Membrane perturbation of macrophages stimulated by bacterial lipopolysaccharide.

Macrophages recovered from regressing Moloney sarcomas lose their capacity to kill tumor cells when held 12–24 h in culture. Exposure of this population to low concentrations (ng/ml range) of bacterial lipopolysaccharide will cause a rapid resurgence of cytolytic activity, however. By using electron spin resonance techniques and either 5- or 12-doxyl stearate spin labels, we have shown that exposure of cultured tumor macrophages to lipopolysaccharide (100 ng/ml) is followed by plasma membrane perturbation. The change was most pronounced 3–4 h after stimulation, and had subsided within 5–6 h. The relationship of the described perturbation to the induction of killing cannot be determined from these studies; however, neither cytolytic activity nor the membrane change developed in other kinds of macrophages exposed to similar concentrations of lipopolysaccharide.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Increased 13-hydroxyoctadecadienoic acid content in lipopolysaccharide stimulated macrophages

Endotoxin-stimulated mouse peritoneal macrophages were found to contain 13-hydroxyoctadecadienoic acid, which was released upon alkaline hydrolysis of the cells. Compared to untreated cells, incubation with LPS increased the content of 13-hydroxyoctadecadienoic acid in macrophage hydrolysates to about 8-fold. Analysis of the material on chiralphase HPLC revealed that it consisted prevalently of 13(S)-hydroxyoctadecadienoic acid. This indicates its enzymatic origine.     

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.     

Hypoxia modulates lipopolysaccharide induced TNF-alpha expression in murine macrophages

The pro-inflammatory activity of Tumor necrosis factor-alpha (TNF-alpha) together with tissue hypoxia determine the clinical outcome in sepsis and septic shock. p38 MAPKinase is the primary intracellular signaling pathway that regulates lipopolysaccharide (LPS)-induced TNF-alpha biosynthesis, however, the effect of hypoxia on LPS mediated activation of p38 is not known. Here we report that SB203580, a specific p38 MAPK inhibitor, which completely abolished LPS-induced TNF-alpha expression by the mouse macrophage cell RAW264.7 in normoxic conditions, lost the inhibitory effect in hypoxic conditions. Hypoxia did not modulate expression of p38 MAPK, but increased that of p-MK2, a downstream target of p38 MAPK. In LPS induced endotoxemia mice model SB203580 had no inhibitory effect on the serum levels of TNF-alpha. Furthermore, hypoxia inducible factor-1alpha (HIF-1alpha) was detected in vivo after LPS administration but its expression was not affected by SB203580. Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-alpha secretion. Furthermore, the induction of HIF-1alpha in mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-alpha expression. These findings provide new insights on the pathophysiological effects of hypoxia in sepsis and septic shock.     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

Intraphagosomal oxygen in stimulated macrophages

A new electron paramagnetic resonance (EPR)-based method was developed to obtain selective information on pO₂ in a specific intracellular compartment (phagosomes). This method did not require the use of a broadening agent thereby eliminating one of the potential sources of experimental error with EPR oximetry. An oxygen-sensitive probe (4-(Trimethylammonium) 2,2,6,6-tetramethylpiperidine-d₁₇-1-oxyl iodide (d-Cat₁)) which has a net positive charge, was incorporated selectively into the phagosomes of macrophages stimulated with zymosan. Extracellular oxygen was measured by addition of a neutral nitroxide (4-oxo-2,2,6,6-tetramethylpiperidine-d₁₆-1-oxyl (¹⁵N PDT)) to this same sample. Measurements based on EPR linewidths showed the average intraphagosomal oxygen concentration to be 11.2 ± 3.4 μM lower than that measured from the extracellular compartment when the sample was perfused with air, and this was increased on stimulation of mitochondrial consumption or by increasing the oxygen concentration in the extracellular compartment. These experiments provide what we believe to be the first reported measurements of the oxygen concentration in a specific intracellular location (intraphagosomal) and its comparison with the oxygen concentration in the extracellular space. The observed gradient cannot be explained in terms of known coefficients of diffusion, and these results are consistent with previous reports that a gradient in oxygen concentration can occur between the average intracellular and extracellular concentration of oxygen. © 1995 Wiley-Liss, Inc.     

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide alters expression of c-fos and c-myc oncogenes

Expression of the c-fos, c-myc, and c-fms proto-oncogenes has been studied in thioglycollate-elicited murine peritoneal macrophages after exposure to lipopolysaccharide (LPS). After incubation with LPS (20 ng/ml), a transient and rapid induction of the expression of c-fos and c-myc oncogenes could be observed, whereas the RNA levels for c-fms were not affected. Treatment with lipid A, the active moiety of the LPS molecule, increased the c-fos and c-myc expression to a comparable degree. Similar induction of c-fos and c-myc was observed after treatment with phorbol myristate acetate, suggesting that this effect of LPS on murine macrophages might be mediated through stimulation of protein kinase C. Under similar experimental conditions, LPS treatment of macrophages did not trigger DNA synthesis. Treatment with LPS blocked DNA synthesis in macrophages treated with L cell-conditioned medium containing colony-stimulating factor. Thus changes in c-fos and c-myc expression may be elements in the complex series of biochemical events that contribute to macrophage activation and are not necessarily related to induction or priming for cellular proliferation.     

Free radical-operated proteotoxic stress in macrophages primed with lipopolysaccharide

The free-radical-operated mechanism of death of activated macrophages at sites of inflammation is unclear, but it is important to define it in order to find targets to prevent further tissue dysfunction. A well-defined model of macrophage activation at sites of inflammation is the treatment of RAW 264.7 cells with lipopolysaccharide (LPS), with the resulting production of reactive oxygen species (ROS). ROS and other free radicals can be trapped with the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a cell-permeable probe with antioxidant properties, which thus interferes with free-radical-operated oxidation processes. Here we have used immuno-spin trapping to investigate the role of free-radical-operated protein oxidation in LPS-induced cytotoxicity in macrophages. Treatment of RAW 264.7 cells with LPS resulted in increased ROS production, oxidation of proteins, cell morphological changes and cytotoxicity. DMPO was found to trap protein radicals to form protein-DMPO nitrone adducts, to reduce protein carbonyls, and to block LPS-induced cell death. N-Acetylcysteine (a source of reduced glutathione), diphenyleneiodonium (an inhibitor of NADPH oxidase), and 2,2'-dipyridyl (a chelator of Fe(2+)) prevented LPS-induced oxidative stress and cell death and reduced DMPO-nitrone adduct formation, suggesting a critical role of ROS, metals, and protein-radical formation in LPS-induced cell cytotoxicity. We also determined the subcellular localization of protein-DMPO nitrone adducts and identified some candidate proteins for DMPO attachment by LC-MS/MS. The LC-MS/MS data are consistent with glyceraldehyde-3-phosphate dehydrogenase, one of the most abundant, sensitive, and ubiquitous proteins in the cell, becoming labeled with DMPO when the cell is primed with LPS. This information will help find strategies to treat inflammation-associated tissue dysfunction by focusing on preventing free radical-operated proteotoxic stress and death of macrophages.     

Induction of expression of protein disulphide-isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide

Protein disulphide-isomerase (PDI) activity, and the level of immunodetectable PDI protein, were monitored in splenic lymphocytes and in BCL1 cells during culture in the presence of various activating factors. Bacterial lipopolysaccharide stimulated induction of PDI in splenic B cells and BCL1 cells. The time-course and specificity of induction indicated that the increase in expression of PDI is closely coupled to the final stages of B cell differentiation into antibody-producing plasma cells. The system will prove valuable in studies on the control of expression of PDI.     

Induced expression of adipophilin mRNA in human macrophages stimulated with oxidized low-density lipoprotein and in atherosclerotic lesions

The RNase activity of barnase mutants obtained by the permutation of modules or secondary structure units was investigated. Four of the 45 mutants had weak but distinct RNase activity, and they had unique optimum pHs and temperatures like natural enzymes. One of the active mutants had an ordered conformation, but the others did not. An active mutant having disordered conformation formed an ordered conformation in the presence of GMP, which is an inhibitor of this mutant. These results indicate that the amino acid sequences derived from barnase have sufficient plasticity to be rearranged into different proteins with basal enzymatic properties.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide.

The synthesis of non-histone chromatin proteins and nucleoplasmic proteins has been followed during lipopolysaccharide-induced division and differentiation of murine B lymphocytes. Synthesis was measured by pulse labelling with [3H]leucine, extraction of proteins was under conditions designed to prevent proteolysis and analysis of labelled proteins was by polyacrylamide gel electrophoresis. The average specific activity of non-histone chromatin proteins increased 3-fold, to a maximum, after culture for 24 h with lipopolysaccharide. Comparison of the relative synthesis of individual proteins (stimulation index) reveals three distinct responses: (1) those in the largest group show low stimulation indices, generally less than two; (2) a group of four proteins have indices between 4 and 5; (3) two proteins (molecular weights 21 000 and 22 000) both show an index of 5 at 24 h rising to between 7 and 8 by 48 h when the average specific activity is falling, coinciding with the period of rapid differentiation to high rate IgM secretion. Additionaly at this time, a newly labelled protein (Mr = 36 500) appears in the nucleoplasm followed by a second protein (Mr = 63 000) appearing between 48 and 72 h. The patterns of change are consistent with an overall increase in non-histone chromatin proteins synthesis, necessary for cell division, with superimposed specific changes in synthesis of non-histone chromatin proteins which could be related to regulation of cell differentiation.     

Interleukin-1beta isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines.

The gilthead seabream IL-1beta gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5' untranslated region (UTR), a single open reading frame of 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3'UTR with a polyadenylation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has the highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1beta sequences. The IL-1beta message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1beta was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation.