The Human G Protein-Coupled ATP Receptor P2Y11 Is Associated With IL-10 Driven Macrophage Differentiation.

The G protein-coupled P2Y11 receptor is known to sense extracellular ATP during inflammatory and immune responses. The dinucleotide NAD+ has also been proposed to be a P2Y11 receptor ligand but its role is less clear. Here, we have examined for the first time human P2Y11 receptor protein levels and show that the receptor was upregulated during polarization of M2 macrophages. IL-10 reinforced P2Y11 receptor expression during differentiation of M2c macrophages expressing CD163, CD16, and CD274 (PD-L1). Nutlin-3a mediated p53 stabilization further increased P2Y11 receptor, CD16, and PD-L1 expression. AMP-activated kinase (AMPK), which mediates anti-inflammatory effects of IL-10, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway, which is under the control of AMPK, were also required for P2Y11 receptor expression. The P2Y11 receptor agonist ATPγS and NAD+ could independently stimulate the production of IL-8 in M2 macrophages, however, only the ATPγS-induced response was mediated by P2Y11 receptor. Both in a recombinant system and in macrophages, P2Y11 receptor-driven IL-8 production predominantly depended on IkB kinase (IKK), and extracellular signal-regulated kinase (ERK). In conclusion, our data indicate that an AMPK-NAMPT-NAD+ signaling axis promotes P2Y11 receptor expression during M2 polarization of human macrophages in response to IL-10. PD-L1 expressing M2c macrophages that secrete the cancer-promoting chemokine IL-8 in response to P2Y11 receptor stimulation may represent an important target in checkpoint blockade immunotherapy.

Subcutaneous administration of a neutralizing IL-1ß antibody prolongs limb allograft survival.

Cytokine-expression profiles revealed IL-1ß highly upregulated in rejecting skin of limb allografts. We investigate the effect of intragraft treatment with a neutralizing IL-1ß antibody in limb transplantation. Following allogenic hind-limb transplantation, Lewis rats were either left untreated or treated with anti-lymphocyte serum + tacrolimus (baseline); baseline immunosuppression + anti-IL-1ß (1 mg/kg once/week, 6-8 subcutaneous injections) into the transplanted or contralateral limb. Endpoint was rejection grade III or day 100. Graft rejection was assessed by histology, immunohistochemistry, flow cytometry phenotyping of immune cells, and monitoring cytokine expression. Anti-IL-1ß injections into the allograft or contralateral limb resulted in a significant delay of rejection onset (controls: 58.60 ± 0.60; group 3: 75.80 ± 10.87, P = .044; group 4: 73.00 ± 6.49, P = .008) and prolongation of graft survival (controls: 64.60 ± 0.87; group 3: 86.60 ± 5.33, P = .002; group 4: 93.20 ± 3.82, P = .002), compared to controls. Although the phenotype of the graft infiltrating immune cells did not differ between groups, significantly decreased skin protein levels of IL-1ß, IL-4, IL-13, IP-10, MCP-1, and MCP-3 in long-term-survivors indicate an overall decrease of chemoattraction and infiltration of immune cells as the immunosuppressive mechanism of anti-IL-1ß. Inhibition of IL-1ß with short-term systemic immunosuppression prolongs limb allograft survival and represents a promising target for immunosuppression in extremity transplantation.

Novel Insights into the PKCß-dependent Regulation of the Oxidoreductase p66Shc.

Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCß-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCß thus may provide a target for therapeutic intervention. We performed a reassessment of PKCß regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCß, they instead provided evidence for Ser139 and Ser213 as PKCß phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCß phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCß-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213.

cJun N-terminal kinase (JNK) phosphorylation of serine 36 is critical for p66Shc activation.

p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants in vivo. Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage.

RAF and antioxidants prevent cell death induction after growth factor abrogation through regulation of Bcl-2 proteins.

We have shown previously that mitochondrial ROS production is essential to turn growth factor (GF) removal into cell death. Activated RAF, AKT, Bcl-2 and antioxidants protected equally well against ROS accumulation and subsequent death. Here we investigated whether protection by survival signaling and antioxidants utilizes shared or distinct targets. Using serum deprivation from NIH 3T3 fibroblasts and IL-3 withdrawal from promyeloid 32D cells, we showed that pro-survival signaling by activated RAF but not AKT prevented the decline in Mcl-1 following GF abrogation. GF starvation increased levels of Bim in both model systems, which was prevented by RAF in 32D cells but not in NIH 3T3 fibroblasts. RAF and AKT suppressed activation and mitochondrial translocation of BAX. Also, antioxidant treatment efficiently prevented BAX activation and death of 32D cells but showed little effect on its mitochondrial translocation. No significant impact of antioxidant treatment on Bim or Mcl-1 expression was observed. ROS produced during GF abrogation also did not alter the activity of intracellular signaling pathways, which have been implicated previously in cell killing by pro-oxidants. Together these data suggest Bcl-2 family proteins as convergence point for RAF and ROS in life and death decisions.

Proliferation arrest in B-Raf mutant melanoma cell lines upon MAPK pathway activation.

Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.

Bilirubin inhibits tumor cell growth via activation of ERK.

Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo, tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.

Continuous administration of heparin in patients with deep vein thrombosis can increase plasma levels of diamine oxidase.

Besides its anticoagulant activity, the sulfated polysaccharide heparin has numerous other biological effects. Especially the antiinflammatory and immunoregulatory properties of heparin may be associated with its ability to release the histamine-degrading enzyme diamine oxidase (DAO) from tissue-bound sites into the circulation. Whereas DAO activity is at the limits of detection in normal human plasma, the application of heparin leads to a significant increase of plasma DAO activity. However, previously, only the effect of bolus injection of unfractionated heparin (UFH) had been studied. To investigate DAO release during continuous heparin infusion, 28 patients with deep vein thrombosis (DVT) undergoing heparin therapy were analyzed. Whereas continuous heparin infusion did not lead to any increase of plasma DAO activity in 12 patients (43%), 6 patients (21%) showed a single elevated and 10 patients (36%) permanently elevated plasma DAO activity. The groups of patients exhibiting different DAO release responses did not differ in age, sex, body weight, concomitant diseases, heparin infusion rates, coagulation indices, location and extension of thrombosis, or clinical outcome. However, the rate of idiopathic DVT was significantly higher in the group of patients releasing DAO. This study shows, for the first time, that continuous heparin infusion can lead to DAO release and that individuals exhibit considerable differences in their release response. Although the significance of heparin-induced DAO release needs further clarification, our results indicate that postheparin plasma DAO activity could be an interesting parameter correlated with idiopathic DVT.