Induction of suppression by a murine nonspecific suppressor-inducer cell line (M1-A5)

A murine nonspecific suppressor-inducer cell line (M1-A5) was established by the limiting dilution method from the spleen cells of a mouse bearing an advanced methylcholanthrene-induced fibrosarcoma. Indirect immunofluorescence studies demonstrated that the M1-A5 cells were Thy-1-, sIg-, Ly-5+, MAC-, and 45% asialo GM1+. The M1-A5 cells were able to activate suppressor cells from unprimed, syngeneic normal spleen cells. These activated cells inhibited antibody production by cocultured syngeneic lymphoid cells. Induction of suppression by the M1-A5 cells was via the release of a suppressor-inducing factor, which was found to be protein in nature. Kinetic studies showed that when M1-A5 cells were separated from NSC by a dialysis tubing in Marbrook vessels, the M1-A5 cells required a minimum of 8 hr incubation period before suppressor cell activity could be demonstrated in precursor cells. On the other hand, induction of suppression by the suppressor-inducing factor required a minimum of 3 hr exposure of the precursor cells to the factor.     

Interrelationships among prostaglandins, vasopressin and cAMP in renal papillary collecting tubule cells in culture

To determine the influence of prostaglandins on cAMP metabolism in renal papillary collecting tubule (RPCT) cells, intracellular cAMP levels were measured after incubating cells with prostaglandins (PGs) alone or in combination with arginine vasopressin (AVP). PGE1, PGE2 and PGI2, but not PGD2 or PGF2 alpha, increased intracellular cAMP concentrations. At maximal concentrations (10(-5) M) the effects of PGE2 plus PGI2 (or PGE1), but not of PGI2 plus PGE1, were additive suggesting that at least two different PG receptors may be present in RPCT cell populations. Bradykinin treatment of RPCT cells caused an accumulation of intracellular cAMP which was blocked by aspirin and was quantitatively similar to that observed with 10(-5) M PGE2. PGs, when tested at concentrations (e.g. 10(-9) M) which had no independent effect on intracellular cAMP levels, did not inhibit the AVP-induced accumulation of intracellular cAMP in RPCT cells. These results indicate that PGs do not block AVP-induced accumulation of intracellular cAMP in RPCT cells at concentrations of PGs which have been shown to inhibit the hydroosmotic effect of AVP on perfused collecting tubule segments. However, at higher concentrations of PGs (e.g. 10(-5) M), the effects of AVP plus PGE1, PGE2, PGI2 or bradykinin on intracellular cAMP levels were not additive. Thus, under certain conditions, there is an interaction between PGs and AVP at the level of cAMP metabolism in RPCT cells.     

Regulation of prostaglandin production by osteoblast-rich calvarial cells

The effect of various factors upon prostaglandin (PG) production by the osteoblast was examined using osteoblast-rich populations of cells prepared from newborn rat calvaria. Bradykinin and serum, and to a lesser extent, thrombin, were all shown to stimulate PGE2 and 6-keto-PGF1 alpha (the hydration product of PGI2) secretion by the osteoblastic cells. Several inhibitors of prostanoid synthesis, dexamethasone, indomethacin, dazoxiben and nafazatrom, were tested for their effects on the calvarial cells. All inhibited PGE2 and PGI2 (the major arachidonic acid metabolites of these cells) production with half-maximal inhibition by all four substances occurring at approximately 10(-7) M. For dazoxiben and nafazatrom, this was in contrast to published results from experiments in vivo which have indicated that the compounds stimulated PGI2 production. Finally, since the osteoblast is responsive to bone-resorbing hormones, these were tested. Only epidermal growth factor (EGF) was shown to modify PG production. At early times EGF stimulated PGE2 release, however, the predominant effect of the growth factor was an inhibition of both PGE2 and PGI2 production by the osteoblastic cells. The present results suggest that the bone-resorbing hormones do not act to cause an increase in PG by the osteoblast and that any increase in PG production by these cells may be in response to vascular agents.     

Augmentation of cytotoxic responses by prostaglandin E2

The cytotoxic-T-lymphocyte activity in the spleen cells after in vivo immunization of C3H mice with allogeneic spleen cells ip was consistently very weak. Substantial cytotoxic responses were obtained, however, when prostaglandins (PGE2, PGE1, or PGI2) were injected ip together with or prior to the immunization. An augmentation of cytotoxic responses against allogeneic stimulator cells was also observed in mixed lymphocyte cultures which were provided with an interleukin 2-containing helper factor. This augmentation was observed when PGE2 was added at the start of the culture but not if added 1 day later. Indomethacin was found to be suppressive in these cultures.     

CREB-dependent cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression is mediated by protein kinase C and calcium

Cellular production of prostaglandins (PGs) is controlled by the concerted actions of cyclooxygenases (COX) and terminal PG synthases on arachidonic acid in response to agonist stimulation. Recently, we showed in an ileal epithelial cell line (IEC-18), angiotensin II-induced COX-2-dependent PGI2 production through p38MAPK, and calcium mobilization (J. Biol. Chem. 280: 1582-1593, 2005). Agonist binding to the AT1 receptor results in activation of PKC activity and Ca2+ signaling but it is unclear how each pathway contributes to PG production. IEC-18 cells were stimulated with either phorbol-12,13-dibutyrate (PDB), thapsigargin (TG), or in combination. The PG production and COX-2 and PG synthase expression were measured. Surprisingly, PDB and TG produced PGE2 but not PGI2. This corresponded to induction of COX-2 and mPGES-1 mRNA and protein. PGIS mRNA and protein levels did not change. Activation of PKC by PDB resulted in the activation of ERK1/2, JNK, and CREB whereas activation of Ca2+ signaling by TG resulted in the delayed activation of ERK1/2. The combined effect of PKC and Ca2+ signaling were prolonged COX-2 and mPGES-1 mRNA and protein expression. Inhibition of PKC activity, MEK activity, or Ca2+ signaling blocked agonist induction of COX-2 and mPGES-1. Expression of a dominant negative CREB (S133A) blocked PDB/TG-dependent induction of both COX-2 and mPGES-1 promoters. Decreased CREB expression by siRNA blocked PDB/TG-dependent expression of COX-2 and mPGES-1 mRNA. These findings demonstrate a coordinated induction of COX-2 and mPGES-1 by PDB/TG that proceeds through PKC/ERK and Ca2+ signaling cascades, resulting in increased PGE2 production.     

Secretion of a suppressor cell inducing factor by an interleukin-3 dependent cell line with natural cytotoxic activity

This report describes the morphology, surface markers, growth requirements, and functional activity of the M1-A5 cell line, which was established by the limiting dilution of spleen cells from a mouse bearing a large methylcholanthrene-induced fibrosarcoma. The M1-A5 cells share many of the morphological features of large granular lymphocytes and, in addition, express asialo GM1 and Ly-5 surface markers which are commonly found on natural killer cells (NK) cells. There is no expression of T-cell differentiation antigens, surface immunoglobulin, or the granulocyte/macrophage marker, MAC-1. M1-A5 cells are dependent on exogenous growth factor(s) for survival and will proliferate if cultured in interleukin 3 (IL-3), but not in interleukin 1 (IL-1), interleukin 2 (IL-2), or granulocyte/macrophage colony stimulating factor (GM-CSF). In addition, the M1-A5 cells do not absorb IL-2. Despite their morphology and surface characteristics, the M1-A5 cells do not lyse NK targets such as YAC-1 and RLM1 in 4- or 18-hr cytotoxic assays but do lyse the natural cytotoxic (NC) susceptible target, WEHI-164, and to a very small extent, the M-1 fibrosarcoma cells, in an 18-hr assay. Thus they exhibit NC-like cytotoxic activity. In addition, the M1-A5 cells secrete a small molecular weight factor which activates suppressor cells capable of inhibiting antibody synthesis by cocultured syngeneic spleen cells.     

Distinctive effect of angiotensin II on prostaglandin production in dog renal and femoral arteries

The effect of angiotensin II (Ang II) on prostaglandin (PG) production in dog renal and femoral vasculature was examined in vivo and in vitro. In pentobarbital anesthetized dogs, the reduction of blood flow induced by intra-arterial infusion of Ang II was potentiated by pre-treatment with indomethacin (5 mg/kg) in the renal but not the femoral vasculature. Isolated renal and femoral arterial strips were incubated and the release of PGE2 and PGI2 (as 6-keto-PGF1 alpha) into the medium was measured by radioimmunoassay. Basal PGE2 and PGI2 production by renal and femoral arterial strips was approximately the same. PGI2 production was predominant for both strips. Ang II stimulated PG production in renal but not femoral arteries. In the renal artery, Ang II-induced PG production was inhibited by indomethacin (10(-6) M), mepacrine (10(-4) M) and saralasin (10(-6) M). These results suggest that Ang II stimulates PG production by the renal artery per se and the Ang II receptor is linked to phospholipase A2 in the renal but not the femoral artery.     

Stimulation of prostaglandin E2 synthesis in cloned osteoblastic cells of mouse (MC3T3-E1) by epidermal growth factor

Prostaglandin (PG) E2, known as a bone-resorption factor, was released as a predominant arachidonate metabolite in the culture medium of an osteoblastic cell line cloned from mouse calvaria (MC3T3-E1). Epidermal growth factor (EGF) (10 ng/ml) prominently enhanced endogenous PGE2 synthesis, requiring the simultaneous presence of unidentified factor(s) contained in bovine serum. PGE2 synthesis increased after a lag phase for 1-2 h and reached a maximum level at about 3 h after EGF addition. EGF-stimulated PGE2 synthesis was almost completely blocked by 10 microM cycloheximide or 1 microM actinomycin D. Furthermore, when the cells were pretreated with EGF, the microsomes exhibited an increased activity of fatty acid cyclooxygenase (arachidonic acid----PGH2), whereas the activity of PGE synthase (PGH2----PGE2) remained unchanged. These results suggested an EGF-mediated induction of cyclooxygenase. Following increased PGE2 synthesis, DNA synthesis increased and alkaline phosphatase activity decreased in a slower response to EGF. PGE2 (above 0.1 microM) added to the cells could replace EGF. However, such effects of EGF on the osteoblasts could not be attributed totally to an autocrine function of PGE2 produced by stimulation with EGF because these effects of EGF were not abolished by indomethacin, which blocked the PGE2 synthesis.     

Formation of prostaglandins by ovarian carcinomas

Tissue contents of prostaglandins (PG) PGE2, PGE2a and 6-keto-PGF1a (degradation product of PGI2) were determined in specimens of advanced human ovarian cancer (n = 11). The PG levels (ng/mg tissue protein) varied widley: PGE2 17-515; PGF2a 2-43 and 6-keto-PGF1a 5-105. Tumors of patients without response to chemotherapy contained more PGE2, PGF2a and 6-keto-PGF1a than did tumors responding to chemotherapy. PG production was investigated in two ovarian carcinoma-derived cell lines. The ability of these cells to synthesize PG varied depending on the cell density. An increase of cell number was associated with a decrease of PG yield. PG formation was inhibited by indomethacin in a concentration-dependent manner. The present study suggests that ovarian carcinoma cells form PG in vivo and vitro.     

Epidermal growth factor (EGF) and tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate PG synthesis and thymidine incorporation in cultured porcine thyroid cells

This is the first report to show that epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulate the production of PGE2 and 6-keto PGF1 alpha, an end metabolite of PGI2, in the thyroid gland. In cultured porcine thyroid cells, EGF and TPA stimulate PGE2 and 6-keto PGF1 alpha production; the maximum PG levels were obtained after 3-4 h incubation with EGF or TPA; the addition of as little as 10(-11) M EGF or 5 X 10(-11) M TPA resulted in increases in PGE2 and 6-keto PGF1 alpha, and the maximum levels were obtained with 10(-8)-10(-7) M EGF or TPA. This report also shows that EGF and TPA stimulate [3H] thymidine incorporation.     

Prostaglandin E1 transported into cells blocks the apoptotic signals induced by nerve growth factor deprivation

Neuronal apoptosis in rat pheochromocytoma PC12 cells, which was confirmed by TUNEL (terminal transferase-mediated dUTP-biotin nick end-labeling) staining and detection of chromatin condensation, appeared within 8 h after nerve growth factor (NGF) deprivation. Prostaglandin (PG) E1 (10(-7)-10(6) M) reduced the incidence of apoptotic cell death in PC12 cells. The genes encoding PG transporter specific to prostaglandins such as PGE2 or PGF2alpha were expressed in the cell lines as shown by RT-PCR. Bromcresol green, an inhibitor of PG transporter, reversed the antiapoptotic effect of PGE1. Moreover, treatment of PC12 cells with an antisense oligonucleotide corresponding to PG transporter cDNA also blocked the inhibitory effects of PGE1 on apoptotic cell death. In addition, PGE1 counteracted the increased activities of stress-activated protein kinase/cJun N-terminal kinase within 1-2 h after NGF deprivation in PC12 cells. These results indicated that the antiapoptotic effect of PGE1 in NGF-deprived PC12 cells was achieved by inhibitory signals following uptake into neurons through the PG transporter.     

Direct evidence for the presence of selective binding sites for (3H) prostaglandin E2 on rat peritoneal macrophages

A method is presented which provides for a simple and rapid determination of PGE2 receptors on viable peritoneal macrophages. Incubation of the harvested cells with (3H)PGE2 revealed specific binding of (3H)PGE2 by use of the Millipore filter assay system. Maximum binding was attained in the presence of 1 mM EDTA. Specific binding was saturable at 65 fmol/mg protein with an equilibrium dissociation constant (Kd) of 3.2 X 10(-8)M. Inhibition of (3H)PGE2 binding with unlabelled prostaglandins revealed a potency series of PGE2 greater than PGE2 greater than PGI2. The PGE2 concentration which displaced 50% of the labelled ligand was 10(-7)M. Comparable kinetic data were obtained for adenylate cyclase stimulation, since the concentration which showed a halfmaximal stimulation of cAMP production was 2 X 10(-7)M of PGE2. Since PGE1 and PGI2 compete with (3H)PGE2 binding in a non-parallel manner compared to PGE2 itself, it is proposed that macrophages possess different types of PG receptors.     

Metabolic activity of cholesteryl esters in aortic smooth muscle cells is altered by prostaglandins I2 and E2

Among the biochemical processes associated with the atherogenic process are increased aortic cholesteryl ester (CE) accumulation and altered prostaglandin (PG) production. The precise physiological role of PG, particularly prostacyclin (PGI2), in the control of CE metabolism in intact aortic smooth muscle cells remains to be fully elucidated. We report here that cytosolic neutral cholesteryl ester hydrolytic activity (NCEH) in intact cultured aortic smooth muscle cells is significantly increased by 75-250 nM PGI2 at the end of a 2-hr incubation period. The effect was mediated by increased intracellular cAMP levels since the effect of PGI2 on NCEH activity was abolished in the presence of an inhibitor of adenylate cyclase activity, viz., dideoxyadenosine (DDA0. Although the addition of 20-100 microM dibutyryl cAMP (Bt2cAMP) and 50-100 microM sodium arachidonate also increased NCEH activity twofold, 6-keto PGF1 alpha, PGE1, and PGE2 did not increase the activity of this enzyme. In contrast to these findings, 75-250 nM PGE2 significantly inhibited CE synthetic activity (ACAT) approximately 60%. Arachidonate or Bt2cAMP did not affect ACAT activity. This decrease in ACAT activity induced by PGE2 does not appear to be mediated by cAMP. Taken together, these findings suggest that PGI2, a well known potent vasodilator and inhibitor of platelet aggregation, and PGE2 may have an important regulatory role in aortic CE metabolism.     

Endothelin-induced prostacyclin production in rat aortic endothelial cells is mediated by protein kinase C.

Endothelin (ET) is a vasoconstrictor peptide released from endothelial cells that is known to cause prostaglandin (PG) release. The mechanism remains unclear. To determine whether the protein kinase C (PKC) signaling pathway is stimulated by endothelin, we pretreated rat aortic endothelial cells with either PKC activator or inhibitors and measured the release of prostacyclin (PGI2) by radioimmunoassay. ET (10(-9) M) produced a 10-fold increase in PGI2 release. Pretreatment with 10(-9) M of three different PKC inhibitors: 1-(5-isoquinolinesulfonyl) piperazine (CL), staurosporine, and 1-(5-isoquinolinesulfonyl-methyl) piperazine (H7) blocked ET induced PGI2 release. ET induced prostacyclin release was also blocked by pretreatment with inhibitors of either phospholipase A2 (7,7,dimethyleicosadienoic acid or trifluoromethyl ketone analogue) (10(-9) M) or cyclooxygenase (indomethacin) (10(-9) M). We conclude that ET activates PKC which activates phospholipase A2 which liberates arachidonic acid which increases PGI2 production and release.     

Human follicular dendritic cells interact with T cells via expression and regulation of cyclooxygenases and prostaglandin E and I synthases

PGE(2) inhibits mature T cell proliferation and protects T cells from activation-induced cell death (AICD). We have previously demonstrated that human follicular dendritic cells (FDC) strongly express PGI synthase. In this study, the hypothesis that FDC have regulatory roles on germinal center T cells by controlling production of PGE(2) and PGI(2) was tested. Confocal microscopic analyses of human tonsil tissues revealed that FDC indeed expressed PGE synthase in addition to PGIS. To confirm these results, we studied the regulation mechanism of PG production in FDC, using an established human FDC-like cell line, HK. Specifically in response to TNF-alpha, TGF-beta, and LPS, protein expression of cyclooxygenase (COX)-2 and downstream PGE synthase was up-regulated with coordinate kinetics, whereas COX-1 and PGIS were constitutively expressed. The increase of these enzymes was reflected in actual production of PGE(2) and PGI(2). Interestingly, IL-4 almost completely abrogated the stimulatory activity of TNF-alpha, TGF-beta, and LPS in PG production. Furthermore, the up-regulation of PGE(2) and PGI(2) production was markedly down-regulated by indomethacin and a selective COX-2 inhibitor. PGI(2) analog and PGE(2) inhibited proliferation and AICD of T cells in dose- and time-dependent manners. Finally, coculture experiments revealed that HK cells indeed inhibit proliferation and AICD of T cells. Put together, these results show an unrecognized pathway of FDC and T cell interactions and differential mechanisms for PGE(2) and PGI(2) production, suggesting an important implication for development and use of anti-inflammatory drugs.     

Endogenous synthesis of prostaglandins E1 and I2 in 3T3 fibroblasts transformed by polyoma virus. Effects on adenosine 3':5'-monophosphate production

Prostaglandins (PG)E1, E2 and I2 were produced by polyoma virus transformed (py) 3T3 fibroblasts. The levels of PGE1, PGE2 and 6-keto-PGF1 alpha (degradation product of PGI2) were 22.7, 225 and 33.2 ng/ml medium, respectively, 72 h after medium change. The stimulatory potencies of exogenous PGE1, PGE2 and PGI2 on adenosine 3':5'-monophosphate (cyclic AMP) formation were similar. Therefore, the prostaglandin mediated increase in cyclic AMP levels observed during growth of these cells (Claesson, H.-E., Lindgren, J.A. and Hammarstr?m, S. (1977) Eur. J. Biochem. 74, 13) is largely (greater than 80%) mediated by PGE2 and to lesser extents by PGE1 and PGI2.     

Prostaglandin synthesis by the cochlea of the guinea pig. Influence of aspirin, gentamicin, and acoustic stimulation

This study describes the synthesis of prostaglandins (PGs) by the vascular structures of the inner ear (lateral wall = stria vascularis and spiral ligament) in vitro. The main PGs produced were PGI2, PGF2 alpha and PGE2. PGI2 and PGF2 alpha were also found in the perilymph. A 350 mg/kg ip injection of aspirin decreased PG synthesis by the lateral wall and PG levels in perilymph. This effect was reversed after 3 days. Gentamicin (10(-9) to 10(-5) M) decreased significantly and reversibly PG synthesis in vitro, as did 100 mg/kg ip injection. Acoustic stimulation increased ex vivo PGI2 and PGE2 synthesis without modifying PG levels in perilymph. Results suggest that PGs could be one humoral mediator of the cochlear microcirculation homeostasis, and, possibly, of the circulatory disturbances reported after acoustic stimulation. The decreased PG synthesis after gentamicin treatment could account for the angiotoxic component observed in aminoglycoside ototoxicity.     

Role of COX-1 and COX-2 on skin PGs biosynthesis by mechanical scratching in mice

We examined the involvement of cyclooxygenase (COX)-1 and COX-2 on mechanical scratching-induced prostaglandins (PGs) production in the skin of mice. The dorsal regions of mice were scratched using a stainless brush. COXs expressions in the skin were analyzed using real-time PCR and Western blotting. The effect of acetylsalicylic acid (ASA) on the ability of PGs production were determined based on skin PGs level induced by arachidonic acid (AA) application. Mechanical scratching increased PGD2, PGE2, PGI2 and PGF(2 alpha). COX-1 was constitutively expressed and COX-2 expression was enhanced by scratching. Intravenous administration of ASA inhibited PGs biosynthesis in the normal skin. PGs levels of the skin 6h after ASA administration (ASA 6 h) were almost equal to those of the skin 10 min after ASA administration (ASA 10 min). In the scratched skin, AA-induced PGE2 and PGI2 of ASA 6 h were significantly higher than those of ASA 10 min. The skin PGD2 and PGF(2 alpha) of ASA 10 min were almost same to those of ASA 6 h. In the normal skin of COX-1-deficient mice, skin PGD2 level was lower than that of wild-type mice, although PGE2, PGI2 and PGF(2 alpha) levels were almost equal to those of wild type. In the scratched skin of COX-1-deficient mice, PGD2, PGE2, PGI2 and PGF(2 alpha) levels were lower than those of wild-type mice. These results suggested that cutaneous PGD2 could be mainly produced by COX-1, and PGE2 and PGI2 could be produced by COX-1 and COX-2, respectively, in mice.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to 3 x 10(-8) M--3 x 10(-4) M of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), prostacyclin (PGI2), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE2, PGF2 alpha, PGI2, and AA. Although preincubation of dispersed adrenal cells with indomethacin (3 x 10(-5) M) markedly inhibited PGE2 synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.