoleic acid did not modify TNF-alpha secretion by cells while five out of eight aldehydes analyzed (2,4-heptadienal, hexanal, 2-nonenal, 2-octenal, 2,4-decadienal) inhibited TNF-alpha secretion (p < .05). These findings demonstrate that ox-HDL, and some of its lipid peroxidation products, plays a role in the modulation of the inflammatory response by macrophages as previously observed for ox-LDL.     

Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma

We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.     

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes

We studied the pathways of macrophage response to lipopolysaccharide (LPS). When mouse macrophages pre-exposed to LPS were restimulated with this agent, reduced tumour necrosis factor-alpha (TNF-alpha) responses (desensitization/endotoxin tolerance) were accompanied by increased (priming) nitric oxide (NO) responses. Priming was also inducible with recombinant interferon-beta (IFN-beta). The requirement of TNF-alpha biosynthesis in the LPS-induced priming was also suggested by the observation that both anti-TNF-alpha serum and pentoxifylline inhibited this effect. However, addition of mouse recombinant TNF-alpha (mrTNF-alpha) did not enhance the priming induced by LPS or IFN-beta, and preincubation with mrTNF-alpha alone, or in association with other cytokines produced by macrophages (interleukin-1 beta, interleukin-6, or leukaemia inhibitory factor), did not induce a priming effect. We found however, that pentoxifylline, which blocked the priming, also decreased the level of membrane-bound TNF-alpha. Furthermore, exposure to compound BB-3103 (a metalloproteinase inhibitor that blocks the processing of membrane-bound TNF-alpha yielding to the secreted cytokine) enhanced the priming effect, the expression of membrane TNF-alpha and the specific binding of LPS. These observations suggest that the membrane form of TNF-alpha is involved in the interaction of LPS with a receptor required for LPS-induced priming.     

Macrophage colony-stimulating factor stimulates synthesis and secretion of a mouse homolog of a human IgE-dependent histamine-releasing factor by macrophages in vitro and in vivo

Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, IFN-gamma, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.     

Expression of intercellular adhesion molecule-1 on macrophages in vitro as a marker of activation

Macrophage activation is a major component of wound healing. It also determines the extent of inflammatory reactions and the response of the body to implanted materials. We have previously shown, using an in vitro model, that the extent of spreading of macrophages on different materials is a marker of activation, and that a soluble inducer has a dose-response effect on the secretion of cytokines in the culture medium. This work investigates the expression of three different cell surface markers [macrophages MAC-1, MAC-3 and intercellular adhesion molecule-1 (ICAM-1)] on macrophages in vitro using confocal microscopy and shows that ICAM-1 is also a marker of macrophage activation in this model. We observed increased amounts of ICAM-1 on activated macrophages compared to unactivated macrophages, whereas MAC-1 and MAC-3 were either expressed constitutively or demonstrated no quantitative change in expression after activation under the same experimental conditions. We also tested the expression of ICAM-1 with various concentrations of soluble inducers (lipopolysaccharide, 0.001, 0.01, 0.1, 1 and 10 micrograms ml-1. S-27609, 0.1, 0.25, 0.5, 1, 2 and 3 micrograms ml-1 and on a sheet of polylactic acid alone or in combination with soluble inducers. All doses of soluble inducers induced the expression of ICAM-1 on cells grown in glass chamber slides. The induction was not dose related but seemed to work rather in an on-off manner. There was no effect of material on ICAM-1 expression on the cell surface when no soluble inducer was added. This was similar to cytokine secretion, which was not induced by our material alone. When either lipopolysaccharide or S-27609 was used in combination with the material, there was an increase in the average measured intensity of ICAM-1. In this in vitro model, ICAM-1 staining as measured by confocal microscopy is a marker for macrophage activation. Our results suggest that the extent of macrophage activation as measured by ICAM-1 and by cytokine secretion is more sensitive to soluble inducers than to the action of the flat sheet of polylactic acid.     

Polyethylene particles of a 'critical size' are necessary for the induction of cytokines by macrophages in vitro

Particulate wear debris from total hip prosthetic components can stimulate macrophages to produce mediators of osteolysis which may cause aseptic implant loosening. This study evaluated the in vitro response of murine peritoneal macrophages to polyethylene particles of definitive size distributions at varying volume doses. Ceridust 3615 polyethylene particles with a mean size of 0.21, 0.49, 4.3 and 7.2 microm and GUR 120 polyethylene resin with a mean size of 88 microm were co-cultured with C3H murine peritoneal macrophages at volume (microm)3 to cell number ratios of 100:1, 10:1, 1:1 and 0.1: 1. The secretion of IL-6, IL-1beta and TNF-alpha was determined by ELISA. Significantly elevated levels of TNF-alpha and IL-1beta were determined at 100:1 ratios when the macrophages were challenged with particles with a mean size of 0.49, 4.3 and 7.2 microm, and at 10:1 ratios for particles with a mean size of 0.49 and 4.3 microm. IL-6 production was significantly elevated at 100:1 ratios for mean particle sizes of 0.49 and 4.3 microm. Particles outside this range produced considerably less cytokine suggesting that both the size and volume (or number) of polyethylene particles are critical factors in macrophage activation. Therefore particles in the phagocytosable size range of 0.3-10 microm appear to be the most biologically active.