Cyclopentenone prostaglandins induce lymphocyte apoptosis by activating the mitochondrial apoptosis pathway independent of external death receptor signaling

15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a naturally occurring cyclopentenone metabolite of PGD(2) that possesses both peroxisome proliferator-activated receptor gamma (PPAR-gamma)-dependent and PPAR-gamma-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ(2) as well as synthetic PPAR-gamma agonists inhibit lymphocyte proliferation. However, only 15d-PGJ(2), but not the specific PPAR-gamma activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain(-/-) or caspase-8(-/-) Jurkat T cells has no effect on apoptosis induction by 15d-PGJ(2). Conversely, overexpression of Bcl-2 or Bcl-x(L) completely inhibits the initiation of apoptosis, indicating that 15d-PGJ(2)-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ(2) induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Deltapsi(m)) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ(2). We also demonstrate that 15d-PGJ(2) potently stimulates reactive oxygen species production in Jurkat T cells, and Deltapsi(m) loss induced by 15d-PGJ(2) is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ(2) may modulate immune responses even independent of PPAR-gamma by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.     

15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone modulate Staphylococcus aureus-dependent astrocyte activation primarily through a PPAR-gamma-independent pathway

Brain abscesses arise from a focal parenchymal infection by various pathogens, particularly Staphylococcus aureus. We have shown that astrocytes are activated upon exposure to S. aureus and may contribute to the excessive tissue damage characteristic of brain abscess. Therefore, modulating astrocyte activation may facilitate a reduction in brain abscess severity. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists are potent inhibitors of microglial activation; however, the effects of these compounds on S. aureus-dependent astrocyte activation have not yet been examined. Here, we demonstrate that two chemically distinct PPAR-gamma agonists, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, suppress the production of several pro-inflammatory molecules in S. aureus-stimulated astrocytes including interleukin-1beta and nitric oxide (NO). Interestingly, 15d-PGJ2 attenuated Toll-like receptor 2 (TLR2) and inducible nitric oxide synthase expression, but failed to modulate macrophage inflammatory protein-2 (MIP-2/CXCL2) production, suggesting that 15d-PGJ2 is not a global inhibitor of astrocyte activation. Another novel finding of this study was the fact that both 15d-PGJ2 and ciglitazone were capable of attenuating pre-existing astrocyte activation, indicating their potential benefit in a therapeutic setting. Importantly, 15d-PGJ2 and ciglitazone were still capable of inhibiting S. aureus-induced pro-inflammatory mediator release in PPAR-gamma-deficient astrocytes, supporting PPAR-gamma-independent effects of these compounds. Collectively, these results suggest that 15d-PGJ2 and ciglitazone exert their anti-inflammatory actions on astrocytes primarily independent of the PPAR-gamma pathway.     

Apoptosis induced by oxidized low density lipoprotein in human monocyte-derived macrophages involves CD36 and activation of caspase-3.

Macrophage death may play a crucial role in the progression of atherosclerotic lesions. Here we present evidence that CD36 is involved in oxidized LDL (OxLDL)-induced apoptosis in human monocyte-derived macrophages. Anti-CD36 mAb SMO and OKM-5 reduced the number of apoptotic cells in OxLDL-treated macrophages by more than 94%, but they did not block ceramide-triggered apoptosis. Thrombospondin inhibited the induction of apoptosis by OxLDL in a dose-dependent manner with an IC50 of 10-30 microM. OxLDL did not induce apoptosis in CD36-negative macrophages, demonstrating the essential role of this scavenger receptor in OxLDL-triggered programmed cell death. Neither anti-CD36 Ig nor thrombospondin triggered programmed cell death suggesting that binding to CD36 alone is not sufficient to initiate apoptosis. However, inhibitors of OxLDL-induced apoptosis did not block the uptake of 3H-labeled OxLDL. In contrast, acetylated LDL and polyinosinic acid, ligands of scavenger receptor A (SRA), inhibited uptake of 3H-labeled OxLDL by 65 and 49%, respectively, but did not block OxLDL-induced apoptosis, indicating that SRA is not involved in this process. OxLDL also stimulated caspase-3 activity in human macrophages. Activation of caspase-3 was blocked by anti-CD36 Ig and the caspase-3 inhibitor Z-DEVD-FMK. These results suggest that binding of OxLDL to CD36 initiates a yet unknown OxLDL-specific signaling event, which leads to the rapid activation of caspase-3 resulting in apoptosis of human macrophages. Our data demonstrate a novel role for CD36 in macrophage biology with likely consequences for the development of atherosclerotic lesions.     

Peroxisome proliferator activator receptor-gamma agonists and 15-deoxy-Delta(12,14)(12,14)-PGJ(2) induce apoptosis in normal and malignant B-lineage cells

The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-gamma (PPAR-gamma) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR-gamma mRNA and, by Western blot analysis, the 67-kDa PPAR-gamma protein. 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), a PPAR-gamma agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [(3)H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Only PPAR-gamma agonists (thiazolidinediones), and not PPAR-alpha agonists, mimicked the effect of 15d-PGJ(2) on B-lineage cells, indicating that the mechanism by which 15d-PGJ(2) negatively affects B-lineage cells involves in part PPAR-gamma. The mechanism by which PPAR-gamma agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF(2alpha), which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR-gamma agonists on B lymphomas. Surprisingly, 15d-PGJ(2) induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF(2alpha) blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR-gamma expression and its significance on B lymphocytes. PPAR-gamma agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE(2), which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ(2), and synthetic PPAR-gamma agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.     

Distinct mechanisms for OxLDL uptake and cellular trafficking by class B scavenger receptors CD36 and SR-BI

Modified forms of LDL, including oxidized low density lipoprotein (OxLDL), contribute to macrophage lipid accumulation in the vessel wall. Despite the pathophysiological importance of uptake pathways for OxLDL, the molecular details of OxLDL endocytosis by macrophages are not well understood. Studies in vitro demonstrate that the class B scavenger receptor CD36 mediates macrophage uptake and degradation of OxLDL. Although the closely related scavenger receptor class B type I (SR-BI) binds OxLDL with high affinity, evidence that SR-BI plays a role in OxLDL metabolism is lacking. In this study, we directly compared OxLDL uptake and degradation by CD36 and SR-BI. Our results indicate that although CD36 and SR-BI internalize OxLDL, SR-BI mediates significantly less OxLDL degradation. Endocytosis of OxLDL by both SR-BI and CD36 is independent of caveolae, microtubules, and actin cytoskeleton. However, OxLDL uptake by CD36, but not SR-BI, is dependent on dynamin. The analysis of chimeric SR-BI/CD36 receptors shows that the CD36 C-terminal cytoplasmic tail is necessary and sufficient for dynamin-dependent OxLDL internalization by class B scavenger receptors. These findings indicate that different mechanisms are involved in OxLDL uptake by SR-BI and CD36, which may segregate these two structurally homologous receptors at the cell surface, leading to differences in intracellular trafficking and degradation.     

15-Deoxy-Delta(12,14)-prostaglandin J2 inhibits the expression of proinflammatory genes in human blood monocytes via a PPAR-gamma-independent mechanism

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in inhibition of the expression of proinflammatory cytokines and inducible enzymes such as cyclooxygenase-2 (COX-2). Using real-time RT-PCR the present study investigates the impact of two PPAR-gamma agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and ciglitazone, on the expression of several proinflammatory genes in lipopolysaccharide (LPS)-stimulated human blood monocytes. Stimulation of cells with LPS resulted in a profound induction of the expression of COX-2, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of cells with 15d-PGJ(2) (10 microM) was associated with a nearly complete inhibition of the expression of all genes that remained unaltered in the presence of the PPAR-gamma antagonist bisphenol A diglycidyl ether (BADGE; 100 microM). By contrast, treatment of cells with another potent PPAR-gamma agonist, ciglitazone (50 microM), and the PPAR-alpha agonist WY-14,643 (100 microM) did not suppress LPS-induced expression of the investigated genes. Stimulation of monocytes with LPS resulted in an 88% inhibition of PPAR-gamma mRNA expression that was fully restored by 15d-PGJ(2) but only to a partial extent by ciglitazone and WY-14,643. Again, BADGE did not alter the effect of 15d-PGJ(2). Collectively, our results show that alterations of gene expression by 15d-PGJ(2) in LPS-stimulated human blood monocytes are mediated by PPAR-gamma-independent mechanisms. Moreover, it is concluded that both inhibition of proinflammatory gene expression and restoration of LPS-induced decrease of PPAR-gamma expression may contribute to the biological action of 15d-PGJ(2).     

Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

Molecular cloning of a novel scavenger receptor for oxidized low density lipoprotein, SR-PSOX, on macrophages

Receptor-mediated endocytosis of oxidized low density lipoprotein (OxLDL) by macrophages has been implicated in foam cell transformation in the process of atherogenesis. Although several scavenger receptor molecules, including class A scavenger receptors and CD36, have been identified as OxLDL receptors on macrophages, additional molecules on macrophages may also be involved in the recognition of OxLDL. From a cDNA library of phorbol 12-myristate 13-acetate-stimulated THP-1 cells, we isolated a cDNA encoding a novel protein designated SR-PSOX (scavenger receptor that binds phosphatidylserine and oxidized lipoprotein), which acts as a receptor for OxLDL. SR-PSOX was a type I membrane protein consisting of 254 amino acids, expression of which was shown on human and murine macrophages with a molecular mass of 30 kDa. SR-PSOX could specifically bind with high affinity, internalize, and degrade OxLDL. The recognition of OxLDL was blocked by polyinosinic acid and dextran sulfate but not by acetylated low density lipoprotein. Taken together, SR-PSOX is a novel class of molecule belonging to the scavenger receptor family, which may play important roles in pathophysiology including atherogenesis.     

Antagonistic effects of oxidized low density lipoprotein and alpha-tocopherol on CD36 scavenger receptor expression in monocytes: involvement of protein kinase B and peroxisome proliferator-activated receptor-gamma

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.     

A redox-sensitive pathway mediates oxidized LDL-induced downregulation of insulin-like growth factor-1 receptor

Oxidized low density lipoprotein (OxLDL) has multiple proatherogenic effects, including induction of apoptosis. We have recently shown that OxLDL markedly downregulates insulin-like growth factor-1 receptor (IGF-1R) in human aortic smooth muscle cells, and that IGF-1R overexpression blocks OxLDL-induced apoptosis. We hypothesized that specific OxLDL-triggered signaling events led to IGF-1R downregulation and apoptosis. We examined OxLDL signaling pathways and found that neither IGF-1R downregulation nor the proapoptotic effect was blocked by inhibition of OxLDL-triggered extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (MAPK), or peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways, as assessed using specific inhibitors. However, antioxidants, polyethylene glycol catalase, superoxide dismutase, and Trolox completely blocked OxLDL downregulation of IGF-1R and OxLDL-induced apoptosis. Nordihydroguaiaretic acid, AA-861, and baicalein, which are lipoxygenase inhibitors and also have antioxidant activity, blocked IGF-1R downregulation and apoptosis as well as reactive oxygen species (ROS) production. These results suggest that OxLDL enhances ROS production possibly through lipoxygenase activity, leading to IGF-1R downregulation and apoptosis. Furthermore, anti-CD36 scavenger receptor antibody markedly inhibited OxLDL-induced IGF-1R downregulation and apoptosis as well as ROS production. In conclusion, our data demonstrate that OxLDL downregulates IGF-1R via redox-sensitive pathways that are distinct from OxLDL signaling through MAPK- and PPARgamma-involved pathways but may involve a CD36-dependent mechanism.     

Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis

Peroxisome proliferator-activated receptors (PPAR) are members of a nuclear hormone receptor superfamily that includes receptors for steroids, retinoids, and thyroid hormone, all of which are known to affect the immune response. Previous studies dealing with PPAR-gamma expression in the immune system have been limited. Recently, PPAR-gamma was identified in monocyte/macrophage cells. In this study we examined the role of PPAR-gamma in experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. The hypothesis we are testing is whether PPAR-gamma plays an important role in EAE pathogenesis and whether PPAR-gamma ligands can inhibit the clinical expression of EAE. Initial studies have shown that the presence of the PPAR-gamma ligand 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ2) inhibits the proliferation of Ag-specific T cells from the spleen of myelin basic protein Ac(1-11) TCR-transgenic mice. 15d-PGJ2 suppressed IFN-gamma, IL-10, and IL-4 production by both Con A- and myelin basic protein Ac(1-11) peptide-stimulated lymphocytes as determined by ELISA and ELISPOT assay. Culture of encephalitogenic T cells with 15d-PGJ2 in the presence of Ag reduced the ability of these cells to adoptively transfer EAE. Examination of the target organ, the CNS, during the course of EAE revealed expression of PPAR-gamma in the spinal cord inflammatory infiltrate. Administration of 15d-PGJ2 before and at the onset of clinical signs of EAE significantly reduced the severity of disease. These results suggest that PPAR-gamma ligands may be a novel therapeutic agent for diseases such as multiple sclerosis.     

The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.     

Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages

Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low density lipoprotein (OxLDL), is found in atherosclerotic lesions, and is upregulated by OxLDL. We tested the effects of alpha-tocopherol (AT) enrichment of human monocyte-derived macrophages on CD36 expression and cholesteryl ester accumulation. Monocytes isolated from normal volunteers were cultured into macrophages. Macrophages were enriched overnight with various doses of AT (25, 50, and 100 microM). LDL from normal volunteers was oxidized or acetylated (AcLDL) and incubated with macrophages for 48 h at a concentration of 50 or 100 microg/ml. CD36 expression was assessed by flow cytometry. Quantitative analysis of scavenger receptor class A (SR-A) activity was performed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled LDL. CD36 expression was maximal after 8;-10 days of culture. AT (> or =50 microM) significantly decreased CD36 expression upregulated by OxLDL and AcLDL (P < 0.01). Other antioxidants (beta- or gamma-tocopherol) or protein kinase C inhibitors failed to decrease CD36 expression. Concomitantly, DiI-AcLDL and DiI-OxLDL uptake was significantly decreased after AT treatment (P < 0.001). Cholesteryl ester accumulation was significantly decreased after AT enrichment (AcLDL + AT, 77% inhibition; OxLDL + AT, 42% inhibition). In conclusion, AT decreases both CD36 and SR-A expression and cholesteryl ester accumulation in human macrophages. This provides additional scientific support for the antiatherogenic properties of AT.