Modulation of monocyte function by activated protein C, a natural anticoagulant

Activated protein C is the first effective biological therapy for the treatment of severe sepsis. Although activated protein C is well established as a physiological anticoagulant, emerging data suggest that it also exerts anti-inflammatory and antiapoptotic effects. In this study, we investigated the ability of activated protein C to modulate monocyte apoptosis, inflammation, phagocytosis, and adhesion. Using the immortalized human monocytic cell line THP-1, we demonstrated that activated protein C inhibited camptothecin-induced apoptosis in a dose-dependent manner. The antiapoptotic effect of activated protein C requires its serine protease domain and is dependent on the endothelial cell protein C receptor and protease-activated receptor-1. In primary blood monocytes from healthy individuals, activated protein C inhibited spontaneous apoptosis. With respect to inflammation, activated protein C inhibited the production of TNF, IL-1beta, IL-6, and IL-8 by LPS-stimulated THP-1 cells. Activated protein C did not influence the phagocytic internalization of Gram-negative and Gram-positive bioparticles by THP-1 cells or by primary blood monocytes. Activated protein C also did not affect the expression of adhesion molecules by LPS-stimulated blood monocytes nor the ability of monocytes to adhere to LPS-stimulated endothelial cells. We hypothesize that the protective effect of activated protein C in sepsis reflects, in part, its ability to prolong monocyte survival in a manner that selectively inhibits inflammatory cytokine production while maintaining phagocytosis and adherence capabilities, thereby promoting antimicrobial properties while limiting tissue damage.     

The modulation of tissue factor by endothelial cells during heat shock

Tissue factor (TF) initiates the extrinsic coagulation cascade on the surface of macrophages and endothelial cells. In septic patients, the extrinsic coagulation cascade is activated. When septic patients are febrile, mortality is decreased. The purpose of this study was to investigate the role of elevated temperatures on TF expression by endothelial cells during a sepsis-like challenge. Human endothelial vein cells (HUVECs) were incubated with lipopolysaccharide (LPS) or interleukin-1 beta (IL-1 beta) for 0, 2, 4, 6, or 8 h. At the 0-h time point, some HUVECs were heat shocked at 43 degrees C for 2 h and then recovered at 37 degrees C for 0, 2, 4, or 6 h. Heat-shocked and non-heat-shocked LPS-stimulated HUVECs were analyzed for TF-specific mRNA expression by ribonuclease protection assay (RPA), surface TF expression by flow cytometry, and TF activity by a two-stage clotting assay. Heat shocked LPS-stimulated HUVECs expressed significantly reduced TF-specific mRNA, TF surface protein levels, and TF surface activity when compared with non-heat-shocked, LPS-stimulated HUVECs (p < 0.0125, p < 0.0125, and p < 0.0001, respectively; repeated measures analysis of variance, ANOVA). If heat shock models elevated core temperature, these results suggest that fever may protect the host during sepsis by reducing TF activity on the surface of endothelial cells.     

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS. IL-9 also down-regulated TNF-alpha and IL-10 release by LPS-stimulated monocytes. In addition, IL-9 strongly up-regulated the production of TGF-beta1 by LPS-stimulated monocytes. The suppressive effect of IL-9 on the respiratory burst and TNF-alpha production in LPS-stimulated monocytes was significantly inhibited by anti-TGF-beta1, but not by anti-IL-10Rbeta mAb. Furthermore, IL-9 inhibited LPS-induced activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases in monocytes through a TGF-beta-mediated induction of protein phosphatase activity. In contrast, IL-4, which exerts a similar inhibitory effect on the oxidative burst and TNF-alpha release by monocytes, acts primarily through a down-regulation of LPS receptors. Thus, IL-9 deactivates LPS-stimulated blood mononuclear phagocytes, and the mechanism of inhibition involves the potentiation of TGF-beta1 production and extracellular signal-regulated kinase inhibition. These findings highlight a new target cell for IL-9 and may account for the beneficial activity of IL-9 in animal models of exaggerated inflammatory response.     

LPS from Neisseria meningitidis is crucial for inducing monocyte- and microparticle-associated tissue factor activity but not for tissue factor expression

Neisseria meningitidis causes sepsis with coagulopathy. The present study evaluated the tissue factor (TF)-inducing capacity of bacterial LPS in different presentation forms, i.e. membrane-bound LPS versus purified LPS, and of non-LPS components of N. meningitidis. By using a wild-type N. meningitidis, a mutant N. meningitidis lacking LPS (LPS-deficient N. meningitidis), purified LPS from N. meningitidis and Escherichia coli, we measured TF-expression and TF-activity on human monocytes and microparticles (MPs). The effect of TF-modulators, such as phosphatidylserine (PS), tissue factor pathway inhibitor (TFPI) and recombinant IL-10 (rhIL-10) was investigated. In plasmas from meningococcal patients, fibrinopeptide A (FPA), LPS and IL-10 were quantified. Monocytes and MPs exposed to purified LPS or wild-type N. meningitidis had much higher TF-activity than monocytes and MPs exposed to LPS-deficient N. meningitidis (clot formation assay). Incubation with wild-type N. meningitidis, but also LPS-deficient N. meningitidis, resulted in TF-expression on monocytes (flow cytometry, qRT-PCR). Increased cellular TF-activity is associated with coincident surface-exposure of PS and the number of monocytes positive for both PS and TF was significantly higher for monocytes exposed to wild-type N. meningitidis (7.6%) compared with monocytes exposed to LPS-deficient N. meningitidis (1.8%). Treatment with rhIL-10 reduced monocyte- and MP-associated TF-activity, the number of monocytes positive for both TF and PS, and microvesiculation. Patients with meningococcal septicemia had significantly higher levels of LPS, FPA and IL-10 than patients with distinct meningitis. Our results indicate that LPS from N. meningitidis is crucial for inducing TF-activity, but not for monocyte- and MP-associated TF-expression. TF-activity seems to require coincident expression of TF and PS on monocytes, and LPS induces such double-positive monocytes.     

Activated protein C attenuates systemic lupus erythematosus and lupus nephritis in MRL-Fas(lpr) mice

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease leading to inflammatory tissue damage in multiple organs (e.g., lupus nephritis). Current treatments including steroids, antimalarials, and immunosuppressive drugs have significant side effects. Activated protein C is a natural protein with anticoagulant and immunomodulatory effects, and its recombinant version has been approved by the U.S. Food and Drug Administration to treat severe sepsis. Given the similarities between overshooting immune activation in sepsis and autoimmunity, we hypothesized that recombinant activated protein C would also suppress SLE and lupus nephritis. To test this concept, autoimmune female MRL-Fas(lpr) mice were injected with either vehicle or recombinant human activated protein C from week 14-18 of age. Activated protein C treatment significantly suppressed lupus nephritis as evidenced by decrease in activity index, glomerular IgG and complement C3 deposits, macrophage counts, as well as intrarenal IL-12 expression. Further, activated protein C attenuated cutaneous lupus and lung disease as compared with vehicle-treated MRL-Fas(lpr) mice. In addition, parameters of systemic autoimmunity, such as plasma cytokine levels of IL-12p40, IL-6, and CCL2/MCP-1, and numbers of B cells and plasma cells in spleen were suppressed by activated protein C. The latter was associated with lower total plasma IgM and IgG levels as well as lower titers of anti-dsDNA IgG and rheumatoid factor. Together, recombinant activated protein C suppresses the abnormal systemic immune activation in SLE of MRL-Fas(lpr) mice, which prevents subsequent kidney, lung, and skin disease. These results implicate that recombinant activated protein C might be useful for the treatment of human SLE.     

The effect of high temperature on the kinetics of lipopolysaccharide (LPS)-induced human monocytes activity in vitro

Human peripheral blood monocytes were stimulated with lipopolysaccharide at different temperatures (34, 37 and 40 °C) and TNF-α, IL-1β and NO production was measured. Levels of TNF-α mRNA and IL-1β mRNA were measured by RT-PCR. Phagocytic activity of LPS-stimulated monocytes in terms of bacterial uptake and intracellular bacterial killing was checked at different conditions in vitro. Early elevation of TNF-α, IL-1β and NO production was found in LPS-stimulated monocytes that incubated at 40 °C followed by cells that incubated at 37 °C and lowest level was detected at 34 °C. Similar results were observed in the phagocytic activity. Expression of TNF-α mRNA and IL-1β mRNA was observed as early as 30 min post exposure to LPS in all studied temperatures and these, decreased sharply after 12 h post exposure to LPS in LPS-stimulated monocytes that incubated at 40 °C only. This report describes the striking effects of incubation temperature on activity of LPS-stimulated monocytes.     

Interleukin-4 downregulates interleukin-6 production by human alveolar macrophages at protein and mRNA levels.

Effects of interleukin-4 (IL-4) on IL-6 production by human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors was examined at the protein and gene levels. IL-6 production was quantitated by enzyme immunoassay (EIA) and bioassay using the IL-6 dependent murine hybridoma cell line MH60.BSF2. Results showed that when activated with LPS, AM released significantly more biologically active IL-6 than blood monocytes. Human rIL-4 significantly suppressed IL-6 production by AM and monocytes stimulated with LPS. Northern blot analysis revealed that IL-4 reduced the expression of IL-6 mRNA in LPS-stimulated AM and monocytes. The inhibitory effect was most pronounced when IL-4 was added with LPS or within the first 4 hr after LPS to AM or monocytes. The suppressive effect of IL-4 was completely neutralized by pretreatment with anti-IL-4 antibody. IL-4 also showed a suppressive effect on IL-6 production by macrophages generated in vitro by maturation of blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF). These observations suggest that IL-4 may play a critical role in in situ regulation of immune responses through suppression of IL-6 production.     

Immunomodulatory cytokines suppress epithelial nitric oxide production in inflammatory bowel disease by acting on mononuclear cells

Inducible nitric oxide synthase (iNOS) activity in colonic epithelial HT-29 cells is modulated by the T-cell-derived cytokines IL-4 and IL-13, but is not affected by IL-10 despite its effect in models of colitis. We studied the effects of these cytokines on nitric oxide (NO) production by colonic tissue. IL-10 and IL-4 but not IL-13 suppressed the NO production and iNOS expression by inflamed tissue and cytokine-stimulated noninflamed tissue from patients with ulcerative colitis, whereas the three cytokines suppressed NO production in cytokine-stimulated biopsies from controls. To examine why colonic biopsies and HT-29 cells respond differently to immunomodulatory cytokines, a coculture of mixed mononuclear monocytes (MMC) and HT-29 cells was studied. Treatment of HT-29 cells with conditioned medium from IFN-γ/LPS-stimulated MMC produced significant amounts of NO, which suggested the presence of an MMC-derived soluble factor modifying epithelial NO production. Pretreatment of IFN-γ/LPS-stimulated MMC with IL-10 and IL-4 but not IL-13 suppressed NO production by HT-29 cells. Interestingly, pretreatment of HT-29 cells with IL-1 receptor antagonist suppressed the IFN-γ/LPS-stimulated MMC-induced NO production. These results suggest that immunomodulatory cytokines might exert an inhibitory effect on NO up-regulation by colonic epithelium via the inhibition of MMC-derived soluble mediators, such as IL-1.     

Purification and characterization of the immunostimulatory properties of recombinant human interleukin-1 beta

In this study we have used a new method for human recombinant IL-1 beta (rIL-1 beta) purification and investigated its immunostimulatory biological activity. The IL-1 beta gene was cloned using a novel mRNA preparation from activated human blood monocytes. The purification protocol consists of extraction and two chromatographic steps using the new Soloza cation exchange resin. The purified protein was characterized electrophoretically, by amino acid analysis and reverse phase chromatography. The protein migrated on SDS-PAGE with a molecular weight of 18.200 but demonstrated the minor presence of aggregates (dimers and trimers). Specific activity of purified rIL-1 beta in comitogenic assay on mouse thymocytes was 10(8) U/mg protein. rIL-1 beta increased in a dose dependent manner proliferation of Con A-stimulated murine thymocytes, splenocytes, PHA-stimulated human peripheral blood lymphocytes and transformed B-cell lines. Comitogenic activity depended on the degree of lymphocyte preactivation and was similar to that of natural human IL-1 beta. rIL-1 beta enhanced IL-2 production by murine spleen cells and EL-4 cell line and IL-2 receptor expression by human peripheral blood mononuclear cells. It induced PGE2 release from human blood monocytes but had no effect on human neutrophil chemotaxis, phagocytosis and respiratory burst.     

Regulation of IL-12 p40 promoter activity in primary human monocytes: roles of NF-kappaB, CCAAT/enhancer-binding protein beta, and PU.1 and identification of a novel repressor element (GA-12) that responds to IL-4 and prostaglandin E(2)

Appropriate regulation of IL-12 expression is critical for cell-mediated immune responses. In the present study, we have analyzed the regulation of IL-12 p40 promoter activity in primary human monocytes in vivo. Accordingly, we analyzed the p40 promoter by in vivo footprinting in resting and activated primary human blood CD14(+) monocytes. Interestingly, footprints at binding sites for trans-activating proteins such as C/EBP, NF-kappaB, and ETS were only found upon stimulation with LPS and IFN-gamma. In contrast, a footprint over a purine-rich sequence at -155, termed GA-12 (GATA sequence in the IL-12 promoter), was observed in resting, but not activated, cells. Further characterization of this site revealed specific complex formation at a protected GATA core motif in unstimulated primary monocytes and RAW264.7 macrophages. Mutagenesis within the GA-12 sequence caused a significant up-regulation of inducible IL-12 p40 promoter activity in both transient and stable transfection systems, suggesting a repressor function of this site. Furthermore, binding activity of the GA-12 binding protein GAP-12 was increased by treatment with two potent inhibitors of IL-12 expression, IL-4 and PGE(2). Finally, we observed that IL-4-mediated repression of IL-12 p40 promoter activity is critically dependent on an intact GA-12 sequence. In summary, our data underline the complex regulation of the human IL-12 p40 promoter and identify GA-12 as a potent, novel repressor element that mediates IL-4-dependent suppression of inducible promoter activity in monocytes. Regulation of GAP-12 binding may thus modulate IL-12 p40 gene expression.     

Immunomodulatory cytokines suppress epithelial nitric oxide production in inflammatory bowel disease by acting on mononuclear cells

Inducible nitric oxide synthase (iNOS) activity in colonic epithelial HT-29 cells is modulated by the T-cell-derived cytokines IL-4 and IL-13, but is not affected by IL-10 despite its effect in models of colitis. We studied the effects of these cytokines on nitric oxide (NO) production by colonic tissue. IL-10 and IL-4 but not IL-13 suppressed the NO production and iNOS expression by inflamed tissue and cytokine-stimulated noninflamed tissue from patients with ulcerative colitis, whereas the three cytokines suppressed NO production in cytokine-stimulated biopsies from controls. To examine why colonic biopsies and HT-29 cells respond differently to immunomodulatory cytokines, a coculture of mixed mononuclear monocytes (MMC) and HT-29 cells was studied. Treatment of HT-29 cells with conditioned medium from IFN-γ/LPS-stimulated MMC produced significant amounts of NO, which suggested the presence of an MMC-derived soluble factor modifying epithelial NO production. Pretreatment of IFN-γ/LPS-stimulated MMC with IL-10 and IL-4 but not IL-13 suppressed NO production by HT-29 cells. Interestingly, pretreatment of HT-29 cells with IL-1 receptor antagonist suppressed the IFN-γ/LPS-stimulated MMC-induced NO production. These results suggest that immunomodulatory cytokines might exert an inhibitory effect on NO up-regulation by colonic epithelium via the inhibition of MMC-derived soluble mediators, such as IL-1.     

Herpes simplex virus-1 up-regulates IL-15 gene expression in monocytic cells through the activation of protein tyrosine kinase and PKC zeta/lambda signaling pathways

IL-15 plays a seminal role in innate immunity through enhancing the cytotoxic function as well as cytokine production by NK and T cells. We have previously shown that exposure of PBMC as well as monocytic cells to different viruses results in immediate up-regulation of IL-15 gene expression and subsequent NK cell activation as an innate immune response of those cells to these viruses. However, no signaling pathway involved in this up-regulation has been identified. Here we show for the first time that HSV-1-induced up-regulation of IL-15 gene expression is independent of viral infectivity/replication. IL-15 gene is up-regulated by HSV-1 in human monocytes, but not in CD3+ T cells. HSV-1 induces the phosphorylation of protein tyrosine kinases (PTKs) and protein kinase C (PKC) for inducing IL-15 expression in monocytic cells. Inhibitors for PTKs reduced HSV-1-induced PTK activity, DNA binding activity of NF-kB as well as IL-15 gene expression. In contrast, an inhibitor for membrane-bound tyrosine kinases had no effect on these events. Experiments using PKC inhibitors revealed that phosphorylation of PKC zeta/lambda (PKC zeta/lambda), DNA binding activity of NF-kB and HSV-1-induced up-regulation of IL-15 were all decreased. Furthermore, we found that HSV-1-induced IL-15 up-regulation was also dependent on PTKs regulation of PKC phosphorylation. Thus, we conclude that IL-15 up-regulation in HSV-1-treated monocytic cells is dependent on the activity of both PTKs and PKC zeta/lambda.     

Induction of interleukin-1 in human monocytes by the superantigen staphylococcal enterotoxin A requires the participation of T cells

Nanogram quantities of the bacterial superantigen Staphylococcal Enterotoxin A (SEA) induced significant amounts of extracellular IL-1 alpha and IL-1 beta in human peripheral blood mononuclear cells. Induction of maximal IL-1 alpha and IL-1 beta levels by lipopolysaccharide (LPS) required microgram quantities. LPS induced detectable extracellular IL-1 content within 3-6 hr and maximal levels were detected already after 12 hr. Induction of IL-1 production by SEA showed a delayed release with peak values after 24-48 hr. IL-1 beta was the major species of IL-1 seen in both SEA- and LPS-stimulated culture supernatants. SEA was in general a relatively stronger inducer of extracellular IL-1 alpha than LPS. SEA-induced extracellular IL-1 production in human monocytes was entirely dependent on the presence of T cells, whereas addition of T cells to LPS-stimulated purified human monocytes only marginally enhanced the extracellular IL-1 production. The capacity to induce extracellular IL-1 production in monocytes in response to SEA was high in the CD4+ 45RO+ memory T cell subset, whereas CD4+ 45RA+ naive T cells and CD8+ T cells had lower IL-1-inducing capacity. The T cell help for IL-1 production could not be replaced by a panel of T cell-derived recombinant lymphokines added to SEA-stimulated monocytes, including IFN-gamma and TNF, indicating the participation of cell membrane-bound ligands or hitherto unidentified soluble mediators.     

Activated protein C inhibits the release of macrophage inflammatory protein-1-alpha from THP-1 cells and from human monocytes

Several lines of evidence have implicated activated protein C (APC) to be an endogenous inhibitor of the inflammatory septic cascade. APC may exhibit direct anti-inflammatory properties, independent of its antithrombotic effects. Chemokines influence the interaction of monocytes at the endothelium during infection and sepsis and are involved in the molecular events leading to an adverse and lethal outcome of sepsis. Defining regulatory mechanisms on the monocytic release profile of the proinflammatory C-C chemokines macrophage inflammatory protein-1-alpha (MIP-1-alpha) and monocyte chemoattractant protein-1 (MCP-1) might have therapeutic implications for the treatment of sepsis. We established a monocytic cell model of inflammation by the addition of lipopolysaccharide (LPS) and examined the effect of human APC on LPS-stimulated chemokine release from the monocytic cell line THP-1. We found that human APC in supra-physiological concentrations of 2.5-10 microg/ml inhibited the LPS-induced release of the chemokines MIP-1-alpha and MCP-1, as measured by enzyme-linked immunosorbent assays (ELISA) at 6 up to 24 h. In addition to experiments on THP-1 cells, recombinant human APC in concentrations of 50 ng/ml was found to have an inhibiting effect on the release of MIP-1-alpha from freshly isolated mononuclear cells of septic patients. The ability of APC to decrease the release of the C-C chemokine MIP-1-alpha from the monocytic cell line THP-1 and from human monocytes may identify a novel immunomodulatory pathway by which APC exerts its anti-inflammatory action and may contribute to control the inflammatory response in sepsis.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.     

IL-1 beta converting enzyme is a target for nitric oxide-releasing aspirin: new insights in the antiinflammatory mechanism of nitric oxide-releasing nonsteroidal antiinflammatory drugs

Caspase-1, the IL-1beta converting enzyme (ICE), is required for intracellular processing/maturation of IL-1beta and IL-18. NO releasing nonsteroidal antiinflammatory drugs (NSAIDs) are a new class of NSAID derivatives that spare the gastric mucosa. Here, we tested the hypothesis that NCX-4016, a NO-aspirin derivative, inhibits proinflammatory cytokine release from endotoxin (LPS)-challenged monocytes. Our results demonstrated that exposing LPS-stimulated human monocytes to NCX-4016 resulted in a 40-80% inhibition of IL-1beta, IL-8, IL-12, IL-18, IFN-gamma, and TNF-alpha release with an EC(50) of 10-20 microM for IL-1beta and IL-18. Incubating LPS-primed monocytes with NCX-4016 resulted in intracellular NO formation as assessed by measuring nitrite/nitrate, intracellular cGMP concentration, and intracellular NO formation. Exposing LPS-stimulated monocytes to aspirin or celecoxib caused a 90% inhibition of prostaglandin E(2) generation but had no effect on cytokine release. NCX-4016, similar to the NO donor S-nitroso-N-acetyl-D-L-penicillamine, inhibited caspase-1 activity with an EC(50) of approximately 20 microM. The inhibition of caspase-1 by NCX-4016 was reversible by the addition of DTT, which is consistent with S-nitrosylation as the mechanism of caspase-1 inhibition. NCX-4016, but not aspirin, prevented ICE activation as measured by assessing the release of ICE p20 subunit. IL-18 immunoneutralization resulted in a 60-80% reduction of IL-1beta, IL-8, IFN-gamma, and TNF-alpha release from LPS-stimulated monocytes. Taken together, these data indicate that incubating human monocytes with NCX-4016 causes intracellular NO formation and suppresses IL-1beta and IL-18 processing by inhibiting caspase-1 activity. Caspase-1 inhibition is a new, cycloxygenase-independent antiinflammatory mechanism of NO-aspirin.