Expression of GTP-binding proteins and prostaglandin E2 receptors during human T cell activation.

GTP-binding proteins (G-proteins) are a family of closely related, yet structurally distinct signal transducing proteins. In this study the presence and relative abundance of several G-proteins and of their corresponding mRNAs were measured in resting and activated human T lymphocytes. We found that T lymphocytes contain RNA coding for Gs, Gi2, and Gi3. No Gi1- and Go-specific RNA could be detected. Membrane fractions of resting and activated lymphocytes were studied in immunoblot experiments. Again, Gs, Gi2, and Gi3, but not Gi1 and Go, were detected. Upon mitogenic activation, a relative increase in mRNA for Gs and Gi3, but not for Gi2 could be demonstrated in Northern blot experiments. Immunoblotting indicated an increase in Gs and Gi3 density in membrane fractions of T cells as well. Paralleling the increase in Gs, we found that activated T cells produce five to seven times more cAMP per cell in response to prostaglandin E2 (PGE2) than resting lymphocytes. Finally, PGE2 binding studies showed that the number of receptors for this hormone increased from 435 +/- 322 to 1035 +/- 357 per cell following in vitro stimulation. We propose that in vitro T cell activation is paralleled by an increase in sensitivity to PGE2-induced cAMP generation. This sensitization is accompanied by both an increase in cell surface PGE2 receptor numbers as well as by increased expression of the signal transducing protein Gs and may physiologically be important for limiting an immune response.     

Interleukin 2 modulation of adenylate cyclase. Potential role of protein kinase C

Interleukin 2 (IL 2) stimulated DNA synthesis of murine T lymphocytes (CT6) in a concentration-dependent manner, over a range of 1-1000 units/ml. This proliferative effect of IL 2 was attenuated by simultaneous exposure to prostaglandin E2 (PGE)2. In intact cells, IL 2 inhibited both basal and PGE2-stimulated cAMP production; the amount of cAMP generated was dependent upon the relative concentrations of IL 2 and PGE2. The effect of IL 2 on CT6 cell proliferation and cAMP production was mimicked by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which, like IL 2, causes a translocation and activation of protein kinase C. While PGE2 stimulated adenylate cyclase activity in membrane preparations, neither IL 2 nor TPA inhibited either basal or stimulated membrane adenylate cyclase activity. However, when CT6 cells were pretreated with IL 2 or TPA and membranes incubated with calcium and ATP, both basal and PGE2-and NaF-stimulated membrane adenylate cyclase activity was inhibited. This inhibition of adenylate cyclase activity was also observed if membranes from untreated cells were incubated with protein kinase C purified from CT6 lymphocytes in the presence of calcium and ATP. The data suggest that the decreased cAMP production which accompanies CT6 cell proliferation results from an inhibition of adenylate cyclase activity mediated by protein kinase C and that these two distinct protein phosphorylating systems interact to modulate the physiological response to IL 2.     

Cell cycle-dependent inhibition of human vascular smooth muscle cell proliferation by prostaglandin E1

We examined the influence of prostaglandins on the initiation of proliferation of growth-arrested human adult aortic and fetal smooth muscle cells. Prostaglandins of the E series (25 nM) exerted a significant (p less than or equal to 0.05) inhibitory effect on DNA synthesis. Inhibition was observed when PGE1 was added in the G1 phase of the cell cycle. PGE1 had no effect when added once DNA synthesis had started. Thus prostaglandins of the E series may inhibit the responsiveness of smooth muscle cells to the mitogenic action of critical growth factors, such as PGDF. This inhibitory response is cell-cycle dependent. Once smooth muscle cells have entered S phase, PGE1 is no longer effective. Our data also suggest that cAMP is involved in the PGE1-induced growth inhibition, since concomitant with PGE1 addition, cAMP levels rose rapidly; addition of the cAMP analogue db-cAMP resulted in a cell-cycle-dependent inhibition pattern comparable to that observed with PGE1.     

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E2 (PGE2) on fibroblast proliferation was examined. The presence of PGE2 for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [3H]thymidine occurred at concentrations greater than 10(-7) M. The inhibitory effect of PGE2 was less potent in exponentially growing cells and was not the result of conversion of PGE2 to PGA2 during incubation in growth medium. The G1 phase was determined to be 12-14 h in untreated cultures. The extent of growth inhibition by PGE2 was similar with addition of PGE2 at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE2-induced growth inhibition. Short-term exposure to PGE2 (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE2 affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE2 for 24 h, indicated that release from PGE2 exposure is associated with prolongation of the G1 phase of the cell cycle.     

Cyclic adenosine 3'5' monophosphate stimulates prostaglandin E production by human adherent synovial cells

Production of prostaglandin E (PGE) by rheumatoid synovium appears important to regulation of the pathologic process in rheumatoid arthritis. Cells derived from human synovium by proteolytic digestion produce large amounts of PGE which in turn can elevate synovial cell cAMP levels and inhibit cell proliferation. Data presented here indicate that cAMP can further increase production of PGE from adherent synovial cells (ASC). PGE production occurs over 12-72 hr and is not due to the ability of cAMP to inhibit cell proliferation. Exposure of cells to cAMP results in increased release of 3H arachidonic acid from precursors but not in activation of the cyclooxygenase enzyme. This phenomenon suggests the presence in adherent synovial cells of a mechanism for amplifying PGE production.     

Prostaglandin E2 inhibits the activation of cloned T cell hybridomas

To minimize complicating interactions inherent in heterogeneous cell populations, we used a panel of cloned murine autoreactive (E8.A1) and antigen-specific (HEL.C10, HEL.B14) T cell hybridomas to examine the effect of prostaglandin E2 (PGE2) on T cell activation. These T cells secrete interleukin 2 (IL 2) when co-cultured with a cloned population of I region-matched stimulator cells (TA3), or with mitogenic signals in the absence of TA3 stimulator cells. Physiologic concentrations of PGE2 inhibited the induction of IL 2 secretion by the T cell hybridomas tested, when they were activated either by TA3 cells or by mitogenic signals. IL 2 production was inhibited in a dose-dependent manner by concentrations of PGE2 between 10(-7) and 10(-11) M, with 50% inhibition occurring at 10(-10) M. Pretreatment of the T hybridoma cells with 10(-7) M PGE2 for 1 hr before culture also resulted in marked inhibition of IL 2 secretion. Similar pretreatment of the TA3 cells did not affect their ability to activate the T cell hybridomas. PGE2 at 10(-8) M induced a 30-fold increase in cAMP levels within 25 min of addition to culture of the E8.A1 T cell hybridoma, but caused no significant elevation of cAMP levels in TA3 cells. The direct addition of dibutyryl cAMP (dcAMP) to cultures of E8.A1 cells resulted in marked inhibition of IL 2 secretion when stimulated by TA3 or by mitogenic signals, with an average of 80% inhibition occurring at 10(-4) M dcAMP. PGE2 and dcAMP also inhibited the growth of E8.A1 cells. Initially, cell growth was virtually halted, but began to recover between 24 and 48 hr after the addition of either PGE2 or dcAMP. Neither PGE2 nor dcAMP inhibited the division of TA3 cells. High affinity binding sites for PGE2 were detected in the E8.A1 T cell hybridomas with an apparent Kd of 7.6 X 10(-10) M, which is consistent with the functional data. No specific binding was detected in the TA3 stimulator cells. These findings suggest that the immunosuppressive effects of PGE2 are localized to the T cell, are receptor regulated, and may be mediated by the associated increase of cAMP levels in the T cell hybridomas.     

17beta-estradiol modulates prostaglandin E2 release from human amnion-derived wish cells

In human amnion-derived WISH cells [(3)H]estradiol-17beta binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17beta-estradiol stimulates prostaglandin (PG)E(2) release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17beta-estradiol modulates PGE(2) release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17beta-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE(2) release observed in the presence of Ro 20-1724.     

Prostaglandin E2 acts at two distinct pathways of T lymphocyte activation: inhibition of interleukin 2 production and down-regulation of transferrin receptor expression

The mechanism by which prostaglandin E2 (PGE2) inhibits human T lymphocyte activation and proliferation was studied. We analyzed the effect of physiologic concentrations of PGE2 on interleukin 2 (IL 2) production, expression of IL 2 receptor (Tac antigen), and expression of the transferrin receptor after in vitro activation with phytohemagglutinin. PGE2 inhibited T lymphocyte proliferation by 80 to 90% of control values. This was associated with a similar degree of inhibition of IL 2 production while the expression of IL 2 receptor was not affected. This was in marked contrast to the expression of the transferrin receptor, which was inhibited 65% after 72 hr of in vitro activation. The addition of exogenous, purified IL 2 reconstituted lymphocyte proliferation to 50% of control values, but had no effect on transferrin receptor expression. Because PGE2 is known to increase the intracellular concentration of 3',5' cyclic adenosine monophosphate (cAMP), we investigated the effect of another adenylate cyclase activator, i.e., isoproterenol, as well as the effect of extracellular administration of the cAMP derivative dibutyryl cAMP (dBcAMP) on IL 2 production, Tac antigen expression, and transferrin receptor expression. It was demonstrated that isoproterenol, as well as dBcAMP, inhibited transferrin receptor expression on PHA-activated T lymphocytes to the same extent as PGE2, and exogenous IL 2 could not counteract the down-regulation of the receptor expression. In contrast, neither isoproterenol nor dBcAMP had any significant effect on IL 2 receptor expression. Prostaglandin F2 alpha (PGF2 alpha), which has been reported to elevate intracellular cyclic GMP levels, had no effect on lymphocyte activation and proliferation, and did not counteract the PGE2-induced depression in IL 2 production. In contrast to its effect on peripheral blood lymphocytes, PGE2 had no effect on transferrin receptor expression or cell proliferation by IL 2-dependent T cell clones and IL 2-independent T cell lines. These studies demonstrate that PGE2 exerts its inhibitory effects on T cell activation and proliferation via two distinct pathways: inhibition of IL 2 production and inhibition of transferrin receptor expression. The transferrin receptor inhibition is mediated via the cAMP pathway and is IL 2-independent.     

Cyclic adenosine 3′5′ monophosphate stimulates prostaglandin E production by human adherent synovial cells

Production of prostaglandin E (PGE) by rheumatoid synovium appears important to regulation of the pathologic process in rheumatoid arthritis. Cells derived from human synovium by proteolytic digestion produce large amounts of PGE which in turn can elevate synovial cell cAMP levels and inhibit cell proliferation. Data presented here indicate that cAMP can further increase production of PGE from adherent synovial cells (ASC). PGE production occurs over 12–72 hr and is not due to the ability of cAMP to inhibit cell proliferation. Exposure of cells to cAMP results in increased release of ³H arachidonic acid from precursors but not in activation of the cyclooxygenase enzyme. This phenomenon suggests that presence in adherent synovial cells of a mechanisms for amplifying PGE production.     

Role of protein kinase C in cyclic AMP-mediated suppression of T-lymphocyte activation following burn injury.

Major burn injury induces T-lymphocyte dysfunction. Previous studies suggest that prostaglandin (PG) E2, which is elevated post-burn, is the causative factor via a cyclic AMP-dependent process. The present study was conducted to elucidate the mechanism by which cAMP induces T-lymphocyte dysfunction following burn injury. Splenocytes were isolated from mice 7 days after receiving a scald burn covering 25% of their total body surface or sham procedure. ConA-induced proliferation by splenocytes from burned mice was significantly suppressed. Macrophage depletion of the splenocyte cultures abrogated the suppression. Concanavalin A-stimulated proliferation by macrophage-depleted splenocytes was suppressed by PGE2 and dibutyryl cAMP in both groups. The IC50 of these cAMP-elevating agents, however, was approximately 100-fold lower for cells from burned mice, indicating an increased sensitivity to cAMP. PGE2 did not suppress PMA/Ca2+ ionophore-induced T-lymphocyte activation. Addition of PMA to ConA-stimulated cultures prevented the suppression of proliferative responses by PGE2, whereas Ca2+ ionophore had no effect. Thus, the suppression of T-lymphocyte activation following burn injury is macrophage-dependent, related to an increased sensitivity to cAMP and due to an uncoupling of cell surface receptors from protein kinase C activation.     

Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

In the presence of ouabain, prostaglandin (PG) E2 stimulated a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells. PGE2 or ouabain alone evoked a marginal secretory response. The synergism of ouabain was also observed with muscarine. PGE2, like muscarine, induced a concentration-dependent formation of inositol phosphates: rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of inositol monophosphate. This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. The potency of PGs (PGE2 greater than PGF2 alpha greater than PGD2) to stimulate catecholamine release was well correlated with that to affect phosphoinositide metabolism and that to increase the level of intracellular Ca2+. PGE2 did not stimulate cAMP generation significantly in bovine chromaffin cells. The effect of PGE2 on catecholamine release was mimicked by 12-O-tetradecanoylphorbol 13-acetate and A23187, but not by the cAMP analogue dibutyryl cAMP nor by forskolin. These results indicate that PGE2 may enhance catecholamine release from chromaffin cells by activating protein kinase C in concert with the increment of intracellular Ca2+.     

Separation of a population of human T lymphocytes that bind prostaglandin E2 and exert a suppressor activity

Prostaglandin E2 (PGE2) is a potent inhibitor of immune functions. Two possible mechanisms of PGE2-mediated suppression have been proposed: one is a direct inhibitory effect exerted on interleukin 2-producing T cells; the second is mediated by the activation of nonspecific suppressor T lymphocytes. We previously showed that PGE2 can directly activate human T lymphocytes to suppress lymphocyte proliferation and B lymphocyte maturation. Herein is described the binding of 10 to 30% of human peripheral blood T lymphocytes to insolubilized PGE2 coated to albumin-Sepharose. The T lymphocytes that bound PGE2 (PGE2(+] could be eluted by the addition of serum and gentle shaking of the beads. The following data indicated the specificity of the binding: i) T lymphocytes after an overnight incubation, a condition known to abolish sensitivity to PGE2, lost their affinity for PGE2; ii) preincubation of T lymphocytes with PGE2 blocked the binding; iii) PGE2(+) T cells bound PGE after a 24-hr incubation, whereas PGE2(-) T cells did not. Few T cells bound albumin, and only a small percentage (7 to 9%) bound 6-keto-prostaglandin F1 alpha-coated beads. Among PGE2(+) T cells, there was a slight increase in the percentage of OKT8+ cells. Although T cells that had no affinity for PGE2 (PGE2(-] proliferated as well as unseparated T lymphocytes when stimulated with mitogens or antigens, the proliferative response of the PGE2(+) subset was poor. Moreover, PGE2(+) T lymphocytes did exert a strong suppressor activity on mitogen- or allogeneic cell-induced lymphocyte proliferation as well as on pokeweed mitogen-driven B cell maturation into Ig-containing cells. PGE2(-) T lymphocytes were shown not to exert a significant suppressor activity in these assays. The PGE2(+) subset-mediated suppression was not secondary to a carry-over of PGE2 released from the beads, because its suppressor activity was not altered by the addition of an anti-PGE2 serum. Moreover, PGE2(-) T lymphocytes were not sensitive to the inhibitory activity on cell proliferation of PGE2. These results indicate that a given functional subset of peripheral blood T lymphocytes binds PGE2, and that at least some of them are activated into suppressor T cells. The relationship between the PGE2-activatable T suppressor subset and other functionally defined suppressor T cells remains to be clarified; it is suggested, however, that PGE2 can act as an immunoregulator through the activation of identifiable suppressor T cells.     

Direct effects of ethanol on bone resorption and formation in vitro

In vitro studies indicate that low concentrations of ethanol can have direct effects on bone formation and resorption. Bone resorption was increased when embryonic chick tibiae were exposed to ethanol at 0.03-0.3% (v/v), and bone formation was inhibited when tibiae were exposed to 0.2% ethanol in the presence of NaF or parathyroid hormone (P less than 0.01 for each). Ethanol also had direct effects on isolated bone cells in vitro, increasing both cAMP and PGE2 production (P less than 0.001 for each), and affecting cell proliferation in a biphasic, time- and dose-dependent manner. After 24 h of exposure, 0.03% ethanol increased bone cell proliferation (P less than 0.001), but 0.3% ethanol was inhibitory (P less than 0.01). Paradoxically, mitogenic doses of ethanol prevented the effects of two other mitogens, NaF and human skeletal growth factor, to increase bone cell proliferation (P less than 0.001). But how were these effects produced? Several observations suggest that these direct effects of ethanol on skeletal tissues in vitro were mediated by changes in bone cell membrane fluidity. (a) Dimethyl sulfoxide, ethylene glycol, and lecithin, which act, like ethanol, to increase membrane fluidity, mimicked the effects of ethanol on bone cell proliferation. Dimethyl sulfoxide also mimicked the effect of ethanol to increase cAMP (P less than 0.001). (b) Cholesterol, which decreases cell membrane fluidity, acted oppositely to ethanol and enhanced the mitogenic response to human skeletal growth factor (P less than 0.001). (c) Preincubation of calvarial cells with ethanol or with cholesterol altered the in situ reaction kinetics of the membrane-bound enzyme, alkaline phosphatase. Together, these data demonstrate that ethanol has direct effects on skeletal tissue in vitro, and suggest that those effects may be secondary to changes in bone cell membrane fluidity.     

Prostaglandin E2 regulates macrophage colony stimulating factor secretion by human bone marrow stromal cells.

Bone marrow stromal cells regulate marrow haematopoiesis by secreting growth factors such as macrophage colony stimulating factor (M-CSF) that regulates the proliferation, differentiation and several functions of cells of the mononuclear-phagocytic lineage. By using a specific ELISA we found that their constitutive secretion of M-CSF is enhanced by tumour necrosis factor-alpha (TNF-alpha). The lipid mediator prostaglandin E2 (PGE2) markedly reduces in a time- and dose-dependent manner the constitutive and TNF-alpha-induced M-CSF synthesis by bone marrow stromal cells. In contrast, other lipid mediators such as 12-HETE, 15-HETE, leukotriene B4, leukotriene C4 and lipoxin A4 have no effect. EP2/EP4 selective agonists (11-deoxy PGE1 and 1-OH PGE1) and EP2 agonist (19-OH PGE2) inhibit M-CSF synthesis by bone marrow stromal cells while an EP1/EP3 agonist (sulprostone) has no effect. Stimulation with PGE2 induces an increase of intracellular cAMP levels in bone marrow stromal cells. cAMP elevating agents (forskolin and cholera toxin) mimic the PGE2-induced inhibition of M-CSF production. In conclusion, PGE2 is a potent regulator of M-CSF production by human bone marrow stromal cells, its effects being mediated via cAMP and PGE receptor EP2/EP4 subtypes.     

Cutting edge: cyclooxygenase-2 activation suppresses Th1 polarization in response to Helicobacter pylori

Helicobacter pylori infection causes a Th1-driven mucosal immune response. Cyclooxygenase (COX)-2 is up-regulated in lamina propria mononuclear cells in H. pylori gastritis. Because COX-2 can modulate Th1/Th2 balance, we determined whether H. pylori activates COX-2 in human PBMCs, and the effect on cytokine and proliferative responses. There was significant up-regulation of COX-2 mRNA and PGE(2) release in response to H. pylori preparations. Addition of COX-2 inhibitors or an anti-PGE(2) Ab resulted in a marked increase in H. pylori-stimulated IL-12 and IFN-gamma production, and a decrease in IL-10 levels. Addition of PGE(2) or cAMP, the second messenger activated by PGE(2), had the opposite effect. Similarly, stimulated cell proliferation was increased by COX-2 inhibitors or anti-PGE(2) Ab, and was decreased by PGE(2). Our findings indicate that COX-2 has an immunosuppressive role in H. pylori gastritis, which may protect the mucosa from severe injury, but may also contribute to the persistence of the infection.     

Prostaglandin E2 promotes IL-4-induced IgE and IgG1 synthesis

PG of the E series are generally known to suppress immune responses, however, we have found that PGE synergizes with IL-4 to induce IgE and IgG1 production in LPS-stimulated murine B lymphocytes. PGE2 and PGE1 (10(-6) to 10(-8) M) significantly increase IgE and IgG1 production (up to 26-fold) at all concentrations of IL-4 tested. In addition to its effects on IgE and IgG1, PGE also causes a significant decrease in IgM and IgG3 synthesis, suggesting that PGE may promote IL-4-induced class switching. The specificity of the E series PG effect is demonstrated by the fact that PGF2 alpha (10(-6) M) does not alter production of any of these isotypes. Because PGE can mediate its effects through cAMP in some cases, we investigated the importance of cAMP levels in regulation of isotype expression. Other agents that increase intracellular cAMP levels (cholera toxin and dibutyryl cAMP) were assessed for their ability to regulate isotype differentiation. Cholera toxin (100 pg/ml) and dibutyryl cAMP (100 microM) significantly enhanced IgE and IgG1 production and diminished IgM and IgG3 synthesis. We also show that PGE and cholera toxin elevate intracellular cAMP in B lymphocytes in a dose-dependent manner. In contrast, PGF2 alpha (10(-6) M) and the B subunit of cholera toxin (100 pg/ml) did not increase cAMP and did not regulate the isotype of Ig produced, reiterating the importance of cAMP in enhancing isotype differentiation. Although PGE is known to inhibit a number of immune responses, our data show that it is not always inhibitory. PGE may play a role in atopy in vivo where PGE-secreting cells such as macrophages, follicular dendritic cells, and fibroblasts can promote IgE synthesis. This research emphasizes the importance of PGE in regulation of the humoral immune response and adds a new stimulatory action to the repertoire of known PGE effects.     

Involvement of inducible isoforms of COX and NOS in streptozotocin-pancreatic damage in the rat: interactions between nitridergic and prostanoid pathway

Streptozotocin-induced pancreatic damage involves nitric oxide (NO) and prostaglandins (PGs) overproduction. In this work we aim to evaluate a putative relationship between the elevated NO levels and the altered prostanoid production in pancreatic tissue from streptozotocin-diabetic rats. Total NOS activity and nitrate/nitrite pancreatic levels in tissues from diabetic rats are decreased when the cyclooxygenase (COX) inhibitor indomethacin (INDO) is added to the incubating medium, while the addition of PGE(2)increases nitrate/nitrite production and NOS levels. INDO and PGE(2)selectively affect Ca(2+)-dependent NOS (iNOS) activity in diabetic tissues, and they have not been able to modify nitrate/nitrite levels, iNOS or Ca(2+)-dependent (cNOS) in control tissues. When the NOS inhibitor L-NMMA was present in the incubating medium, control pancreatic [(14)C]-Arachidonic Acid ([(14)C]-AA) conversion to 6-keto PGF(1 alpha)and to TXB(2)was lower, and PGF(2 alpha), PGE(2)and TXB(2)production from diabetic tissues diminished. The NO donors, spermine nonoate (SN) and SIN-1, enhanced TXB(2)levels in control tissues, while PGF(2 alpha), PGE(2)and TXB(2)levels from diabetic tissues were increased. PGE(2)production from control and diabetic tissues was assessed in the presence of the NO donor SN plus INDO or NS398, a specific PG synthase 2 inhibitor. When SN combined with INDO or NS398 was added, the increment of PGE(2)production was abolished by both inhibitors in equal amounts, indicating that the activating effect of nitric oxide is exerted on the inducible isoform of cyclooxygenase. In the diabetic rat, prostaglandins and NO seem to stimulate the generation of each other, suggesting a lack of regulatory mechanisms that control the levels of vasoactive substances in acute phase of beta-cell destruction.     

Prostaglandin E2 and cAMP inhibit B lymphocyte activation and simultaneously promote IgE and IgG1 synthesis.

Macrophage-secreted prostaglandins of the E series inhibit numerous immunologic events, including IgM secretion by B lymphocytes. In this study, we investigated whether PGE also regulates the activation of normal quiescent murine B cells and subsequent isotype differentiation to IgE and IgG1 production. PGE2 and PGE1 were found to inhibit cellular enlargement induced by IL-4 or bacterial LPS, IL-4 and LPS, or anti-mu and IL-4 by approximately 75%, and completely inhibit enlargement in response to anti-mu antibody. PGE2 also suppresses activation-induced class II MHC up-regulation by 35% and expression of the low affinity IgE receptor, Fc epsilon RII/CD23, by 30%. Interestingly, PGE completely inhibits a fraction of cells from these activation events, while other cells fully respond to activation stimuli, even in the presence of high PGE2 concentrations. Therefore, a PGE-resistant subset of B lymphocytes may exist. A closely related PG, PGF2 alpha, had no immunoregulatory effect in these systems. Because PGE induces production of cAMP in B cells, we determined whether other agents that increase cAMP could inhibit B cell activation. Cholera toxin and dibutyryl cAMP mimicked the ability of PGE2 to inhibit B cell enlargement, and class II MHC and Fc epsilon RII induction, suggesting that PGE2 signaling occurs via cAMP. In addition, cholera toxin and dibutyryl cAMP inhibited B cell activation much more potently (90-100% inhibition) than PGE, indicating that whereas all B cells are cAMP-sensitive only some are PGE-sensitive. Although PGE inhibits activation-associated events, we previously reported that PGE enhances IL-4 and LPS-induced differentiation to IgE and IgG1 synthesis. To investigate the relationship between the cells that are activation-inhibited and those that are differentiation-enhanced by PGE, we sorted B cell subsets by FACS and determined their relative abilities to produce IgM, IgG1, and IgE in response to IL-4 and LPS in the presence of PGE. The population of lymphocytes that was unaffected by PGE in terms of class II hyperexpression was also unaffected by PGE for Ig synthesis, again indicating a PGE-resistant subpopulation of B cells. Furthermore, the PGE activation-inhibited subset of B cells was responsive to PGE enhancement of IL-4-induced class switching, reducing IgM synthesis and inducing a sevenfold increase in IgE and IgG1 synthesis compared with other sort groups. These results are consistent with the hypothesis that the B lymphocytes that are PGE activation-inhibited are the same cells that are PGE differentiation-enhanced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Agents that raise cAMP in human T lymphocytes release an intracellular pool of calcium in the absence of inositol phosphate production

Prostaglandins (PGs) of the E series are recognized by specific receptors on T lymphocytes which lead to an increase in cAMP. The role of cAMP in modulation of T lymphocyte function is unknown. Here, we demonstrate that agents which increase cAMP in human T cells raise the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i occurred following receptor stimulation with PGEs or by bypassing the receptor with the cell-permeant analog 8-(4-chlorophenylthio)-cAMP or forskolin, a direct activator of adenylyl cyclase. The calcium response to a submaximally stimulatory concentration of PGE2 was potentiated by the cAMP phosphodiesterase inhibitor isobutylmethylxanthine. A time course of cAMP production in response to PGE2 stimulation closely resembled the calcium response and suggested that the two events were coincident. The PGE2 concentrations required to achieve 50% maximum effect of cAMP production and increases in [Ca2+]i were similar, 0.07 and 0.15 microM respectively. Chelation of extracellular Ca2+ did not abolish the PGE2-stimulated Ca2+ response, suggesting that an intracellular source of calcium was sensitive to cAMP. Significant inositol phosphate production was not detected in response to PGE2 over a wide concentration range. The PGE2-induced calcium response curves were of lesser magnitude with shorter times to peak than those of a known inositol 1,4,5 trisphosphate-producing agonist, anti-CD3, suggesting distinct Ca2+ release mechanisms. However, the cAMP-releasable store appeared to be contained within the inositol trisphosphate-releasable store since no response could be seen with cAMP-elevating agents following emptying of the inositol trisphosphate-sensitive pool of Ca2+.