NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 μM) or with lipopolysaccharide, interferon-γ, and NG-monomethyl-l-arginine (LPS/IFN-γ/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-γ/NMMA prestimulation activated nuclear factor (NF)-κB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-κB and activator protein-1 (AP-1). NF-κB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-κB site derived from the murine Cox-2 promoter, confirmed NF-κB activation after NO addition. An NF-κB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-γ/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-κB activation, a low-level NO–elicited Cox-2 response required activation of both NF-κB and AP-1.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3

Two important cytokines mediating inflammation are tumor necrosis factor α (TNFα) and IL-1β, both of which require conversion to soluble forms by converting enzymes. The importance of TNFα-converting enzyme and IL-1β-converting enzyme in the production of circulating TNFα and IL-1β in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFα and/or IL-1β by neutrophil-derived proteinases, immunoreactive TNFα and IL-1β release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFα and IL-1β release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.

Calcineurin and vacuolar-type H+-ATPase modulate macrophage effector functions

While effector molecules produced by activated macrophages (including nitric oxide, tumor necrosis factor α, interleukin 1, etc.) help to eliminate pathogens, high levels of these molecules can be deleterious to the host itself. Despite their importance, the mechanisms modulating macrophage effector functions are poorly understood. This work introduces two key negative regulators that control the levels and duration of macrophage cytokine production. Vacuolar-type H+-ATPase (V-ATPase) and calcineurin (Cn) constitutively act in normal macrophages to suppress expression of inflammatory cytokines in the absence of specific activation and to inhibit macrophage cytokine responses induced by bacterial lipopolysaccharide (V-ATPase), interferon γ (V-ATPase and Cn), and calcium (Ca2+) flux (Cn). Cn and V-ATPase modulate effector gene expression at the mRNA level by inhibiting transcription factor NF-κB. This negative regulation by Cn is opposite to its crucial positive role in T cells, where it activates NFAT transcription factor(s) leading to expression of interleukin 2, tumor necrosis factor α, and other cytokine genes. The negative effects of V-ATPase and Cn on NF-κB-dependent gene expression are not limited to the macrophage lineage, as similar effects have been seen with a murine fibroblast cell line and with primary astrocytes.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: Molecular basis for serotype specificity

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab–Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: A possible link between inflammatory cytokines and atherogenesis

Inflammation plays a critical role in atherogenesis, yet the mediators linking inflammation to specific atherogenic processes remain to be elucidated. One such mediator may be secretory sphingomyelinase (S-SMase), a product of the acid sphingomyelinase gene. The secretion of S-SMase by cultured endothelial cells is induced by inflammatory cytokines, and in vivo data have implicated S-SMase in subendothelial lipoprotein aggregation, macrophage foam cell formation, and possibly other atherogenic processes. Thus, the goal of this study was to seek evidence for S-SMase regulation in vivo during a physiologically relevant inflammatory response. First, wild-type mice were injected with saline or lipopolysaccharide (LPS) as a model of acute systemic inflammation. Serum S-SMase activity 3 h postinjection was increased 2- to 2.5-fold by LPS (P < 0.01). To determine the role of IL-1 in the LPS response, we used IL-1 converting enzyme knockout mice, which exhibit deficient IL-1 bioactivity. The level of serum S-SMase activity in LPS-injected IL-1 converting enzyme knockout mice was ≈35% less than that in identically treated wild-type mice (P < 0.01). In LPS-injected IL-1-receptor antagonist knockout mice, which have an enhanced response to IL-1, serum S-SMase activity was increased 1.8-fold compared with LPS-injected wild-type mice (P < 0.01). Finally, when wild-type mice were injected directly with IL-1β, tumor necrosis factor α, or both, serum S-SMase activity increased 1.6-, 2.3-, and 2.9-fold, respectively (P < 0.01). These data show regulation of S-SMase activity in vivo and they raise the possibility that local stimulation of S-SMase may contribute to the effects of inflammatory cytokines in atherosclerosis.

Toll-related receptors and the control of antimicrobial peptide expression in Drosophila

Insects defend themselves against infectious microorganisms by synthesizing potent antimicrobial peptides. Drosophila has appeared in recent years as a favorable model to study this innate host defense. A genetic analysis of the regulation of the antifungal peptide drosomycin has demonstrated a key role for the transmembrane receptor Toll, which prompted the search for mammalian homologs. Two of these, Toll-like receptor (TLR)2 and TLR4, recently were shown to play a critical role in innate immunity against bacteria. Here we describe six additional Toll-related genes (Toll-3 to Toll-8) in Drosophila in addition to 18-wheeler. Two of these genes, Toll-3 and Toll-4, are expressed at a low level. Toll-6, -7, and -8, on the other hand, are expressed at high levels during embryogenesis and molting, suggesting that, like Toll and 18w, they perform developmental functions. Finally, Toll-5 is expressed only in larvae and adults. By using chimeric constructs, we have tested the capacity of the signaling Toll/IL-1R homology domains of these receptors to activate antimicrobial peptide promoters and found that only Toll and Toll-5 can activate the drosomycin promoter in transfected cells, thus demonstrating specificity at the level of the Toll/IL-1R homology domain. In contrast, none of these constructs activated antibacterial peptide promoters, suggesting that Toll-related receptors are not involved in the regulation of antibacterial peptide expression. This result was independently confirmed by the demonstration that a dominant-negative version of the kinase Pelle can block induction of drosomycin by the cytokine Spaetzle, but does not affect induction of the antibacterial peptide attacin by lipopolysaccharide.

Human interleukin-10-related T cell-derived inducible factor: Molecular cloning and functional characterization as an hepatocyte-stimulating factor

IL-10-related T cell-derived inducible factor (IL-TIF or IL-21) is a new cytokine structurally related to IL-10 and originally identified in the mouse as a gene induced by IL-9 in T cells and mast cells. Here, we report the cloning of the human IL-TIF cDNA, which shares 79% amino acid identity with mouse IL-TIF and 25% identity with human IL-10. Recombinant human IL-TIF was found to activate signal transducer and activator of transcription factors-1 and -3 in several hepatoma cell lines. IL-TIF stimulation of HepG2 human hepatoma cells up-regulated the production of acute phase reactants such as serum amyloid A, α1-antichymotrypsin, and haptoglobin. Although IL-10 and IL-TIF have distinct activities, antibodies directed against the β chain of the IL-10 receptor blocked the induction of acute phase reactants by IL-TIF, indicating that this chain is a common component of the IL-10 and IL-TIF receptors. Similar acute phase reactant induction was observed in mouse liver upon IL-TIF injection, and IL-TIF expression was found to be rapidly increased after lipopolysaccharide (LPS) injection, suggesting that this cytokine contributes to the inflammatory response in vivo.

Origin of a chloroplast protein importer

During evolution, chloroplasts have relinquished the majority of their genes to the nucleus. The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes. The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system. A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares ≈22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome. Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes. These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane. Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.

Chronic elevation of plasma thioredoxin: Inhibition of chemotaxis and curtailment of life expectancy in AIDS

Thioredoxin (Trx) is an intracellular redox protein with extracellular cytokine-like and chemokine-like activities. We show here that, although plasma Trx levels are unrelated to survival of HIV-infected individuals with CD4 cell counts above 200/μl blood, survival is significantly impaired (P = 0.003) when plasma Trx is chronically elevated in HIV-infected subjects with CD4 T cell counts below this level (i.e., with Centers for Disease Control (CDC)-defined AIDS). Relevant to the mechanism potentially underlying this finding, we also present data from experimental studies in mice showing that elevated plasma Trx efficiently blocks lipopolysaccharide (LPS)-induced chemotaxis, an innate immune mechanism that is particularly crucial when adaptive immunity is compromised. Thus, we propose that elevated plasma Trx in HIV-infected individuals with low CD4 T cell counts directly impairs survival by blocking pathogen-induced chemotaxis, effectively eliminating the last (innate) barrier against establishment of opportunistic and other infections in these immunodeficient individuals.

Mobilization of MHC class I molecules from late endosomes to the cell surface following activation of CD34-derived human Langerhans cells

Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as “sentinels” of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses and are able to present both endogenous and exogenous antigen onto class I molecules. Here, we study the MHC class I presentation pathway following activation of immature, CD34-derived human Langerhans cells by lipopolysaccharide (LPS). LPS induces an increase in all components of the MHC class I pathway including the transporter for antigen presentation (TAP), tapasin and ERp57, and the immunoproteasome subunits LMP2 and LMP7. Moreover, in CD34-derived Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation is because of mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the CD34-derived Langerhans cells and suggest potential sites for antigen loading of class I proteins.

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

Stimulation of endothelial cells by various inflammatory mediators leads to release of Weibel–Palade bodies and therefore to exocytosis of both P-selectin (adhesion receptor for leukocytes) and von Willebrand factor (vWf) (platelet ligand). The potential role of vWf in leukocyte recruitment was investigated with the use of vWf-deficient mice. We report a strong reduction of leukocyte rolling in venules of vWf-deficient mice. Similarly, vWf deficiency led to a decrease in neutrophil recruitment in a cytokine-induced meningitis model as well as in early skin wounds. In all instances with an antibody that preferentially recognizes plasma membrane P-selectin, we observed a dramatic reduction in P-selectin expression at the cell surface of vWf-deficient endothelium. With confocal microscopy, we found that the typical rodlike shape of the Weibel–Palade body is missing in vWf −/− endothelial cells and that part of the P-selectin content in the vWf −/− cells colocalized with LAMP-1, a lysosomal marker. However, intracellular P-selectin levels were similar in tumor necrosis factor α- and lipopolysaccharide-activated cells of both genotypes. We conclude that the absence of vWf, as found in severe von Willebrand disease, leads to a defect in Weibel–Palade body formation. This defect results in decreased P-selectin translocation to the cell surface and reduced leukocyte recruitment in early phases of inflammation.

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. NG-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1–3 μM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as α-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.

Highly expressed and alien genes of the Synechocystis genome

Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the Synechocystis PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In Synechocystis, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO2 fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.