No abstract

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...

Incubation of ex vivo cultured mature B cells in the presence of nitric oxide or nitric oxide-donor substances delays programmed cell death as determined by the appearance of DNA laddering in agarose gel electrophoresis or by flowcytometry analysis of DNA. Nitric oxide also rescues B cells from antigen-induced apoptosis but fails to provide a costimulatory signal that converts the signal elicited by the antigen into a proliferative response. The protective effects of nitric oxide against programmed cell death can be reproduced by treatment of the cells with permeant analogues of cyclic GMP. Regarding the mechanisms by which nitric oxide prevents apoptosis in B cells, we have observed that nitric oxide release prevents the drop in the expression of the protooncogene bcl-2, both at the mRNA and protein levels, suggesting the existence of an unknown pathway that links nitric oxide signaling with Bcl-2 expression. (J. Clin.

Nitric oxide (NO) induces apoptosis in thymocytes, peripheral T cells, myeloid cells and neurons.Here we show that NO is highly efficient in inducing mitochondrial permeability transition, thereby causing the liberation of apoptogenic factors from mitochondria which can induce nuclear apoptosis (DNA condensation and DNA fragmentation) in isolated nuclei in vitro. In intact thymocytes, NO triggers disruption of the mitochondrial transmembrane potential, followed by hypergeneration of reactive oxygen species, exposure of phosphatidyl serine on the outer plasma membrane leaflet, and nuclear apoptosis. Inhibitors of mitochondrial permeability transition such as bongkrekic acid and a cyclophilin D-binding cyclosporin A derivative, /V-methylVal-4-cyclosporin A, prevent the mitochondrial as well as all post-mitochondrial signs of apoptosis induced by NO including nuclear DNA fragmentation and exposure of phosphatidylserine residues on the cell surface. These findings indicate that NO can cause apoptosis via triggering of permeability transition.

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

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.

Activation of the macrophage cell line RAW 264.7 with LPS and IFN-γ induces apoptosis through the synthesis of high concentrations of NO due to the expression of NO synthase-2. In addition to NO, activated macrophages release other molecules involved in the inflammatory response, such as reactive oxygen intermediates and PGs. Treatment of macrophages with cyclopentenone PGs, which are synthesized late in the inflammatory onset, exerted a negative regulation on cell activation by impairing the expression of genes involved in host defense, among them NO synthase-2. However, despite the attenuation of NO synthesis, the percentage of apoptotic cells increased with respect to activated cells in the absence of cyclopentenone PGs. Analysis of the mechanisms by which these PGs enhanced apoptosis suggested a potentiation of superoxide anion synthesis that reacted with NO, leading to the formation of higher concentrations of peroxynitrite, a more reactive and proapoptotic molecule than the precursors. The effect of the cyclopentenone 15-deoxy-Δ12,14-PGJ2 on superoxide synthesis was dependent on p38 mitogen-activated protein kinase activity, but was independent of the interaction with peroxisomal proliferator-activated receptor γ. The potentiation of apoptosis induced by cyclopentenone PGs involved an increase in the release of cytochrome c from the mitochondria to the cytosol and in the nitration of this protein. These results suggest a role for cyclopentenone PGs in the resolution of inflammation by inducing apoptosis of activated cells.

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.