Lipopolysaccharide (LPS) and zymosan-resistant mutant isolated from a macrophage-like cell line, WEHI-3, with a defective response to LPS under serum-free conditions.

A LPS-resistant mutant, W3SF-1, was isolated from a murine macrophage-like cell line, WEHI-3. The W3SF-1 mutant did not produce a significant amount of nitric oxide (NO) or TNF-alpha even with high concentrations of LPS in the presence or absence of FCS, whereas the parental WEHI-3 cells produced them in response to LPS. The parental cells expressed a significant level of TNF-alpha mRNA after LPS stimulation, whereas the mutant cells did not. This defective response of the mutant cells to LPS was neither dependent on the concentration or chemical structure of LPS, nor on the time of LPS treatment. The mutant cells also showed a defective response to zymosan, suggesting that the defect in the mutant cells is common to LPS and zymosan in the signal transduction pathways. The parental and mutant cells showed similar levels of Mac1, F4/80 and CD14, suggesting that these surface markers of macrophages are not linked directly to the defective responses of the mutant to LPS. The treatment of mutant cells with IFN-gamma did not restore the defect of NO or TNF-alpha production on LPS treatment. Binding experiments with 125I-labelled LPS showed a similar binding affinity for LPS in the parental and the mutant cells. These results suggest that the defect in the W3SF-1 mutant cells may not reside in the LPS binding but rather in the early step of signal transduction pathways in the cells after LPS binding.

Regulation of SulA cleavage by Lon protease by the C-terminal amino acid of SulA, histidine.

SulA protein, a cell division inhibitor in Escherichia coli, is degraded by Lon protease. The C-terminal eight residues of SulA have been shown to be recognized by Lon; however, it remains to be elucidated which amino acid in the C-terminus of SulA is critical for the recognition of SulA by Lon. To clarify this point, we constructed mutants of SulA with changes in the C-terminal residues, and examined the accumulation and stability of the resulting mutant SulA proteins in vivo. Substitution of the extreme C-terminal histidine residue with another amino acid led to marked accumulation and high stability of SulA in lon(+) cells. A SulA mutant in which the C-terminal eight residues were deleted (SulAC161) showed high accumulation and stability, but the addition of histidine to the C-terminus of SulAC161 (SulAC161+H) made it labile. Similarly, SulAC161+H fused to maltose-binding protein (MBP-SulAC161+H) formed a tight complex with and was degraded rapidly by Lon in vitro. Histidine competitively inhibited the degradation of MBP-SulA by Lon, while other amino acids did not. These results suggest that the histidine residue at the extreme C-terminus of SulA is recognized specifically by Lon, leading to a high-affinity interaction between SulA and Lon.

Inhibition of prostaglandin synthesis by nitric oxide in RAW 264.7 macrophages.

Multiple effects of nitric oxide (NO) were revealed on the inhibition of prostaglandin (PG) synthesis by a macrophage-like cell line, RAW 264.7 cells, treated with lipopolysaccharide (LPS). NO-generating reagent, N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)ethanamine (NOC 12), inhibited the release of PG from cells with LPS treatment at higher concentrations although it stimulated the release at 50 microM. PGH synthase (PGHS) activity in the microsome fraction of the LPS-treated cells was inhibited by (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamine (NOR 1), another NO-generating reagent, dose dependently. NOC 12 also dose dependently inhibited PG synthesis from exogenous arachidonic acid in those cells. On the other hand, NOC 12 increased PGHS-2 mRNA, while it increased the PGHS-2 protein at concentrations lower than 200 microM or decreased it at higher concentrations. These results suggest that the effect of NO on PGs synthesis in LPS-treated macrophage cells is mainly due to the balance of its stimulations of the transcriptional and/or translational expression of PGHS-2 and the inhibition of the induced PGHS-2 activity.

Salivary gland extract of Rhipicephalus appendiculatus ticks inhibits in vitro transcription and secretion of cytokines and production of nitric oxide by LPS-stimulated JA-4 cells.

There is increasing evidence that compounds in tick saliva and salivary gland extract (SGE) have a suppressive effect on host immunity and that tick-borne pathogens exploit this situation to their benefit thus causing diseases. We have demonstrated that SGE derived from Rhipicephalus appendiculatus ticks has a suppressive effect on a macrophage like cell line, JA-4, in terms of secretion as well as mRNA transcription of three cytokines. Percent suppression of cytokine secretion by JA-4 cells cultured in the presence of lipopolysaccharide (LPS) and SGE in comparison to JA-4 cells cultured in the presence of LPS alone was 67.8, 89.1 and 82.0% for IL-1alpha, TNF-alpha and IL-10, respectively (P<0.05). A similar pattern of results was demonstrated in terms of mRNA transcription where SGE-induced suppression was 36.9% for IL-1alpha, 25.0% for TNF-alpha and 31.5% for IL-10 (P<0.05). In addition, we have demonstrated that SGE partially inhibited nitric oxide production by JA-4 activated with LPS. The results of the present study suggest that tick salivary gland compounds may exert their effect in vivo by blocking the functions of macrophages in the transcription of cytokines and production of nitric oxide. This SGE-induced immunomodulation may comprise a major gateway in the facilitation of tick feeding and transmission of pathogens in hosts.

The expression of prostaglandin E receptors EP2 and EP4 and their different regulation by lipopolysaccharide in C3H/HeN peritoneal macrophages.

The expression and regulation of the PGE receptors, EP(2) and EP(4), both of which are coupled to the stimulation of adenylate cyclase, were examined in peritoneal resident macrophages from C3H/HeN mice. mRNA expression of EP(4) but not EP(2) was found in nonstimulated cells, but the latter was induced by medium change alone, and this induction was augmented by LPS. mRNA expression of EP(4) was down-regulated by LPS but not by medium change. PGE(2) increased the cAMP content of both LPS-treated and nontreated cells. ONO-604, an EP(4) agonist, also increased cAMP content in nonstimulated cells and in cells treated with LPS for 3 h, but not for 6 h. Butaprost, an EP(2) agonist, was effective only in the cells treated with LPS for 6 h. The inhibitory effects of ONO-604 on TNF-alpha and IL-12 production were equipotent with PGE(2) at any time point, but the inhibitory effects of butaprost were only seen from 14 h after stimulation. PGE(2) or dibutyryl cAMP alone, but not butaprost, reduced EP(4) expression, and indomethacin reversed the LPS-induced down-regulation of EP(4), indicating that the down-regulation of EP(4) is mediated by LPS-induced PG synthesis and EP(4) activation. Indeed, when we used C3H/HeJ (LPS-hyporesponsive) macrophages, such reduction in EP(4) expression was found in the cells treated with PGE(2) alone, but not in LPS-treated cells. In contrast, up-regulation of EP(2) expression was again observed in LPS-treated C3H/HeJ macrophages. These results suggest that EP(4) is involved mainly in the inhibition of cytokine release, and that the gene expression of EP(2) and EP(4) is differentially regulated during macrophage activation.

Lipopolysaccharide (LPS)-induced cell death of C3H mouse peritoneal macrophages in the presence of cycloheximide: different susceptibilities of C3H/HeN and C3H/HeJ mice macrophages.

Lipopolysaccharide (LPS) induced cytotoxicity toward mouse peritoneal macrophages from C3H/HeN mice but not C3H/HeJ mice in vitro in the presence of cycloheximide (CHX). More than 1 ng/ml LPS induced a significant time-dependent release of a cytoplasmic enzyme, lactate dehydrogenase (LDH), while even 1000 ng/ml LPS failed to induce it in LPS-non-responsive C3H/HeJ mouse macrophages. Although similar LPS-induced cytotoxicity was observed in a murine macrophage-like cell line, J774.1, but not in an LPS-resistant mutant of J774.1, the LPS1916 cell line, these results suggest that the induction of this cytotoxicity is linked to the LPS-sensitivity of mouse macrophages. A recombinant TNF-alpha (rTNF-alpha) at 100 ng/ml augmented LDH release from both C3H/HeN and C3H/HeJ macrophages treated with LPS and CHX, while rTNF-alpha alone or in combination with LPS or CHX failed to induce LDH release. These results suggest that this cytotoxicity might be partially regulated by high concentrations of exogenous TNF-alpha in both C3H/HeN and C3H/HeJ macrophages, implying a possibility of paracrine regulation of TNF-alpha in mice toward LPS-treated macrophages under impaired protein synthesis.

A selective inhibitor of p38 MAP kinase, SB202190, induced apoptotic cell death of a lipopolysaccharide-treated macrophage-like cell line, J774.1.

A selective p38 MAP kinase (p38 MAPK) inhibitor, SB202190, induced apoptotic cell death of a macrophage-like cell line, J774.1, in the presence of lipopolysaccharide (LPS), as judged by DNA nicks revealed by terminal deoxy transferase (TdT)-mediated dUTP nick end labeling (TUNEL), activation of caspase-3, and subsequent release of lactate dehydrogenase. This cytotoxicity was dependent on both LPS and SB202190, and such inhibitors of the upstream LPS-signaling cascade as polymyxin B and TPCK blocked this macrophage cell death. SB202190 suppressed the kinase activity of p38, leading to inhibition of activation of MAPKAPK2 and then the subsequent phosphorylation of hsp27 in LPS-treated macrophages both in vitro and in vivo, but an inactive analog of SB202190, SB202474, did not. There was a threshold of the time of addition of SB202190 to LPS-treated macrophages to induce apoptosis, which was before full transmission of p38 activity to a direct downstream kinase, MAPKAPK2. Besides, localization of phosphorylated hsp27 in Golgi area of the LPS-treated macrophages was suppressed by SB202190, while it was not by SB202474. These results suggest that selective inhibition of p38 MAPK activity in LPS-induced MAP kinase cascade leads to apoptosis of macrophages.

Regulatory role of C-terminal residues of SulA in its degradation by Lon protease in Escherichia coli.

The SulA protein is a cell division inhibitor in Escherichia coli, and is specifically degraded by Lon protease. To study the recognition site of SulA for Lon, we prepared a mutant SulA protein lacking the C-terminal 8 amino acid residues (SA8). This deletion protein was accumulated and stabilized more than native SulA in lon(+) cells in vivo. Moreover, the deletion SulA fused to maltose binding protein was not degraded by Lon protease, and did not stimulate the ATPase or peptidase activity of Lon in vitro, probably due to the much reduced interaction with Lon. A BIAcore study showed that SA8 directly interacts with Lon. These results suggest that SA8 of SulA was recognized by Lon protease. The SA8 peptide, KIHSNLYH, specifically inhibited the degradation of native SulA by Lon protease in vitro, but not that of casein. A mutant SA8, KAHSNLYH, KIASNLYH, or KIHSNAYH, also inhibited the degradation of SulA, while such peptides as KIHSNLYA did not. These results show that SulA has the specified rows of C-terminal 8 residues recognized by Lon, leading to facilitated binding and subsequent cleavage by Lon protease both in vivo and in vitro.

Changes of caspase activities involved in apoptosis of a macrophage-like cell line J774.1/JA-4 treated with lipopolysaccharide (LPS) and cycloheximide.

The addition of lipopolysaccharide (LPS) together with cycloheximide (CHX) induced apoptosis in a subline of a J774.1 macrophage-like cell line, JA-4, as judged by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL)-staining and poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP)-cleavage. Caspase activities were examined in these macrophages in vitro using fluorogenic substrates such as acetyl-DEVD-aminomethyl coumarine (Ac-DEVD-AMC, caspase-3-like), acetyl-YVAD-aminomethyl coumarine (Ac-YVAD-AMC, caspase-1-like), acetyl-VEID-aminomethyl coumarine (Ac-VEID-AMC, caspase-6-like), and carbobenzoxy-IETD-aminofluoro coumarine (Z-IETD-AFC; caspase-8-like). Kinetic studies revealed these caspase activities with different Km and Vmax values in extracts of apoptotic macrophages. In the course of apoptosis, caspase-3-like activity increased first at 75 min, simultaneously with the appearance of TUNEL staining and prior to PARP cleavage, and then caspase-6 and 8-like activities increased at 90 and 105 min, respectively. However, caspase-1-like activity did not change throughout the experiment. Furthermore, removal of LPS and CHX by extensive washing of the cells for 60 min completely abolished the apoptosis and the subsequent release of lactate dehydrogenase (LDH) during additional incubation until 4 h after LPS addition. However, washing of the cells after 75 min or later resulted in the progress of apoptosis and LDH release, which was coordinated with the elevation of caspase-3-like activity at 60 min and that of caspase-6 or 8-like activity at 90 min, but not with that of caspase-1-like activity. These results suggest that caspase-3-like activity represents the most apical caspase among these caspases in terms of the intiation of apoptosis in macrophages treated with LPS and CHX. In the present study, we also provide evidence on the relatively low specificities of a series of caspase inhibitors other than acetyl-DEVD-aldehyde (Ac-DEVD-CHO) which specifically inhibited the caspase-3-like activity.

Inhibition of listeriolysin O-induced hemolysis by bovine lactoferrin.

Lactoferrin (LFR) plays an important role in the anti-microbial defense through iron binding, lipopolysaccharide binding and immunomodulation. In this study, we demonstrate that bovine LFR specifically inhibits the hemolytic activity of listeriolysin O (LLO) produced by Listeria monocytogenes. The hemolytic activity of LLO was completely inhibited in the presence of bovine LFR that was highly purified on two cation-exchange columns, whereas that of streptolysin O or perfringolysin O was not inhibited at all. A rabbit anti-LFR antibody canceled this inhibitory activity of bovine LFR. Although human transferrin exhibits 62% amino acid identity with bovine LFR, human apo-transferrin could not inhibit LLO-induced hemolysis. An increase in the concentration of FeCl3 or the Fe3+-saturation of bovine LFR, however, slightly reduced its inhibition of the hemolysis. The inhibitory activity of bovine LFR was dependent on pH, since it was observed under neutral and alkali conditions, but not under acidic conditions. These results suggest that the inhibition of LLO-induced hemolysis by bovine LFR is influenced by pH and iron ions, both of which may lead to conformational changes of LFR.

Epigallocatechin gallate and gallocatechin gallate in green tea catechins inhibit extracellular release of Vero toxin from enterohemorrhagic Escherichia coli O157:H7.

We studied the effects of six catechin derivatives (catechin, epigallocatechin, epicatechin, epicatechin gallate, epigallocatechin gallate (EGCg) and gallocatechin gallate (GCg)) in green tea on the production and extracellular release of Vero toxins (VTs) from enterohemorrhagic Escherichia coli (EHEC) cultured at 37 degrees C for 24 h. EGCg and GCg in the culture medium markedly inhibited extracellular VTs release from EHEC cells into the culture supernatant fluid at concentrations of 0.05 mg/ml or higher, as estimated by both the reversed passive latex agglutination assay and cytotoxic assay using Vero cells. Production and extracellular release of maltose binding protein, a periplasmic protein, into the culture supernatant were also inhibited by EGCg and GCg, indicating that their inhibitory effect on release from periplasm into the outer milieu is not specific to VTs, but general to the proteins accumulated in EHEC periplasm.

Suppression of prostaglandin synthesis by arachidonic acid or eicosapentaenoic acid in a macrophage-like cell line, RAW 264.7, treated with LPS.

In a mouse macrophage-like cell line, RAW 264.7, increasing the arachidonic acid (AA) content, by culturing cells with AA, caused profound AA release, irrespective of the lipopolysaccharide (LPS)-treatment, while lowering the AA content, by culturing cells with eicosapentaenoic acid (EPA), decreased it compared with the level in non-modified control cells. However, the release of prostaglandin D2 (PGD2), which had been generated from AA in response to LPS-treatment, was significantly decreased in both AA- and EPA-treated cells. Furthermore, although the amount of PG endoperoxide synthase-2 (PGHS-2) increased following LPS-treatment in all cases, both AA- and EPA-treatment caused a reduction of PGHS activity in LPS-treated cell lysates. Also, the addition of EPA or preincubation with AA in an in vitro PGHS assay system involving an LPS-treated, un-modified macrophage lysate, resulted in rapid inhibition of PGHS activity. These results suggest that both AA- and EPA-treatment inhibit PGD2 synthesis by inactivating PGHS-2 without affecting induction of the protein, and that the increase or decrease in AA content following AA- or EPA-treatment correlates simply with the level of AA release but not with that of PGD2 formation.

LPS-induced signals in activation of caspase-3-like protease, a key enzyme regulating apoptotic cell damage into a macrophage-like cell line, J774.1, in the presence of cycloheximide.

The earliest observed apoptotic change in a macrophage-like cell line, J774.1, treated with lipopolysaccharide (LPS) in the presence of cycloheximide (CHX) was a selective increase in caspase-3-like activity. The addition of polymyxin B, TPCK, herbimycin A, or genistein, all of which inhibited LPS-induced tumor necrosis factor alpha (TNF-alpha) production by macrophages, suppressed the activation of the caspase-3-like protease in these macrophages treated simultaneously with CHX. However, SB202190 and SB203580, inhibitors of MAP kinase, and PD98059, an inhibitor of MAP-kinase kinase (MEK), showed no effect on the activation of the caspase-3-like protease or on the cell damage of the macrophages treated with LPS and CHX, whereas they inhibited LPS-induced TNF-alpha production. These results suggest that some of the early signals in LPS-treated macrophages are common to the subsequent pathways for TNF-alpha production and caspase-3-like protease activation, but the later signals, like MAP-kinase kinase or MAP-kinase, are not involved in the pathways for caspase-3-like protease activation.

Inhibitory effects of hydrolyzable tannins from Melastoma dodecandrum Lour. on nitric oxide production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide and interferon-gamma.

An extract of Melastoma dodecandrum LOUR. with 80% aqueous acetone (MDL) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW264.7, activated with lipopolysaccharide (LPS) and recombinant mouse interferon-gamma (IFN-gamma). On further fractionation of the extract, the majority of the inhibitory activity was recovered in the 50% methanol extracts, which contained hydrolyzable tannins. Among the latter, casuarinin, casuarictin, pedunclagin and nobotannin B exhibited strong inhibitory activities toward NO production, with ID50 values between 2.0 and 5.1 microM. Both MDL and the purified tannins significantly reduced the induction of the inducible nitric oxide synthase (iNOS) protein in the course of macrophage activation with LPS and IFN-gamma. In addition, the NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by these tannins, with IC50 values around 30-130 microM, but not by MDL. These results suggest that MDL has the pharmacological ability to suppress NO production by activated macrophages and that the hydrolyzable tannins have major inhibitory activities.

Suppressive effects of serum on the LPS-induced production of nitric oxide and TNF-alpha by a macrophage-like cell line, WEHI-3, are dependent on the structure of polysaccharide chains in LPS.

The effect of serum on LPS-induced activation of a murine macrophage-like cell line, WEHI-3, was examined. Foetal calf serum strongly inhibited the production of nitric oxide (NO) and TNF-alpha by LPS-stimulated WEHI-3 cells, while it enhanced the production of both by other macrophage-like cell lines, J774.1 and BAM3, on treatment with LPS. This suppressive effect of serum on WEHI-3 cells was most remarkable when the cells were stimulated with rough-chemotype LPS, Ra LPS, Rc LPS and Rd2 LPS. Foetal calf serum also inhibited TNF-alpha production by the same cells stimulated with high concentrations of smooth-form LPS (S LPS; > 1000 ng/mL). Serum-mediated suppression was also observed for expression of the TNF-alpha gene in Rc LPS-stimulated WEHI-3 cells. This suppressive effect of FCS was most remarkable during the 1-2 h before the addition of LPS, but it was not observed when FCS was added at 1 h after the addition of LPS, suggesting dependence on the time of FCS addition to LPS-stimulated cells. No significant difference was observed in the expression of CD14 on WEHI-3 cells cultured in the presence and absence of serum, suggesting that CD14 is not involved in the serum-mediated suppression of these LPS-responses. On the contrary, FCS showed enhancing effects on the production of NO and TNF-alpha by WEHI-3 cells stimulated with low concentrations (< 100 ng/mL) of S LPS and rough mutant Salmonella minnesota Re LPS. These results suggest that the ability of FCS to suppress LPS-induced activation of WEHI-3 cells in mainly dependent on the structure of polysaccharide chains and also on the concentration of LPS employed.

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.

Characterization of the LPS-stimulated expression of EP2 and EP4 prostaglandin E receptors in mouse macrophage-like cell line, J774.1.

The expression of prostaglandin (PG) E receptor subtypes were characterized in J774.1, a mouse macrophage-like cell line. EP2- and EP4-mRNAs were found to be expressed. The expression of EP2 mRNA increased by the addition of lipopolysaccharide (LPS) in a dose-dependent manner. EP2 mRNA rapidly increased by more than 5-fold of the control level at 1 h, and decreased after 4 h. EP4 mRNA increased by only 2-fold of the control at 2 h. Gamma interferon inhibited both basal and LPS-induced expression of EP2 mRNA but did not affect the expression level of EP4 mRNA. When tumor necrosis factor-alpha (TNF-alpha) accumulation was measured after the treatment ofthe cells with LPS for 90 min, PGE2 was found to inhibit this accumulation, but butaprost, an EP2-selective agonist, did not. When TNF-alpha release was measured after the treatment of the cells with LPS for 8 h, accumulation was inhibited by butaprost as well as PGE2. These results indicated that the inhibitory effects of PGE2 on TNF-alpha production are mediated by EP2 and EP4 in macrophages, and that expression regulation of EP2 and EP4 in macrophages is quite different.

Defect in the induction of nitric oxide synthase by lipopolysaccharide (LPS) in an LPS-resistant mutant of a murine macrophage-like cell line, J774.1.

Nitric oxide (.NO)-generating activity was examined in a lipopolysaccharide (LPS)-resistant mutant of a murine macrophage-like cell line, J774.1, treated with LPS or LPS and interferon-gamma (IFN-gamma). This mutant, an LPS1916 cell line, showed no NO2- accumulation in the culture medium, and no expression of NOS activity in the cell extract, .NO synthase (NOS(II)) protein or NOS(II) mRNA on treatment with up to 10(4) ng/ml LPS, although the parental cell line, JA-4, showed significant .NO production. The addition of 10 U/ml IFN-gamma, together with more than 1 ng/ml LPS to JA-4 cells, increased .NO production remarkably, while IFN-gamma did not reverse the defect of .NO production in LPS1916 cells when they were treated with less than 10 ng/ml LPS; however, it induced .NO production by the mutant cells with more than 100 ng/ml LPS. Analysis of NOS activity, NOS(II) protein and NOS(II) mRNA revealed that LPS1916 cells are not devoid of the NOS(II) gene, but are rather defective in transcription of the gene in response to LPS, and this defect is partly reversed by IFN-gamma with higher LPS doses at more than 100 ng/ml. In addition, the delay of NOS(II) mRNA induction in LPS1916 cells, compared to that in JA-4 cells, treated with LPS+IFN-gamma seems to suggest some additional inducer(s) of NOS(II) transcription, followed by LPS signaling.

Apoptotic changes preceding necrosis in lipopolysaccharide-treated macrophages in the presence of cycloheximide.

Apoptotic changes occurred specifically in a macrophage-like cell line, J774.1, treated with lipopolysaccharide (LPS) and cycloheximide (CHX) prior to the release of lactate dehydrogenase (LDH). The addition of 100 ng/ml LPS and 10 microg/ml CHX induced both the formation of DNA nicks and elevation of caspase-3-like activity (DEVDase) after 75 min, and then the cleavage of poly(ADP-ribose) polymerase (PARP) into 28-kDa fragments, formation of apoptotic bodies, and DNA ladder formation. These apoptotic changes were reversible until 60 min, however, later than 75 min after LPS and CHX addition, the apoptosis proceeded normally even on extensive washing of the macrophages, which removed the LPS and CHX. These results suggest that there is a "point of no return" in the apoptotic processes in macrophages induced by LPS and CHX and that DNA nicks and activation of DEVDase are critical for these processes.