Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages

Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.     

Expression of class A scavenger receptor is enhanced by high glucose in vitro and under diabetic conditions in vivo: one mechanism for an increased rate of atherosclerosis in diabetes

In the early stage of atherosclerosis, macrophages take up chemically modified low density lipoproteins (LDL) through the scavenger receptors, leading to foam cell formation in atherosclerotic lesions. To get insight into a role of the scavenger receptors in diabetes-enhanced atherosclerotic complications, the effects on class A scavenger receptor (SR-A) of high glucose exposure in vitro as well as the diabetic conditions in vivo were determined in the present study. The in vitro experiments demonstrated that high glucose exposure to human monocyte-derived macrophages led to an increased SR-A expression with a concomitant increase in the endocytic uptake of acetylated LDL and oxidized LDL. The endocytic process was significantly suppressed by an anti-SR-A neutralizing antibody. Stability analyses revealed a significant increased stability of SR-A at a mRNA level but not a protein level, indicating that high glucose-induced up-regulation of SR-A is due largely to increased stability of SR-A mRNA. High glucose-enhanced SR-A expression was prevented by protein kinase C and NAD(P)H oxidase inhibitors as well as antioxidants. High glucose-enhanced production of intracellular peroxides was visualized in these cells, which was attenuated by an antioxidant. The in vivo experiments demonstrated that peritoneal macrophages from streptozotocin-induced diabetic mice increased SR-A expression when compared with those from nondiabetic mice. Endocytic degradation of acetylated LDL and oxidized LDL were also increased with these macrophages but not with the corresponding macrophages from diabetic SR-A knock-out mice. These in vitro and in vivo results probably suggest that reactive oxygen species generated from a protein kinase C-dependent NAD(P)H oxidase pathway plays a role in the high glucose-induced up-regulation of SR-A, leading to the increased endocytic degradation of modified LDL for foam cell formation. This could be one mechanism for an increased rate of atherosclerosis in patients with diabetes.     

IL-2 induces IL-6 production in human monocytes.

IL-2 is a potent activator of effector and secretory activities of human monocytes. Since monocytes are an important source of IL-6, we investigated whether IL-2 can induce IL-6 production and whether regulatory circuits can modulate this process. We found that stimulation of monocytes with IL-2 induced expression of IL-6 mRNA and bioactivity in a dose-dependent manner. Production of IL-6 in monocytes can be induced by other cytokines such as IL-1 beta. By using mAb alpha-IL-1 beta we showed that IL-2-induced IL-6 production is not mediated by the autocrine stimulation of IL-1 beta elicited by IL-2. IL-6 induction by monocytes is not a common response to activating signals because IFN-gamma did not induce IL-6 expression under conditions in which it elicits tumoricidal activity. In contrast, IFN-gamma could completely abrogate the induction of IL-6 expression by IL-1 beta but did not affect the levels of mRNA and the secretion of IL-2-elicited IL-6. We have previously reported that transforming growth factor-beta inhibits IL-6 production in response to IL-1 beta. Studies on the inhibitory activity of transforming growth factor-beta demonstrated that this cytokine differs from IFN-gamma because it inhibited both IL-1- and IL-2-induced IL-6 expression. These data demonstrate that, in human monocytes, both IL-1 and IL-2 stimulate IL-6 expression by independent mechanisms that can be dissociated by the susceptibility to the inhibitory effect of IFN-gamma. IL-6 production is also down-regulated by TGF-beta, whose inhibitory activity is stimulus-unrelated.     

Bovine dialyzable leukocyte extract modulates cytokines and nitric oxide production in lipopolysaccharide-stimulated human blood cells

BACKGROUND: In the current study, we determined whether bovine dialyzable leukocyte extract (bDLE) modulates lipopolysaccharide (LPS)-induced nitric oxide and cytokine overproduction. METHODS: Human whole blood cells were treated with LPS (50 ng) + bDLE (1 U). RESULTS: The bDLE treatment decreased nitric oxide as well as TNF-alpha, IL-6 and IL-10 (P <0.01) cytokine production. In addition, it decreased TNF-alpha, IL-1beta and IL-6 mRNA expression and suppressed IL-10 and IL-12p40 mRNA expression, but did not modulate IL-8 mRNA expression in LPS-stimulated human blood cells. DISCUSSION: Our results suggest that bDLE may effectively modulate the fatal symptoms of hypotensive shock associated with endotoxin (LPS)-induced nitric oxide and cytokine production, and this may offer therapeutic potential for the treatment of endotoxic shock.     

Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria

The use of lipoproteins has been suggested as a treatment for Gram-negative sepsis because they inhibit lipopolysaccharide (LPS)-mediated cytokine production. However, little is known about the neutralizing effects of lipoproteins on cytokine production by meningococcal LPS or whole Gram-negative bacteria. We assessed the neutralizing effect of LDLs, HDLs, and VLDLs on LPS- or whole bacteria-induced cytokines in human mononuclear cells. A strong inhibition of Escherichia coli LPS-induced interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and IL-10 by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, Neisseria meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a longer interaction time, a lower concentration of LPS, or higher concentrations of lipoproteins. The difference in neutralization was independent of the saccharide tail, suggesting that the lipid A moiety accounted for the difference. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These results indicate that efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low LPS concentration, or high lipoprotein concentration also inhibited cytokines by the less efficiently neutralized N. meningitidis LPS. Irrespective of these differences, whole bacteria showed no neutralization by lipoproteins.     

Induction of interleukin 1 beta expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid.

Oxidatively modified low density lipoproteins (LDL) have recently been proposed to play a role in atherogenesis by promoting foam cell formation and endothelial cell toxicity. The purpose of the present study was to determine whether modified LDL could also induce macrophage release of interleukin 1 beta (IL-1 beta), a cytokine which enhances vascular smooth muscle cell proliferation, another feature of the atherosclerotic process. LDL were oxidatively modified by incubation with either Cu2+ (Cu(2+)-LDL) or human peripheral blood monocyte-derived macrophages (M-LDL). Incubation of these modified LDL with macrophages (6 x 10(6) cells/culture) resulted in a dose-dependent induction of IL-1 beta release. At 300 micrograms protein/ml, Cu(2+)-LDL and M-LDL induced 422 and 333 pg of IL-1 beta/culture, respectively. Saponified Cu(2+)-LDL and M-LDL were shown to contain 9- and 13-hydroxyoctadecadienoic acid (HODE), lipid oxidation products of linoleate. When tested for activity in macrophage culture (3 x 10(6) cells/culture), it was found that 9-HODE and 13-HODE (final concentration 33 microM) induced the release of 122 and 43 pg of IL-1 beta/culture, respectively, whereas untreated cells released only 4 pg of IL-1 beta/culture. Incubation of macrophages with cholesteryl-9-HODE also induced IL-1 beta release; however, the degree of induction of IL-1 beta release by 9-HODE or its cholesteryl ester relative to modified LDL suggests that other components in oxidized LDL may also contribute to IL-1 beta induction. 9-HODE was rapidly taken up by macrophages, and the kinetics were similar to IL-1 beta release. A 1.5- to 6-fold increase in the level of IL-1 beta mRNA was detected as little as 3-h post-9-HODE treatment. The induction of IL-1 beta release from human monocyte-derived macrophages by 9-HODE and cholesteryl-9-HODE suggests a role for modified LDL, and its associated linoleate oxidation products, in vascular smooth muscle cell proliferation.     

Atherogenic effects of TNF-α and IL-6 via up-regulation of scavenger receptors

Patients with chronic inflammatory disorders such as rheumatoid arthritis (RA) have a high risk of developing cardiovascular disease. We evaluated the effects of TNF-α and IL-6 on foam cell formation, a pivotal process in atherogenesis. Accumulation of intracellular oxidized LDL (oxLDL) was induced when THP-1/macrophages were stimulated with TNF-α or IL-6. TNF-α induced the expressions of scavenger receptors SR-A and LOX-1, and IL-6 induced SR-A expression. Inhibition of the NF-κB signaling markedly decreased TNF-α-induced foam cell formation and SR-A expression. Serum from RA patients, but not healthy subjects, induced foam cell formation, which was partially reversed by either IL-6 or TNF-α blockade in conjunction with inhibiting the induction of scavenger receptors. The present study clearly showed that in patients with chronic inflammation mediated by TNF-α and IL-6, these cytokines are directly implicated in atherosclerotic plaque formation.     

Immunomodulatory effects of glycine on LPS-treated monocytes: reduced TNF-alpha production and accelerated IL-10 expression.

Cytokines play a pivotal role in the pathogenesis of septic shock. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) stimulate the progression of septic shock whereas the anti-inflammatory cytokine IL-10 has counterregulative potency. The amino acid glycine (GLY) has been shown to protect against endotoxin shock in the rat by inhibiting TNF-alpha production. In the current study we investigated the role of GLY on lipopolysaccharide (LPS) -induced cell surface marker expression, phagocytosis, and cytokine production on purified monocytes from healthy donors. GLY did not modulate the expression of HLA-DR and CD64 on monocytes, whereas CD11b/CD18 expression (P<0.05) and E. coli phagocytosis (P<0.05) decreased significantly. GLY decreased LPS-induced TNF-alpha production (P<0.01) and increased IL-10 expression of purified monocytes. Similarly, in a whole blood assay, GLY reduced TNF-alpha (P<0.0001) and IL-1beta (P<0.0001) synthesis and increased IL-10 expression (P<0.05) in a dose-dependent manner. The inhibitory effects of GLY were neutralized by strychnine, and the production of IL-10 and TNF-alpha was augmented by anti-IL-10 antibodies. Furthermore, GLY decreased the amount of IL-1beta and TNF-alpha-specific mRNA. Our data indicate that GLY has a potential to be used as an additional immunomodulatory tool in the early phase of sepsis and in different pathophysiological situations related to hypoxia and reperfusion.     

Site-specific modulation of LPS-induced fever and interleukin-1 beta expression in rats by interleukin-10

Bacterial lipopolysaccharide (LPS) induces fever that is mediated by pyrogenic cytokines such as interleukin (IL)-1 beta. We hypothesized that the anti-inflammatory cytokine IL-10 modulates the febrile response to LPS by suppressing the production of pyrogenic cytokines. In rats, intravenous but not intracerebroventricular infusion of IL-10 was found to attenuate fever induced by peripheral administration of LPS (10 microg/kg iv). IL-10 also suppressed LPS-induced IL-1 beta production in peripheral tissues and in the brain stem. In contrast, central administration of IL-10 attenuated the febrile response to central LPS (60 ng/rat icv) and decreased IL-1 beta production in the hypothalamus and brain stem but not in peripheral tissues and plasma. Furthermore, intravenous LPS upregulated expression of IL-10 receptor (IL-10R1) mRNA in the liver, whereas intracerebroventricular LPS enhanced IL-10R1 mRNA in the hypothalamus. We conclude that IL-10 modulates the febrile response by acting in the periphery or in the brain dependent on the primary site of inflammation and that its mechanism of action most likely involves inhibition of local IL-1 beta production.     

Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

Cytokine responses of human gingival fibroblasts to Actinobacillus actinomycetemcomitans cytolethal distending toxin

Actinobacillus actinomycetemcomitans is implicated in the pathogenesis of localized aggressive periodontitis, and has the capacity to express a cytolethal distending toxin (Cdt). Gingival fibroblasts (GF) are resident cells of the periodontium, which can express several osteolytic cytokines. The aims of this study were a) to investigate the role of Cdt in A. actinomycetemcomitans-induced expression of osteolytic cytokines and their cognate receptors in GF and b) to determine if the previously demonstrated induction of receptor activator of NFkappaB ligand (RANKL) by A. actinomycetemcomitans is mediated by these pro-inflammatory cytokines or by prostaglandin E(2) (PGE(2)). A. actinomycetemcomitans clearly induced interleukin (IL)-6, IL-1beta, and to a minimal extent, tumor necrosis factor (TNF)-alpha mRNA expression. At the protein level, IL-6 but not IL-1beta or TNF-alpha expression was stimulated. The mRNA expression of the different receptor subtypes recognizing IL-6, IL-1beta and TNF-alpha was not affected. A cdt-knockout strain of A. actinomycetemcomitans had similar effects on cytokine and cytokine receptor mRNA expression, compared to its parental wild-type strain. Purified Cdt stimulated IL-6, but not IL-1beta or TNF-alpha protein biosynthesis. Antibodies neutralizing IL-6, IL-1 or TNF-alpha, and the PGE(2) synthesis inhibitor indomethacin, did not affect A. actinomycetemcomitans-induced RANKL expression. In conclusion, a) A. actinomycetemcomitans induces IL-6 production in GF by a mechanism largely independent of its Cdt and b) A. actinomycetemcomitans-induced RANKL expression in GF occurs independently of IL-1, IL-6, TNF-alpha, or PGE(2).     

Expression and regulation of histidine decarboxylase mRNA expression in the uterus during pregnancy in the mouse

It has been hypothesized that hormonally regulated histamine production plays a role in preparation of the uterus for implantation. Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine production. The current study was designed to determine intrauterine expression of HDC mRNA expression during pregnancy in the mouse. High levels of HDC mRNA expression were observed in the preimplantation mouse uterus with peak expression occurring on day 4. High levels of HDC mRNA expression were also detected in the post-implantation uterus. In an effort to determine whether HDC mRNA is regulated by pro-inflammatory cytokines, the HDC mRNA pattern was compared to intrauterine expression of mRNA's for interleukin-1alpha (IL-1alpha), IL-1beta, macrophage chemotactic protein-1 (MCP-1) and RANTES (regulated on activation, normal T expressed and secreted) during the peri-implantation period. IL-1beta, MCP-1 and RANTES mRNA levels were increased in the uterus on days 1-2 and on days 4-5. Increased expression of IL-1alpha mRNA was observed on days 1-2 and days 5-7. There was no clear relationship between HDC mRNA expression and cytokine/chemokine mRNA expression. Progesterone-stimulated intrauterine expression of HDC mRNA. Intrauterine cytokine/chemokine mRNA was also hormonally regulated. This data allowed the possibility that one or more of these pro-inflammatory cytokines could be involved in regulating intrauterine HDC mRNA production. Recombinant IL-1alpha, IL-1beta, MCP-1 and RANTES all failed to induce HDC mRNA expression in the preimplantation uterus in a mouse pseudopregnancy model. At the same time, IL-1beta induced the expression of mRNA for each of the four cytokines/chemokines. Despite the fact that these were also produced in the uterus during pregnancy and were hormonally regulated, none of these cytokines induced intrauterine HDC mRNA expression. The data suggest that progesterone is involved in the regulation of HDC mRNA expression in the preimplantation uterus, but IL-1alpha/beta, MCP-1 and RANTES, which have been reported to regulate histamine synthesis during inflammatory processes, do not appear to play a role.     

Anti-atherosclerotic effects of sirolimus on human vascular smooth muscle cells

Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine IL-1 beta. We explored the effect of sirolimus on the lipid accumulation of VSMCs in the presence of IL-1 beta, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of sirolimus on the gene and protein expression of lipoprotein receptors and ATP binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and Western blotting, respectively. Furthermore, the effect of sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1 beta was also investigated using [(3)H] cholesterol efflux. Finally, we examined the effect of sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1 beta possibly due to the reduction of expression of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1 beta. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1 beta. We further confirmed that sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, tumor necrosis factor-alpha, IL-8, and monocyte chemoattractant protein-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of sirolimus on VSMCs.