Secretion of IL-1: role of protein kinase C.

In an attempt to define the mechanism by which endotoxin induces its biologic activity, LPS was incorporated into phospholipid vesicles (liposomes) and compared with free LPS for ability to stimulate human monocytes. Activation of human monocytes by free LPS caused the translocation of protein kinase C (PKC) from the cytosol to the plasma membranes, the production of both IL-1, alpha and beta, and IL-1 secretion. Activation by LPS presented in multilamellar vesicles (MLV)-LPS caused IL-1 production but not IL-1 secretion. Moreover, MLV-LPS did not induce PKC translocation. MLV themselves did not inhibit monocyte stimulation by LPS, since LPS presented at the surface of lyophilized liposomes behaved like free LPS in cell activation. In contrast, MLV-LPS primed monocytes for subsequent LPS stimulation. When monocytes were activated by LPS in the presence of PKC inhibitors, no plasma membrane-associated PKC or IL-1 secretion was detected, whereas IL-1 production was observed. PKC inhibitors did not affect IL-1 alpha and IL-1 beta production, showing that PKC is not involved in the production of either IL-1. It can be concluded that IL-1 production and secretion are induced independently, and that IL-1 secretion involves PKC.     

Immune interferon enhances the production of interleukin 1 by human endothelial cells stimulated with lipopolysaccharide

Interferon-gamma (IFN-gamma) is a macrophage-activating factor that has also been shown to act on endothelial cells (EC). Interleukin 1 (IL 1), first described as a monocyte product, is also produced by EC after stimulation by lipopolysaccharide (LPS). In this study, the effect of IFN-gamma on the release of IL 1 by EC stimulated with LPS has been investigated. Although IFN-gamma did not stimulate the release of IL 1 or increase the apparent intracellular pool of IL 1 when incubated with EC, there was an increase in the amount of IL 1 released when cells preincubated with IFN-gamma were stimulated with LPS. The effect of IFN-gamma increased with concentration (1 to 1000 U/ml) and with duration of preincubation (24 to 96 hr). The presence of IFN-gamma was not required during the stimulation with LPS. When EC were cultured without IFN-gamma for increasing time periods up to 96 hr, the amount of IL 1 released by EC on subsequent stimulation with LPS progressively decreased. Addition of as little as 1 U/ml of IFN-gamma, however, prevented the loss in capacity of EC to secrete IL 1 when stimulated with LPS. In vivo, EC are involved in the emigration of mononuclear cells from the blood to inflammatory sites. Because IL 1 is chemotactic for lymphocytes and also increases the binding of lymphocytes to EC, activation of EC by T cell-derived factors such as IFN-gamma may augment lymphocyte emigration by increasing the release of IL 1 at the blood-tissue interface.     

Induction by lipopolysaccharide of intracellular and extracellular interleukin 1 production: analysis with synthetic models

An attempt was made to identify the molecular structures that are present in bacterial LPS and induce the production of intracellular and extracellular pools of IL 1 by peritoneal macrophages of the mouse and by human monocytes. Activities of glycolipids and carbohydrates prepared by synthesis, and structurally related to the hydrophobic (Lipid A) and to the polysaccharide (PS) regions of LPS were compared with those induced by Bordetella pertussis endotoxin and by fragments derived therefrom. Both isolated regions of this LPS (PS and Lipid A) were able to induce IL 1 synthesis by monocytes and macrophages. Among the synthetic glycolipids employed, propyl-2-deoxy-2-[(3R)-3-hydroxytetrade-canamido]-4-O-pho sph ono-6-O-tetradecanoyl-beta-D-glucopyranoside (glycolipid M9) induced IL 1 secretion more efficiently than Lipid A and LPS, whereas the amounts of intracellular IL 1 produced upon induction by these three substances were comparable. Macrophages from C3H/HeJ mice were unresponsive to Lipid A and to glycolipid M9, but produced IL 1 when incubated with PS or with a hydrophilic fragment isolated after methanolysis of the endotoxin. However, all synthetic derivatives of 3-deoxy-D-manno-2-octulosonic acid (KDO) used in this study failed to induce IL 1 production by both mouse macrophages and human monocytes. The implications of these findings for a more precise comprehension of the molecular mechanism of LPS-induced activation of macrophages, and the relations between the molecular structures required for the induction of IL 1 production vs cytostatic activity in macrophages, are discussed.     

Adenosine 3',5'-cyclic monophosphate stimulates secretion of alpha-melanocyte-stimulating hormone from permeabilized cells of the intermediate lobe of the rat pituitary gland

The effect of Mg-ATP and cyclic AMP on the secretion of alpha-melanocyto-stimulating hormone (alpha-MSH) from electrically permeabilized cells of rat intermediate lobe (IL) were investigated. Addition of exogenous Ca2+ stimulated alpha-MSH secretion in a concentration- (EC50 = 4.8 microM) and temperature-dependent manner. This Ca2+-evoked secretion was further enhanced by Mg-ATP and cyclic AMP. Mg-ATP was required for the fully secretory response in the electrically permeabilized IL cells and the maximal secretion was reached at 1 mM. Cyclic AMP in the presence of GTP gamma S also potentiated Ca2+-evoked alpha-MSH secretion to the same magnitude as Mg-ATP. In the absence of Ca2+ both the cyclic AMP and Mg-ATP did not stimulate alpha-MSH secretion from IL cells. The data suggest that Mg-ATP and cyclic AMP may modulate directly the secretory components rather than change intracellular concentration of free Ca2+.     

Bacterial lipopolysaccharide, phorbol myristate acetate, and muramyl dipeptide stimulate the expression of a human monocyte surface antigen, Mo3e

Exposure of mononuclear phagocytes to bacterial lipopolysaccharide (LPS), phorbol myristate acetate (PMA), or muramyl dipeptide (MDP) is known to stimulate a variety of cellular activities that include increases in phagocytosis, oxidative metabolism, synthesis and secretion of monokines, and cytotoxicity of microbes and tumor cells. We now report that culture of human peripheral blood monocytes in medium containing LPS, phorbol compounds, or MDP also results in the acquired expression of a plasma membrane antigen. Mo3e, as identified by a murine monoclonal antibody. Mo3e is barely detectable (by immunofluorescence flow cytometry) on freshly isolated monocytes, but is expressed in high antigen density after exposure of cells to E. coli, Salmonella minnesota, or Serratia marcescens LPS (at concentrations exceeding 0.1 ng/ml), PMA (and other biologically active phorbol compounds) (0.5 to 1 X 10(-8) M), or MDP (0.01 to 1 X 10(-6) M). Mo3e expression stimulated by LPS is prevented by pretreatment of LPS with polymyxin B, suggesting that the lipid A portion of LPS is responsible for Mo3e induction (polymyxin B has no effect on Mo3e expression stimulated by PMA or MDP). Culture of monocytes in medium containing protein synthesis inhibitors (or at 4 degrees C) blocks the acquisition of Mo3e. Recombinant IFN-gamma, which is also known to "activate" mononuclear phagocytes, does not stimulate Mo3e expression, although both LPS and IFN induce enhanced expression of monocyte Ia antigen. Analogous to their stimulatory effect on monocytes, LPS and PMA induce Mo3e expression by the human monocytic cell line, U-937. On the basis of these observations, Mo3e may represent an immunologic marker for monocyte activation stimulated in vitro by LPS, PMA (and related compounds), and MDP.     

Regulation of interleukin 1 production by mouse peritoneal macrophages. Effects of arachidonic acid metabolites, cyclic nucleotides, and interferons

The effects of prostaglandin E2 (PGE2), cyclic nucleotides, leukotriene B4 (LTB4), and interferons on interleukin 1 (IL 1) production by lipopolysaccharide (LPS)-stimulated C3H/HeNCrl mouse peritoneal macrophages were studied. IL 1 production was inhibited by PGE2, the adenosine 3':5'-monophosphate analog dibutyryl cAMP, the cAMP agonist isoproterenol, and the phosphodiesterase inhibitor isobutylmethylxanthine. These agents were more inhibitory when added early in the latent phase of IL 1 synthesis following stimulation with LPS rather than just prior to release of IL 1 into the medium. Production of both the intracellular and extracellular forms of IL 1 was blocked by PGE2 and cAMP. Suppression of LPS-induced IL 1 production by PGE2 was prevented by leukocyte alpha-interferon. Moreover, alpha-interferon augmented LPS-induced IL 1 production but did not stimulate IL 1 production in the absence of LPS. Immune gamma-interferon markedly inhibited LPS-stimulated IL 1 production. The lipoxygenase inhibitor eicosa-5,8,11,14-tetraynoic acid suppressed, whereas 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline augmented, LPS-induced IL 1 production. The opposing effects of these agents suggested that lipoxygenase metabolites do not act as inducers of IL 1 production. Purified LTB4 did not stimulate base-line or augment LPS-induced IL 1 production (both intracellular and extracellular forms). Moreover, calcium ionophore A23187 (a lipoxygenase activator) did not stimulate IL 1 production, alone or in combination with LTB4. Thus, net IL 1 production by macrophages may be regulated by a balance between the effects of PGE2, cAMP, alpha-interferon, and gamma-interferon, but not LTB4.     

Forskolin potentiates calcium-dependent amylase secretion from rat pancreatic acinar cells

The cellular and molecular effects of forskolin, a direct, nonhormonal activator of adenylate cyclase, were assessed on the enzyme secretory process in dispersed rat pancreatic acinar cells. Forskolin stimulated adenylate cyclase activity in the absence of guanyl nucleotide. It promoted a rapid and marked increase in cellular accumulation of cyclic AMP alone or in combination with vasoactive intestinal peptide (VIP) but was itself a weak pancreatic agonist and did not increase the secretory response to VIP or other cyclic AMP dependent agonists. Somatostatin was a partial antagonist of forskolin stimulated cyclic AMP synthesis and forskolin plus cholecystokinin-octapeptide (CCK-OP) induced amylase release. Forskolin potentiated amylase secretion in response to calcium-dependent agonists such as CCK-OP, carbachol and A-23187, but did not affect the ability of CCK-OP and (or) carbachol to mobilize 45Ca from isotope preloaded cells; forskolin alone did not stimulate 45Ca release. In calcium-poor media, the secretory response to forskolin and CCK-OP was reduced in a both absolute and relative manner. The data suggests that calcium plays the primary role as intracellular mediator of enzyme secretion and that the role of cyclic AMP may be to modulate the efficiency of calcium utilization.     

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1 microM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 +/- 39% and synthesis by 87 +/- 27% whereas at 0.1 microM a decrease of IL1 release of 52 +/- 9% and synthesis of 46 +/- 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase of inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1 microM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocytes functions, possibly via specific binding sites.     

Liposomes That Provide T-Dependent Help to Weak Antigens (T-Independent Antigens)

The design of an adjuvant for eliciting a thymus-dependent response to LPS, a well-defined thymus-independent antigen, is presented. Hybrid liposomes containing LPS and HA2 peptide from the hemagglutinin protein of influenza virus within the liposome bilayer were prepared (LPS/HA2 liposomes). The HA2 polypeptide contains epitopes recognized by T-helper lymphocytes and T-cytotoxic lymphocytes. Outbred mice immunized with LPS/HA2 liposomes produced anti-LPS-specific IgG responses. IgG subclass analysis indicated that IgG1, IgG2, and IgG3 antibodies were produced by these animals. LPS liposomes (liposomes without HA2) stimulated a T-independent response only. This was demonstrated by the detection of IgG3 but not IgG1 or IgG2 in serum of mice immunized with LPS liposomes. These results support the concept that the simultaneous incorporation into liposomes of a polypeptide with T-cell recognition sites along with a T-independent antigen can lead to the generation of cognate T-cell help for the T-independent antigen. The synthesis and characterization of a neo-lipopolysaccharide T-independent antigen for incorporation in hybrid HA2 liposomes are also presented. Findings are discussed relative to the liposome model used and implications for development of vaccines for use in humans.     

Natural and recombinant human interleukin 1-beta is cytotoxic for human melanoma cells

Human peripheral blood monocytes stimulated with LPS were found to release an activity that was cytotoxic for the A375 melanoma. Biochemical and immunological characterization of the activity indicated that IL 1-beta was the cytotoxic agent. Human recombinant IL 1-beta, purified to homogeneity, was directly cytotoxic for A375. Tumor necrosis factor, also released by activated monocytes, was not cytotoxic for the A375 melanoma.     

LPS-neutralizing peptides reduce outer membrane vesicle-induced inflammatory responses

Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1β and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1β secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1β secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.     

Synergistic increases in IL-1 synthesis by the human monocytic cell line THP-1 treated with PAF and endotoxin

The capacity to stimulate cytokine release may be important to the long-term effects of platelet-activating factor (PAF), which has a very short half-life. Previous studies have shown that PAF stimulates interleukin 1 (IL-1) release by human monocytes. IL-1 and other cytokines produced in response to PAF may be important to the long-term effects of this short-lived lipid. The THP-1 human monocytic leukemia cell line, was used to study the mechanism by which PAF stimulates IL-1 release. PAF stimulates the release of IL-1 beta activity into THP-1 cell supernatants with a multiphasic dose-response curve very similar to that for monocytes. When THP-1 cells are treated with PAF and LPS in combination, these two stimuli interact synergistically to greatly increase the release of IL-1 activity. To assess the effect of PAF on IL-1 beta synthesis, THP-1 cell pellet proteins were separated by SDS-PAGE, blotted, and immunostained to detect IL-1 beta. Immunostaining revealed that PAF increases intracellular IL-1 beta precursor and that the combination of PAF and LPS increases IL-1 beta precursor synergistically. PAF increases IL-1 beta release mainly by increasing IL-1 beta synthesis.     

Characterization of a 30,000-35,000 m.w. monokine involved in the specific immune response: intracellular production, secretion and partial purification

Peritoneal macrophages from normal mice which do not secrete interleukin 1 (IL 1) spontaneously release a factor with an approximate m.w. of 30,000-35,000. In contrast, in the presence of silica only IL 1 is produced. IL 1 as well as this macrophage-replacing factor (MRF) can restore the antibody response of macrophage-depleted spleen cells. IL 1, however, is the only one that has the capacity to increase the proliferation of thymocytes. In every strain of mice studied, including nude mice, we observed a spontaneous release of MRF and a silica-induced shift to the secretion of IL 1, except in lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ mice. Macrophages from these mice are unable to secrete MRF spontaneously, or IL 1 when stimulated with silica. The kinetics of the secretion of MRF and IL 1 appear to be similar. Macrophages, regardless of whether they have been stimulated to secrete IL 1, produce an intracellular IL 1-like activity with an approximate m.w. of 15,000. In contrast, the intracellular PFC-restoring activity is widely distributed in the 15,000-60,000 m.w. range; one of these compounds could be related to IL 1 precursor and/or to MRF itself. Chromatofocusing and chromatography on Blue Trisacryl have led to a partial purification and resolution from a possible contamination by IL 1. Purified MRF induces, in conjunction with lymphokines, the differentiation of B cells into antibody-forming cells.     

C3a(C3adesArg) induces production and release of interleukin 1 by cultured human monocytes

Purified human C3a(C3adesArg) induced dose-dependent generation of intracellular IL 1 activity and release of IL 1 in cultures of human mononuclear adherent cells in serum-free conditions. Concentrations of C3a(C3adesArg) of 10(-8) M and 6 hr of culture were sufficient to induce production of cell-associated IL 1, as detected in monocyte lysates. Ten- to 100-fold higher concentrations of C3a(C3adesArg) and 24 hr of culture were required for induction of IL 1 release. Release of IL 1 induced by suboptimal amounts of C3a(C3adesArg) was greatly enhanced by the addition of indomethacin to the culture medium. Contamination with C5a of the C3a(C3adesArg) preparation did not account for C3a(C3adesArg)-induced IL 1 production. Induction of IL 1 activity by C3a(C3adesArg) was not due to contaminating LPS, as indicated by the following observations: the amount of contaminating LPS in C3a(C3adesArg) was below that which could induce IL 1 release from human monocytes in serum-free conditions; induction of IL 1 by C3a(C3adesArg) was not suppressed by polymyxin B; kinetics of IL 1 production and release in the presence of C3a(C3adesArg) differed from those observed in the presence of LPS; and sialated gangliosides, which inhibit IL 1 release induced by LPS, had no effect on the induction of IL 1 by C3a(C3adesArg). The C3a(C3adesArg) preparation used in this study mostly contained the desArg derivative, suggesting that, in contrast with the requirement for an intact C-terminal arginyl residue for the spasmogenic activity of C3a, both C3a and its C3adesArg derivative may interact with receptors on human monocytes. By inducing IL 1 production and release, C3a(C3adesArg) may contribute to the generation of the inflammatory process and the regulation of the immune response.     

Ultrastructural localization of Interleukin 1 in human peripheral blood monocytes; evidence for IL-1β in mitochondria

Summary The pathway of interleukin 1 (IL-1) secretion from the cell remains unclear. IL-1β is the major form produced by human monocytes, and is synthesized as a precursor of 35kDa which is processed to the extracellular biologically active 17kDa form. We have examined the intracellular localization of IL-1β in lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes, by immunocytochemistry and immunoprecipitation of subcellular fractions. LPS treatment slightly damaged the cells. Unstimulated cells showed very little immunolabelling. In contrast, there was heavy immunolabelling on LPS stimulated cells. Immunolabelling occured within the cytoplasm, nucleus and mitochondria. There was no immunolabelling on the membranous secretory organelles and the plasma membrane. Blebs of cytoplasm budding from the cell surface were immunolabelled, suggesting an alternative route of secretion of IL-1β from the cell. Immunoprecipitation studies confirmed these results.     

Effect of platelet-activating factor (PAF-acether) and its specific receptor antagonist, BN 52021, on interleukin 1 (IL1) release and synthesis by rat spleen adherent monocytes

PAF-acether, at doses ranging from 1pM to 0.1μM did not induce a significative release and/or synthesis of IL1 from monocytes. In contrast, depending upon the dose of the mediator, adverse effects on the lipopolysaccharide (LPS)-induced IL1 release and synthesis were observed. PAF-acether at 1pM increased IL1 release by 120 ± 39% and synthesis by 87 ± 27% whereas at 0.1μM a decrease of IL1 release of 52 ± 9% and synthesis of 46 ± 6% were observed. BN 52021, a specific PAF-acether receptor antagonist, reversed by more than 70% the increase or inhibition of LPS-induced IL1 release and synthesis induced by 1pM and 0.1μM of the autacoid, respectively. No direct effect of BN 52021 on IL1 release and synthesis from adherent monocytes was noted. These results indicate that PAF-acether modulates monocyte functions, possibly specific binding sites.     

Lipopolysaccharide-induced production of cytokines by bone marrow-derived macrophages: dissociation between intracellular interleukin 1 production and interleukin 1 release

We investigated the capacity of mouse bone marrow-derived macrophages (BMDM) to produce interleukin 1 (IL 1), interleukin-6 (IL 6), and tumor necrosis factor (TNF) upon lipopolysaccharide (LPS) stimulation. BMDM were allowed to differentiate either in the presence of conditioned medium (from WEHI-3 or L cells), or in the presence of recombinant cytokines (IL 3, macrophage-colony stimulating factor [M-CSF], or granulocyte/macrophage-colony stimulating factor [GM-CSF]). Cells were maintained in culture up to 3 weeks and tested at different times. Significant spontaneous cytokine production was never observed. BMDM rapidly acquired the capacity to elaborate cytokine upon LPS activation. LPS-triggered BMDM were able to produce IL 1, IL 6, and TNF, throughout the culture period, although 2- to 3-week-old cells lost their ability to release IL 1 while accumulation of intracellular IL 1 remained unchanged. The dissociation between synthesis and release of IL 1 was not correlated with a significant modification of the specific binding of LPS onto the cell surface.     

Physiological mechanisms contributing to increased interleukin-1 secretion

Interleukin-1 (IL-1) is a monocyte-derived polypeptide that mediates many host defense adaptations to environmental and infectious stresses. This investigation was intended to characterize further IL-1 activity found in human plasma following exercise (3) and to identify physiological initiators of IL-1 secretion. IL-1 activity was measured by the ability of plasma fractions to stimulate lymphocyte proliferation. This activity appeared in plasma several hours after exercise on a cycle ergometer (1 h at 60% of aerobic capacity, n = 8 subjects) and was neutralized with a specific antiserum to human IL-1. The hypothesis that IL-1 release from monocytes was initiated by phagocytosis of material from cells damaged by exercise was tested. The increase in IL-1 activity did not correlate significantly (r = 0.55) with creatine kinase activity, a marker for release of intracellular proteins into the circulation, and IL-1 secretion by monocytes was not stimulated by incubation with red blood cell lysates in vitro. Thus the stimulus for IL-1 secretion did not appear to be related to a scavenging function of monocytes. The possibility that IL-1 secretion may be mediated by stress hormones associated with exercise was examined. IL-1 secretion by monocytes was increased up to 48 +/- 18% (P less than 0.01) by addition of physiological concentrations of epinephrine in vitro. Low concentrations of hydrocortisone (1 ng/ml) also augmented IL-1 secretion by 58 +/- 20%. Higher concentrations in the physiological range had no effect, and combinations of epinephrine and hydrocortisone suppressed IL-1 secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.     

Adult human vascular endothelial cells express the IL6 gene differentially in response to LPS or IL1

We investigated the regulation of IL6 biological activity, de novo synthesis, and mRNA levels in adult vascular endothelial cells (EC) by bacterial endotoxin or inflammatory cytokines. Cells incubated without stimulus released scant IL6 activity. IFN gamma, IL2, or PDGF did not augment IL6 release from EC. LPS, lipid A, and TNF increased IL6 release modestly (5 to 20-fold), while recombinant IL1s (rIL1s) stimulated this process 100 to 400-fold. Differential release of IL6 from EC treated with LPS or rIL1 continued for at least 144 hr. Exposure to LPS or rIL1 caused EC to synthesize IL6 de novo. EC secreted the newly synthesized IL6 into the supernatant, rather than retaining it within or bound to cells. EC accumulated IL6 mRNA after 3 hr of exposure to rIL1. However, we could only detect IL6 message in cells incubated with LPS under "superinduction" conditions with cycloheximide, consistent with lower levels of IL6 biological activity in response to LPS compared to IL1 stimulation. We propose that local production of IL6 by vascular EC, which comprise the barrier between tissues and the blood, may influence regional immune and inflammatory responses.