Macrophage activation in response to proinflammatory cytokines and bacterial cell wall products constitutes a key component of the immune response (23,31,50). Resolution of the process occurs after removal of the proinflammatory stimuli and through the action of negative regulators of the activationsignaling pathways, among them interleukin-10 (IL-10), IL-13, alpha/beta interferons (IFN-␣/␤), and more recently several cyclopentenone prostaglandins (PGs) (8,21,35,36,49). In particular, 15-deoxy-⌬ 12,14 -prostaglandin J 2 (15dPGJ 2 ) has been shown to exert important anti-inflammatory effects on several cell types such as monocytes/macrophages and microglia (4,16,35,36). Controversy exists about the identification of intracellular targets involved in the mechanism of action of cyclopentenone PGs: some of these effects have been explained through the transcriptional inhibition exerted by 15dPGJ 2 -activated peroxisome proliferator receptor gamma (PPAR␥) (12,14,36,39); however, other data suggest a main contribution of PPAR␥-independent mechanisms on the antiinflammatory action of this PG, in view of the lack of effect of synthetic PPAR␥ ligands such as thiazolidinediones (17,35).It has been shown that 15dPGJ 2 inhibits the expression of genes requiring the activation of the transcription factors NF-B, AP-1, and Stat1 (17, 35, 36), which are involved in the induction of several enzymes participating in the development of the inflammatory process, such as type 2 nitric oxide synthase (NOS-2) and cyclooxygenase 2 (COX-2) (7,42,51). In macrophages, activated NF-B complexes are composed mainly of p50 and p65 subunits that translocate to the nucleus in response to cell stimulation with lipopolysaccharide (LPS) and proinflammatory cytokines (13,45,48). This activation of NF-B requires phosphorylation by IB kinase (IKK) of IB proteins in specific serine residues that target these proteins for ubiquitin conjugation and degradation by the 26S proteasome (26, 45). The IKK complex contains two catalytic subunits, IKK1 and IKK2, and a regulatory subunit termed NF-B essential modulator (10,54,56). In turn, activation of IKK is mediated by phosphorylation through NF-B-inducing kinase, which acts preferentially over IKK1, and MEK kinase 1 (MEKK1), which phosphorylates IKK2 (6, 30). Biochemical and genetic data indicate that IKK1 and IKK2, despite the sequence similarity, have different functions (15,55). IKK1 participates in differentiation of various cell types (20), whereas IKK2 is involved in LPS signaling in monocytes/macrophages and in general the response to proinflammatory stimuli (34, 55). IKK2 is rapidly activated after cell challenge with LPS, IL-1␤, or tumor necrosis factor alpha (TNF-␣) and progressively undergoes phosphorylation at multiple serine residues that decreases the kinase activity and therefore contributes to the transient activation of this enzyme (6). In this regard, we have investigated the possibility of early effects of 15dPGJ 2 on LPS and IFN-␥ (collectively termed LPS/IFN-␥) cooperative signaling in R...

Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-γ, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-γ, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.

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Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS-induced NF-jB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF-a and IL-6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF-jB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF-jB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IjB kinase a/b complex and the expression of some NF-jB family members. Treatment of macrophages with the c-secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF-jB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF-a and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage-dependent inflammatory response by enhancing NF-jB signaling.Key words: Macrophages . NF-kB . Notch IntroductionMacrophages are essential cells for the innate immune response. They discriminate between pathogens and self through signals triggered by TLR, which recognize different pathogens' components, such as LPS, lipoproteins, or dsRNA, among others [1]. Activation of most TLR on the macrophage surface triggers a complex signaling pathway, which involves NF-kB activation (reviewed in [2]). In the classical NF-kB pathway, a ternary IkB kinase (IKK) complex, formed by IKK-a, IKK-b, and NF-kB essential modulator, is responsible for inducing IkB phosphorylation, allowing the release of sequestered cytoplasmic NF-kB from IkB and its translocation to the nucleus. Once in the nucleus, NF-kB controls the expression of multiple genes implicated in the inflammatory response, including cytokines, effector enzymes such as iNOS and COX-2, and adhesion molecules [2].Notch proteins encompass a family of transmembrane receptors composed of an extracellular subunit linked to a transmembrane and intracellular subunit via heterodimerization domains [3]. Ligand binding induces proteolytic cleavage of the transmembrane and intracellular receptor subunit by several proteases, including g-secretase [4], allowing the release of the intracellular domain of Notch (NIC), which then translocates to the nucleus and converts the CBF1 factor from a repressor to a transcriptional activator. Some NIC target genes have been characterized, including basic-helix-loop-helix transcription factors belonging to 2556the hairy/enhancer of split (HES) gene family [3]. Although some CBF1-independent Notch signaling can occur, its mechanism of action is not well characterized yet [5].Notch signaling is an evolutionarily conserved pathway that controls different aspects of tissue development and homeostasis [6]. In cells o...

The protein DLK2, highly homologous to DLK1, belongs to the EGF-like family of membrane proteins, which includes NOTCH receptors and their DSL-ligands. The molecular mechanisms by which DLK proteins regulate cell differentiation and proliferation processes are not fully established yet. In previous reports, we demonstrated that DLK1 interacts with itself and with specific EGF-like repeats of the NOTCH1 extracellular region involved in the binding to NOTCH1 canonical ligands. Moreover, the interaction of DLK1 with NOTCH1 caused an inhibition of basal NOTCH signaling in preadipocytes and mesenchymal multipotent cells. In this work, we demonstrate, for the first time, that DLK2 interacts with itself, with DLK1, and with the same NOTCH1 receptor region as DLK1 does. We demonstrate also that the interaction of DLK2 with NOTCH1 similarly results in an inhibition of NOTCH signaling in preadipocytes and Mouse Embryo fibloblasts. In addition, we demonstrate that a membrane DLK1 variant, lacking the sequence recognized by the protease TACE, also inhibits NOTCH signaling. Furthermore, both DLK1 and DLK2 are able to decrease NOTCH activity also when triggered by specific NOTCH ligands. However, the decrease in NOTCH signaling induced by overexpression of Dlk2 is reversed by the overexpression of Dlk1, and viceversa. We conclude that DLK1 and DLK2 act as inhibitory non-canonical protein ligands for the NOTCH1 receptor that modulate NOTCH signaling.

The induction of hepatic nitric oxide synthase (NOS) and the biosynthesis of nitric oxide (NO) were studied in liver after partial hepatectomy (PH). NOS activity in the liver remnant was observed 4 to 6 hours after PH, and no differences were evidenced between the proximal and distal surgical areas. The form of NOS expressed in liver was independent of calcium and calmodulin, and the messenger RNA levels were first detected 2 hours after hepatectomy using a probe corresponding to the cytokine-induced macrophage NOS. The seric concentration of nitrites remained unchanged after hepatectomy, whereas the content in nitrates and in S-nitrosylated proteins progressively increased in parallel with the NOS activity. The spectra of hemoglobin in the 400-to 460-nm region failed to exhibit the characteristic shift caused by the formation of the nitrosyl-hemoglobin complex, suggesting that NO was rapidly metabolized in liver. Treatment of the animals with substrate analogue NOS inhibitors blocked the pattern of DNA ploidy elicited after hepatectomy, suggesting a role for NO in the regenerative process. Peritoneal resident macrophages were used as an alternative reporter cell system for the assessment of NOS expression. Incubation ex vivo of peritoneal macrophages from animals that underwent hepatectomy induced the expression of NOS in a cytokine-modulated fashion, suggesting that macrophages were primed as a result of the hepatectomy. When peritoneal macrophages from control rats were incubated with the sera of animals that underwent hepatectomy, a time-dependent induction of NOS was observed, with a maximal induction corresponding to sera collected 2 hours after PH. These results indicate that NO might be involved in the control of early responses after PH.

Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-C ysSerLys4 or S(-)[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-++ +CysAlaLys4, two synthetic bacterial lipopeptides, promoted the expression of the inducible form of nitric oxide synthase, exhibiting a temporal pattern of nitric oxide release that was delayed with respect to the induction elicited by bacterial lipopolysaccharide. Treatment of macrophages with genistein blocked the nitric oxide synthesis triggered by the lipopeptides or lipopolysaccharide. Simultaneous incubation with lipopolysaccharide and lipopeptide resulted in an antagonistic effect on nitric oxide synthase mRNA levels and on nitrite plus nitrate release to the medium. Triggering with bacterial lipopeptides induced macrophage programmed cell death. In macrophages activated with lipopeptide, apoptosis was observed even in the absence of nitric oxide synthesis, therefore indicating the existence of alternative pathways in the control of programmed cell death in these cells.

Macrophages activated through Toll receptor triggering increase the expression of the A 2A and A 2B adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A 2A and A 2B receptors, we demonstrate that the A 2A receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A 2B receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1␣ is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A 2 receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.