TGFß induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy.

PurposeOne of the earliest hallmarks of diabetic retinopathy is the loss of retinal pericytes. However, the mechanisms that promote pericyte dropout are unknown. In the present study, we propose a novel pathway in which pericyte apoptosis is mediated by macrophages, TGFß and pro-apoptotic BIGH3 (TGFß-induced Gene Human Clone 3) protein.Patients and methodsTo elucidate this pathway, we assayed human retinal pericyte (HRP) apoptosis by TUNEL assay, BIGH3 mRNA expression by qPCR, and BIGH3 protein expression by western blot analysis. HRP were treated with BIGH3 protein, TGFß1 and TGFß2 and inhibition assays were carried out by blocking with antibodies against BIGH3. The distribution of BIGH3 and CD68+ macrophages were compared in a post-mortem donor eye with 7-year history of Type II diabetes and histopathogically confirmed non-proliferative diabetic retinopathy (NPDR).ResultsTGFß induced a significant increase in BIGH3 mRNA and protein expression, and HRP apoptosis. BIGH3 treatment showed HRP undergo apoptosis in a dose-dependent manner. At 5 µg/ml, BIGH3 induced 3.5-times more apoptosis in HRP than in retinal endothelial cells. TGFß induced apoptosis was inhibited by blocking with antibodies against BIGH3. In an example of NPDR, BIGH3 accumulated within the walls of the inner retina arterioles. Macrophage infiltrates were frequently associated with these vessels and the inner nuclear layer.ConclusionTogether with our previously published results on macrophage-induced retinal endothelial cell apoptosis, the present study supports a novel inflammatory pathway mediated by macrophages and the BIGH3 protein leading to HRP apoptosis. As shown in human post-mortem globes, these observations are clinically relevant, suggesting a new mechanism underlying pericyte dropout during NPDR.

Glutaredoxin 2a overexpression in macrophages promotes mitochondrial dysfunction but has little or no effect on atherogenesis in LDL-receptor null mice.

AIMS: Reactive oxygen species (ROS)-mediated formation of mixed disulfides between critical cysteine residues in proteins and glutathione, a process referred to as protein S-glutathionylation, can lead to loss of enzymatic activity and protein degradation. Since mitochondria are a major source of ROS and a number of their proteins are susceptible to protein-S-glutathionylation, we examined if overexpression of mitochondrial thioltranferase glutaredoxin 2a (Grx2a) in macrophages of dyslipidemic atherosclerosis-prone mice would prevent mitochondrial dysfunction and protect against atherosclerotic lesion formation. METHODS AND RESULTS: We generated transgenic Grx2aMac(LDLR-/-) mice, which overexpress Grx2a as an EGFP fusion protein under the control of the macrophage-specific CD68 promoter. Transgenic mice and wild type siblings were fed a high fat diet for 14 weeks at which time we assessed mitochondrial bioenergetic function in peritoneal macrophages and atherosclerotic lesion formation. Flow cytometry and Western blot analysis demonstrated transgene expression in blood monocytes and peritoneal macrophages isolated from Grx2aMac(LDLR-/-) mice, and fluorescence confocal microscopy studies confirmed that Grx2a expression was restricted to the mitochondria of monocytic cells. Live-cell bioenergetic measurements revealed impaired mitochondrial ATP turnover in macrophages isolated from Grx2aMac(LDLR-/-) mice compared to macrophages isolated from non-transgenic mice. However, despite impaired mitochondrial function in macrophages of Grx2aMac(LDLR-/-) mice, we observed no significant difference in the severity of atherosclerosis between wildtype and Grx2aMac(LDLR-/-) mice. CONCLUSION: Our findings suggest that increasing Grx2a activity in macrophage mitochondria disrupts mitochondrial respiration and ATP production, but without affecting the proatherogenic potential of macrophages. Our data suggest that macrophages are resistant against moderate mitochondrial dysfunction and rely on alternative pathways for ATP synthesis to support the energetic requirements.

Amifostine prior to lethal irradiation prevents allogeneic bone marrow engraftment in mice.

We and others have demonstrated that the milieu created by ionizing radiation (IR) used for conditioning plays a major role in the development of acute graft-versus-host disease (aGVHD). We reasoned that antioxidants that could inhibit IR induction of inflammatory cytokines and/or apoptosis might reduce the incidence or severity of aGVHD. Therefore, BALB/c mice were treated with amifostine, n-acetyl cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) prior to transplantation with allogeneic C57Bl/6 bone marrow and spleen cells. None of 30 amifostine-pretreated mice developed weight loss or other signs of aGVHD and they rejected their allogeneic transplants. However, pretreatment to groups of five mice each with molar equivalent doses of NAC or PDTC accelerated death, and lower doses did not prevent aGVHD. In vitro tests demonstrated that PDTC and NAC acted as pro-oxidants when incubated with isolated normal mouse lymphocytes, whereas amifostine and its active metabolite WR-1065 did not. The conclusion that amifostine protected immune function from IR in vivo was further supported by the fact that amifostine and WR-1065 preserved the response of radiated normal lymphocytes to respond to PHA and both stimulated growth of non-radiated, non-PHA-treated normal lymphocytes in vitro. Taken together, these data caution the use of amifostine in allogeneic transplantation.

The isoflavone genistein inhibits LPS-stimulated TNFalpha, but not IL-6 expression in monocytes from hemodialysis patients and healthy subjects.

BACKGROUND: Whole blood and peripheral blood mononuclear cells from hemodialysis (HD) patients show increased production and secretion of inflammatory cytokines. We determined the contribution of blood monocytes to the production of inflammatory cytokines in whole blood from HD patients. METHODS: Whole blood and isolated mononuclear cells from HD patients and healthy control subjects were preincubated with the isoflavone genistein and stimulated with LPS. TNFalpha, IL-6 and IL-10 formation in the whole blood was measured with ELISA and intracellular cytokine formation in CD 14-positive monocytes was determined by flow cytometry. RESULTS: Unstimulated blood levels of TNFalpha, IL-6 and IL-10 were significantly elevated in HD patients compared to controls, but intracellular monocyte content of these cytokines was identical between groups. LPS induced a robust TNFalpha response in both whole blood and monocytes, and TNFalpha formation was 2.3-fold higher in blood from HD patients compared to controls. A similar trend was observed in monocytes. Conversely, LPS stimulation increased IL-6 levels >1000-fold in whole blood, albeit without a noticeable difference between groups. Only minor increases in monocyte IL-6 content were observed. The isoflavone genistein did not inhibit IL-6 formation and did not alter basal TNFalpha levels, but genistein selectively blocked LPS-induced TNFalpha formation in whole blood and monocytes from both groups. CONCLUSION: Intracellular levels of TNFalpha, IL-6 and IL-10 in monocytes are indistinguishable between HD patients and healthy controls. However, monocytes from HD patients are selectively primed for enhanced TNFalpha secretion in response to LPS. The selective inhibition of monocyte TNFalpha production by genistein may explain the anti-inflammatory action of this phytochemical observed in vivo.

Vitamin E and the prevention of atherosclerosis.

Successful strategy for the prevention of coronary heart disease (CHD) in particular of atherosclerosis, require a detailed understanding of the underlying mechanism. It is now being recognised that dietary antioxidants, in particular vitamin E, will play an important role in designing future strategies. Although more and more beneficial effects of vitamin E on atherosclerosis are being described, the biochemical and cell biological mechanism underlying these benefits are not yet fully understood, preventing the use of vitamin E as therapeutic agent. Recent new findings have shed new light on the physiological role of vitamin E and suggest that it has a much broader array of biological activities than originally expected. In addition to its well described role as an antioxidant, it is becoming evident that vitamin E also can modulate the immune system, suppress local and chronic inflammation, reduce blood coagulation and thrombus formation, and enhance cell function and survival. This review summarises new findings from in vitro studies and discusses their potential relevance in human atherosclerosis.

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.

Vitamin E supplementation of human macrophages prevents neither foam cell formation nor increased susceptibility of foam cells to lysis by oxidized LDL.

Several studies in macrophage cell lines, rodent macrophages, and animal models of atherosclerosis suggest that vitamin E may prevent the formation of foam cells. We tested this hypothesis in a recently developed, fully autologous in vitro model of human foam cell formation. During maturation, macrophages continuously increased their alpha-tocopherol/total cholesterol ratio, demonstrating that these cells accumulate alpha-tocopherol at an even higher rate than cholesterol. In the presence of unsupplemented serum, we observed no correlation between serum vitamin E levels and the increase in the cellular alpha-tocopherol/total cholesterol ratio. In contrast, under supplemented conditions, a 3.1-fold increase in the mean serum alpha-tocopherol/total cholesterol ratio resulted in a corresponding mean 3.5-fold increase in the cellular alpha-tocopherol/total cholesterol ratio. Vitamin E loading had no effect on the lipid composition of macrophages and did not affect their growth. Foam cell formation was stimulated in mature unsupplemented and vitamin E-loaded macrophages for 1 week with 50 microg autologous aggregated low density lipoprotein (LDL) in the presence of unsupplemented and vitamin E-loaded serum, respectively. We observed no effect of vitamin E supplementation on the formation of foam cells. However, foam cell formation resulted in a 36% and 44% reduction in the cellular alpha-tocopherol/total cholesterol ratio in unsupplemented and vitamin E-supplemented foam cells, respectively. The loss of vitamin E was accelerated with increasing concentrations of aggregated LDL and was accompanied by an increase in the susceptibility of these foam cells to succumb to the cell lytic effects of oxidized LDL (OxLDL). However, vitamin E supplementation did not protect macrophages or foam cells from OxLDL-mediated cell lysis, suggesting that vitamin E loss in foam cells is not the cause of their increased susceptibility to cell lysis. Our results suggest that the beneficial effects of vitamin E on cardiovascular disease observed in humans are due neither to a reduction in the propensity of macrophages to form foam cells nor to an increased resistance of these cells to cytolytic OxLDL.

Comparison of lipid-mediated and adenoviral gene transfer in human monocyte-derived macrophages and COS-7 cells.

Lipid-mediated transfection was compared to adenoviral-mediated gene transfer in COS-7 cells as well as human monocyte-derived macrophages (HMDM). For this purpose, we monitored enhanced green fluorescent protein (EGFP) expression by fluorescence microscopy and quantified gene transfer by competitive PCR. Transfection of COS-7 cells with a novel lipid formulation for DNA transfer was highly effective in COS-7 cells. On average, 30% of the cells were fluorescent 48 h after transfection. In HMDM, the same formulation resulted in the expression of EGFP in less than 0.5% of cells. We measured plasmid DNA by quantitative PCR in lipid-transfected macrophages and found that each macrophage contained on average 2 fg of plasmid DNA 24 h after transfection, that is, more than 400 molecules of plasmid DNA entered each cell. Despite the high level of reporter DNA in lipid transfected cells, expression of the fluorescent protein was suppressed in more than 99.5% of the macrophages. We also used adenoviral gene transfer to introduce the foreign DNA into both COS-7 cells and HMDM. Even though the multiplicity of infection was less than 30, expression of EGFP was observed in nearly all COS-7 cells and in more than 80% of HMDM 48 h after transfection. Despite major advances in the field of lipid-mediated transfection of HMDM, the lipid formulations that are available commercially cannot compete with the efficiency of adenoviral gene transfer.

Large variations in human foam cell formation in individuals: a fully autologous in vitro assay based on the quantitative analysis of cellular neutral lipids.

The transformation of monocyte-derived macrophages into lipid-laden foam cells constitutes a characteristic and crucial event in the development of the earliest atherosclerotic lesions. We investigated whether the propensity to form foam cells varies among individuals. We developed a fully autologous foam cell assay based on a recently developed novel culture technique for human monocyte-derived macrophages (Wintergerst ES, Jelk J, Asmis, R. Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity, Histochem. Cell Biol. 1998;110:231-241). Thin layer chromatography and laser densitometry were used to determine cholesterol, triglyceride and cholesteryl ester levels in human macrophages. Aggregated LDL obtained by vortexing was found to be a reproducible stimulus of foam cell formation in human macrophages. In our hands, Cu(2+)-oxidized LDL also induced cholesteryl ester accumulation, but only when vortexed. We found that foam cell formation in an individual varied by less than 25% over a 10-month period. In contrast, we observed a sevenfold difference in foam cell formation among eight male volunteers. The transfer of foam cells into culture medium with freshly thawed autologous serum resulted in a 75% regression within 1 week, independent of the amount of cellular cholesteryl esters accumulated. Foam cell formation correlated neither to serum nor to cellular cholesterol and triglyceride levels. The propensity to form foam cells could therefore represent a novel indicator of individual risk of atherogenesis.

Differential expression of CD14, CD36 and the LDL receptor on human monocyte-derived macrophages. A novel cell culture system to study macrophage differentiation and heterogeneity.

Macrophages are key players in many aspects of human physiology and disease. It has been hypothesized that in a given microenvironment monocytes differentiate into specific subpopulations with distinct functions. In order to study the role of macrophage heterogeneity in atherogenesis, we established a novel isolation and culture technique for human monocyte-derived macrophages. The present technique does not select for monocyte subpopulations prior to the onset of differentiation. Monocytes were cultured for 2 weeks in the presence of autologous lymphocytes before being plated quantitatively. They differentiated into mature macrophages in terms of morphology, lipid composition, and biological activity. Based on phagocytic activity as well as on the expression of CD14, CD36, and the low-density lipoprotein (LDL) receptor, we have identified macrophage subpopulations that may play distinct roles in atherogenesis. While virtually all adherence-purified monocytes expressed CD14, CD36, and the LDL-R, we characterized three subpopulations of macrophages based on the expression of these antigens: CD36+CD14-LDL-R-(58+/-12%), CD36+CD14+LDL-R+(18+/-5%), the remaining cells being CD36-CD14- LDL-R-. The first two subsets decreased in size during further differentiation (51+/-12% and 8+/-3%, respectively). Our culture technique may also serve as a good model for studying the implications of macrophage heterogeneity in diseases other than atherosclerosis.

Dehydroascorbic acid prevents apoptosis induced by oxidized low-density lipoprotein in human monocyte-derived macrophages.

Human low-density lipoprotein (LDL) oxidized with Cu2+ or the radical generator 2,2'-azobis(2-methyl-propionamidine) hydrochloride (AAPH) induces apoptosis in mature human monocyte-derived macrophages as assessed by staining with fluorescein-isothiocyanate-labeled annexin V, by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and by staining of the 7A6 mitochondrial antigen. Oxidized LDL-induced apoptosis was dose and time dependent and clearly distinct from apoptosis induced by serum deprivation. Human autologous serum and lipoprotein-deficient human serum prevented apoptosis induced by oxidized LDL. Supplementation of serum-free culture medium with 25 microM ascorbic or isoascorbic acid only partially protected macrophages from apoptosis, whereas dehydroascorbic acid (DHAA) completely inhibited apoptosis induced by either Cu2+- or AAPH-oxidized LDL. Apoptosis was also inhibited by the structural analogue alloxan. Both cyclic multiketones dose-dependently inhibited oxidized LDL-induced apoptosis with IC50 in the submicromolar range. Prior loading of macrophages with ascorbic acid did not prevent the induction of apoptosis. Apoptosis was reduced by more than 90% after treatment of oxidized LDL with DHAA, whereas after incubation with either ascorbic or isoascorbic acid there was no such reduction. Removal of free DHAA by gel filtration did not reverse the inactivation. Parameters of LDL oxidation such as electrophoretic mobility, alpha-tocopherol content, thiobarbituric-acid-reactive subtances and lipid peroxide levels did not correlate to apoptotic activity. Also, binding and uptake of Texas-red-labeled oxidized LDL was not prevented by DHAA. Dithiothreitol-treatment of oxidized LDL, however, reduced the apoptotic activity by 76%. Our results suggest that oxidized thiols on apoB may be essential for the induction of apoptosis by oxidized LDL in human macrophages.

Concurrent quantification of cellular cholesterol, cholesteryl esters and triglycerides in small biological samples. Reevaluation of thin layer chromatography using laser densitometry.

Absolute specificity and high accuracy is required for the quantitation of cholesterol, cholesteryl esters and triglycerides in small biological samples, particularly in a limited number of cells. Both can be achieved through thin-layer chromatography and molybdatophosphoric acid staining, while the shortcomings of traditional spot detection are overcome by laser densitometry. The major advantage of the proposed technique is the concurrent assay of nanogram quantities of cholesterol, cholesteryl esters and triglycerides. Our assay is at least ten-fold more sensitive than common thin-layer chromatography-based techniques and at least four-fold more sensitive than common enzymatic methods. The present low-cost assay is highly reproducible and may be particularly suitable for the routine lipid analysis of a 10% aliquot of relatively small tissue and cell samples, equivalent, for instance, to > or = 10(4) human monocytes.

Physical partitioning is the main mechanism of alpha-tocopherol and cholesterol transfer between lipoproteins and P388D1 macrophage-like cells.

The regulation of cellular vitamin E concentration was studied in P388D1 macrophage-like cells. Cellular alpha-tocopherol levels increased more than 5000-fold over constitutive levels without reaching saturation when P388D1 cells were cultured in vitamin-E-supplemented fetal calf serum. The uptake of alpha-tocopherol was accompanied by accumulation of alpha-[3H]tocopherol and [14C]cholesterol in these cells. Human unmodified low-density lipoprotein (LDL) inhibited the uptake of alpha-[3H]tocopherol and [14C]cholesterol in a dose-dependent manner and with very similar IC50. Acetylated, Cu2+-oxidized and aggregated human LDL and human very-low-density-lipoprotein (VLDL) were similarly potent, whereas human HDL was at least tenfold less effective than human LDL when inhibitory activity was correlated to lipoprotein protein levels. The rate of vitamin E uptake by P388D1 cells, however, always correlated with the extracellular alpha-tocopherol/cholesterol ratio. Efflux of alpha-[3H]tocopherol from labeled P388D1 cells required extracellular acceptors and was accompanied by the concomitant release of [14C]cholesterol. Both human LDL and HDL could serve as acceptors. Changes in the cellular alpha-tocopherol level appear to be the direct consequence of changes in the extracellular alpha-tocopherol/cholesterol ratio due to a rapid exchange of lipids between P388D1 cells and their extracellular environment. While the transfer of alpha-tocopherol from LDL, VLDL, and fetal calf serum into P388D1 cells appears to occur mainly by diffusion, HDL-stimulated efflux of alpha-tocopherol may underlie a different mechanism. The alpha-tocopherol/cholesterol ratio of the extracellular environment may be a critical factor in determining cellular vitamin E levels in vivo.

Prevention of cholesteryl ester accumulation in P388D1 macrophage-like cells by increased cellular vitamin E depends on species of extracellular cholesterol. Conventional heterologous non-human cell cultures are poor models of human atherosclerotic foam cell formation.

Since the cellular role of the antioxidative vitamins in the formation of foam cells has not yet been studied in detail, we investigated the effect of alpha-tocopherol and ascorbic acid loading of P388D1 macrophage-like cells on their cholesterol and cholesteryl ester levels and their response to the exposure to different lipoproteins. alpha-Tocopherol loading, but not ascorbic acid loading, of P388D1 cells strongly reduced their cellular cholesteryl ester/cholesterol ratio (the crucial indicator of foam cell formation) when fetal calf serum was the only extracellular source of cholesterol. Balance studies suggest that this effect of alpha-tocopherol was mainly due to a reduced uptake of fetal-calf-serum-derived cholesterol. alpha-Tocopherol loading, however, did not reduce the cholesteryl ester/cholesterol ratio when human unmodified low-density lipoprotein (LDL) was added to culture medium containing fetal calf serum. Thus, the uptake of fetal-calf-serum-derived cholesterol was competitively reduced by human LDL, the uptake of which remained unaffected by alpha-tocopherol. Similarly, alpha-tocopherol loading did not prevent cholesteryl ester formation induced by human LDL either oxidized with Cu2+, ultraviolet light or HOCl, or modified by acetylation, aggregation or by malondialdehyde treatment. The present experimental conditions lacked any pro-oxidative burden, since (a) ascorbic acid, either alone or combined with alpha-tocopherol, did not affect cellular cholesteryl ester levels, (b) foam cell formation was not a linear function of the degree of oxidative LDL modification, and (c) alpha-tocopherol lacked specific effects on oxidatively modified LDL. Thus, the reduction of cellular cholesteryl esters by alpha-tocopherol in the absence of human unmodified LDL was hardly due to common antioxidative properties of vitamin E. In conclusion, the present observation that a desirable alpha-tocopherol effect on the cholesteryl ester balance in mouse-tumor-derived P388D1 cells strongly depended on the species of extracellular cholesterol carrier, cautions against premature generalizations of conventional non-human cell culture data.

PAF stimulates cAMP formation in P388D1 macrophage-like cells via the formation and secretion of prostaglandin E2 in an autocrine fashion.

The role of cAMP in the formation of prostaglandin E2 (PGE2) was investigated in bacterial lipopolysaccharide (LPS)-primed P388D1 macrophage-like cells stimulated with platelet activating factor (PAF). cAMP levels and PGE2 secretion were correlated with stimulation by PAF or ionomycin. Indomethacin inhibited cAMP formation induced by PAF, but not PGE2-stimulated cAMP production. Inositol (1,4,5)-trisphosphate levels were strongly reduced by exogenous PGE2 and increased by H-89, an inhibitor of PKA. However, exogenous PGE2 did not affect PAF-stimulated PGE2 formation. These results suggest that cAMP levels in P388D1 cells are regulated by PGE2 in an autocrine fashion. Evidence is presented that this feedback mechanism regulates inositol (1,4,5)-triphosphate levels in these cells, while PGE2 formation is not affected.

Intracellular Ca2+, inositol 1,4,5-trisphosphate and additional signalling in the stimulation by platelet-activating factor of prostaglandin E2 formation in P388D1 macrophage-like cells.

In the P388D1 macrophage-like cell line, phospholipase A2 activity and prostaglandin production are stimulated by platelet-activating factor (PAF) and bacterial lipopolysaccharide (LPS). We have investigated the role of Ins(1,4,5)P3 and Ca2+ in signal transduction of PAF-induced prostaglandin E2 (PGE2) formation in these cells. The role of Ca2+ in the activation mechanism was studied by fluorescence imaging of intracellular Ca2+ in individual adherent cells and by determining the PGE2 production in the same population of cells. This new approach enabled us to correlate directly events on the single-cell level with a physiologically relevant response of the cell population. Priming the cells with LPS was required for PAF to stimulate PGE2 formation, yet LPS affected neither the intracellular free Ca2+ concentration ([Ca2+]i) nor the PAF-induced rise in [Ca2+]i. In addition, basal and PAF-stimulated Ins(1,4,5)P3 levels were not affected by LPS priming. However, the Ca2+ transient, the release of Ins(1,4,5)P3 and the formation of PGE2 induced by PAF were inhibited in cells pretreated with pertussis toxin. Buffering the [Ca2+]i with intracellular BAPTA [bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid] blocked the PAF-stimulated rise in [Ca2+]i and PGE2 formation. Removal of extracellular Ca2+ during PAF stimulation prevented the influx of Ca2+, but did not affect the initial [Ca2+]i transient, nor did it inhibit PGE2 formation. Under the same conditions, ionomycin stimulated an identical [Ca2+]i transient, but, in contrast with PAF stimulation, no PGE2 formation was observed. PGE2 production could be rescued by prompt subsequent addition of PAF, which caused no further [Ca2+]i change on its own. These results show that the transient initial rise in [Ca2+]i, produced either by PAF via the formation of Ins(1,4,5)P3 or directly by ionomycin, is necessary, but not sufficient for the formation of PGE2 in LPS-primed P388D1 cells. Furthermore, we have demonstrated for the first time that PAF triggers a second signal that is not mediated by a change in [Ca2+]i. However, both signals are required to induce PGE2 formation.

Bacterial lipopolysaccharide priming of P388D1 macrophage-like cells for enhanced arachidonic acid metabolism. Platelet-activating factor receptor activation and regulation of phospholipase A2.

P388D1 cells are stimulated by platelet-activating factor (PAF) to release arachidonic acid metabolites (Lister, M. D., Glaser, K. B., Ulevitch, R. J., and Dennis, E. A. (1989) J. Biol. Chem. 264, 8520-8528). While the release of prostaglandin E2 (PGE2) in response to PAF is only two to three times the constitutive PGE2 production, bacterial lipopolysaccharides (LPS) are able to prime P388D1 cells for enhanced arachidonic metabolism, increasing PAF-stimulating PGE2 production to 9-12 times the constitutive PGE2 production. The extent and rate of [3H]arachidonic acid release from prelabeled P388D1 cells are also increased in primed cells relative to unprimed cells in response to PAF-stimulation. LPS from either Salmonella Re595 or Escherichia coli 0111:B4 prime P388D1 cells in a concentration-dependent manner but have themselves no ability to stimulate arachidonic acid metabolism. LPS priming is sensitive to inhibition by actinomycin D, while primed PAF-stimulation of PGE2 production is blocked by cyclohexamide which implicates a protein which is rapidly turning over. Primed PAF stimulation is also inhibited by the phospholipase A2 inhibitor manoalogue and the tyrosine-specific protein kinase inhibitor genistein, but not by the kinase inhibitor H-7. These results suggest that priming amplifies signal transduction pathways for PAF, which results in increased arachidonate availability. The multiple levels at which primed PAF-stimulated PGE2 production appears to be regulated are discussed.

Calcium-ionophore-induced formation of platelet-activating factor and leukotrienes by horse eosinophils: a comparative study.

Horse eosinophils preincubated with 3H-labelled acetate and stimulated with the Ca2+ ionophores ionomycin or A23187 form a radioactive compound, which we have shown to be 1-O-alkyl-2-[3H]acetyl-sn-glycero-3-phosphocholine (platelet-activating factor). We could detect no 1-O-acyl-2-[3H]acetyl-glycero-3-phosphocholine in the radioactive fraction. The formation of platelet-activating factor was strongly correlated to the generation of leukotriene C4, the main arachidonate metabolite in horse eosinophils, suggesting that platelet-activating factor and leukotriene C4 have a common precursor pool (1-O-alkyl-2-arachidonyl-glycero-3-phosphocholine) and a common regulation of synthesis. Even though both ionomycin and A23187 are described as Ca2+ ionophores, they have a series of significantly different effects on the eosinophils with respect to formation of platelet-activating factor and leukotriene C4. While A23187 induces an asymptotic maximum of mediator formation at concentrations higher than 20 microM, ionomycin expressed a narrow optimum at 2 microM. The effects of exogenous pH on the release of mediators also differ strongly between the two ionophores: for A23187 the effects are the same for both mediators but when ionomycin is used as stimulant, generation of platelet-activating factor and leukotriene C4 are affected differently.