Norbert Gualde

Cyclooxygenase-2-Issued Prostaglandin E2 Enhances the Production of Endogenous IL-10, Which Down-Regulates Dendritic Cell Functions

PGE2 is a well-known immunomodulator produced in the immune response by APCs, such as dendritic cells (DCs), the most potent APC of the immune system. We investigated the PGE2 biosynthetic capacity of bone marrow-derived DC (BM-DC) and the effects of PG on the APC. We observed that BM-DC produce PGE2 and other proinflammatory mediators, such as leukotriene B4 and NO, after LPS exposure. Constitutively present in BM-DC, cyclooxygenase (COX)-1 did not contribute significantly to the total pool of PGE2 compared with the LPS-induced COX-2-produced PGE2. Treatment of BM-DC with exogenous PGE2 induced the production of large amounts of IL-10 and less IL-12p70. In addition, selective inhibition of COX-2, but not COX-1, was followed by significant decrements in PGE2 and IL-10, a concomitant restoration of IL-12 production, and an enhancement of DC stimulatory potential. In contrast, we found no demonstrable role for leukotriene B4 or NO. In view of the potential of PGE2 to stimulate IL-10, we examined the possibility that the suppressive effect of PGE2 is mediated via IL-10. We found that exogenous IL-10 inhibits IL-12p70 production in the presence of NS-398, a COX-2 selective inhibitor, while the inhibitory effects of PGE2 were totally reversed by anti-IL-10. We conclude that COX-2-mediated PGE2 up-regulates IL-10, which down-regulates IL-12 production and the APC function of BM-DC.

Arachidonic-acid-derived eicosanoids: roles in biology and immunopathology

Arachidonic acid (AA)-derived eicosanoids belong to a complex family of lipid mediators that regulate a wide variety of physiological responses and pathological processes. They are produced by various cell types through distinct enzymatic pathways and act on target cells via specific G-protein-coupled receptors. Although originally recognized for their capacity to elicit biological responses such as vascular homeostasis, protection of the gastric mucosa and platelet aggregation, eicosanoids are now understood to regulate immunopathological processes ranging from inflammatory responses to chronic tissue remodelling, cancer, asthma, rheumatoid arthritis and autoimmune disorders. Here, we review the major properties of eicosanoids and their expanding roles in biology and medicine.

Association between immune activation and early depressive symptoms in cancer patients treated with interleukin-2-based therapy.

The relationship between immune activation and the development of early depressive symptoms were studied in 33 cancer patients undergoing cytokine therapy. Patients were treated either with subcutaneous IL-2 administered alone (n=13) or in association with IFN-alpha (n=5), or with IFN-alpha alone administered subcutaneously at low doses (n=5) or intravenously at high doses (n=10). The intensity of depressive symptoms was assessed during a clinical interview carried out before the start of cytokine therapy and five days later using the Montgomery and Asberg Depression Rating Scale (MADRS). On the same days, blood samples were collected for each patient to measure serum concentrations of cytokines (IL-6, IL-10, IL-1ra) and cytokine-receptors (sIL-2R, LIF-R). Results showed that patients treated with IL-2 or IL-2+IFN-alpha displayed concomitant mood symptoms and increased serum cytokine levels during treatment. In these patients, the intensity of depressive symptoms at endpoint was positively correlated with the increases measured in serum levels of IL-10 between baseline and endpoint. IL-10 is an anti-inflammatory cytokine that is produced in response to the production of pro-inflammatory cytokines, and thereby reflects an inflammatory response. These results support the hypothesis of close relationship between depressive symptoms and the activation of the cytokine network.

Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes

We have reported previously that PGE2 inhibits dendritic cells (DC) functions. Because E prostanoid receptor (EPR) subtypes involved in this action are unknown, expression and functions of these receptors were examined in DC. Western blot and flow cytometry analyses showed that all EPRs were coexpressed in DC. In a dose‐dependent manner, lipopolysaccharide (LPS) enhanced EP2R/EP4R but not EP1R/EP3R expressions. NS‐398, a cyclooxygenase (COX)‐2‐selective inhibitor, suppressed LPS‐enhanced EP2R/EP4R expression, suggesting that COX‐2‐issued prostaglandin E2 (PGE2) modulates DC function through stimulation of specific EPR subtypes. Using selective agonists, we found that butaprost, an EP2R agonist, and PGE1 alcohol, an EP2R and EP2R/EP4R agonist, inhibited major histocompatibility complex class II expression and enhanced interleukin‐10 production from DC. However, no effect was observed with sulprostone and 17‐phenyl‐ω‐trinor‐PGE2, selective agonists for EP1R and EP1R/EP3R, respectively. Treatment of DC with dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, mimics PGE2‐induced, inhibitory effects. Taken together, our data demonstrate that EP2R/EP4R are efficient for mediating PGE2‐induced modulation of DC functions.

Prostanoids and their receptors that modulate dendritic cell-mediated immunity.

Dendritic cells (DC) are essential for the initiation of immune responses by capturing, processing and presenting antigens to T cells. In addition to their important role as professional APC, they are able to produce immunosuppressive and pro‐inflammatory prostanoids from arachidonic acid (AA) by the action of cyclooxygenase (COX) enzymes. In an autocrine and paracrine fashion, the secreted lipid mediators subsequently modulate the maturation, cytokine production, Th‐cell polarizing ability, chemokine receptor expression, migration, and apoptosis of these extremely versatile APC. The biological actions of prostanoids, including their effects on APC‐mediated immunity and acute inflammatory responses, are exerted by G protein‐coupled receptors on plasma membrane. Some COX metabolites act as anti‐inflammatory lipid mediators by binding to nuclear receptors and modulating DC functions. Although the role of cytokines in DC function has been studied extensively, the effects of prostanoids on DC biology have only recently become the focus of investigation. This review summarizes the current knowledge about the role of prostanoids and their receptors in modulating DC function and the subsequent immune responses.

Lowered Serum Dipeptidyl Peptidase IV Activity is Associated with Depressive Symptoms and Cytokine Production in Cancer Patients Receiving Interleukin-2-Based Immunotherapy

There is some evidence that treatment with interleukin-2 (IL-2) and interferon-alpha (IFNα) frequently induces depressive symptoms and activation of the inflammatory response system (IRS). There is evidence that major depression is accompanied by lowered serum activity of dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5), a membrane-bound serine protease which catalyses the cleavage of some cytokines and neuro-active peptides and which modulates T cell activation and the production of cytokines, such as IL-2. This study was carried out to examine the effects of immunochemotherapy with IL-2 and IFNα, alone and together, in cancer patients on serum DPP IV activity in relation to changes in depressive symptoms and the IRS. The Montgomery and Asberg Rating Scale (MADRS), serum DPP IV activity, and the serum IL-6, and IL-2 receptor (IL-2R) concentrations were measured in 26 patients with metastatic cancers before and three and five days after treatment with IL-2 and IFNα, alone or together. Treatment with IL-2 with or without IFNα significantly suppressed serum DPP IV activity. The MADRS scores were significantly elevated by treatment with IL-2 with or without IFNα, but not IFNα alone. The immunochemotherapy-induced decreases in serum DPP IV were significantly and inversely correlated with the increases in the MADRS. Treatment with IL-2 alone or combined with IFNα also elevated serum IL-6 and IL-2R. There were significant and inverse correlations between the immuchemotherapy-induced decreases in serum DPP IV and the elevations in serum IL-6 or IL-2R. In conclusion, treatment with IL-2/IFNα decreases serum DPP IV activity within 3–5 days and the immunochemotherapy-induced decreases in serum DPP IV activity are significantly and inversely related to treatment-induced increases in severity of depression and signs of activation of the IRS.

CD40 engagement on dendritic cells induces cyclooxygenase-2 and EP2 receptor via p38 and ERK MAPKs

We have previously reported that cyclooxygenase (COX)‐2‐derived prostaglandin (PG)E2 critically regulates dendritic cell (DC) inflammatory phenotype and function through EP2/EP4 receptor subtypes. As genes activated by CD40 engagement are directly relevant to inflammation, we examined the effects of CD40 activation on inflammatory PGs in murine bone marrow‐derived DC (mBM‐DC). We showed for the first time that activation of mBM‐DC with agonist anti‐CD40 monoclonal antibody (anti‐CD40 mAb) dose dependently induces the synthesis of significant amounts of PGE2 via inducible expression of COX‐2 enzyme, as NS‐398, a COX‐2‐selective inhibitor reduces this upregulation. In contrast to lipopolysaccharide, which upregulates mBM‐DC surface levels of EP2 and EP4 receptors, CD40 crosslinking on mBM‐DC increases EP2, but not EP4, receptor expression. Flow cytometry analysis and radioligand‐binding assay showed that EP2 was the major EP receptor subtype, which binds to PGE2 at the surface of anti‐CD40‐activated mBM‐DC. Upregulation of COX‐2 and EP2 levels by CD40 engagement was accompanied by dose‐dependent phosphorylation of p38 and ERK1/2 mitogen‐activated protein kinase (MAPK) and was abrogated by inhibitors of both pathways. Collectively, we demonstrated that CD40 engagement on mBM‐DC upregulates COX‐2 and EP2 receptor expression through activation of p38 and ERK1/2 MAPK signaling. Triggering the PGE2/EP2 pathway by anti‐CD40 mAb resulted on the induction of Th2 immune response. Thus, CD40‐induced production of PGE2 by mBM‐DC could represent a negative feedback mechanism involving EP2 receptor and limiting the propagation of Th1 responses. Blocking CD40 pathway may represent a novel therapeutic pathway of inhibiting COX‐2‐derived prostanoids in chronically inflamed tissues (that is, arthritis).

EFFECT OF PGE2 ON THE CELL SURFACE MOLECULE EXPRESSION IN PMA TREATED THYMOCYTES

PGE2 is produced by cells of the thymic microenvironment. The effects of PGE2 are mediated by cAMP through binding to its intracellular receptor protein kinase A (PKA). Phorbol 12-myristate 13-acetate (PMA) is known to modulate CD molecule expression on thymocytes, probably through activation of protein kinase C (PKC). We have hypothesized that cross-talk between these two signalling pathways may affect modulation of the CD molecules on the cell surface of thymocytes. For this purpose, we compare the effects of PMA alone or combined with PGE2 on CD3, CD4 and CD8 expression on mouse thymocytes by flow-cytometric analysis. PMA treatment almost completely abolished CD4 expression and slightly decreased CD3 and CD8 expression. PGE2 alone did not change the CD3, CD4 and CD8 molecule expression. Combined with PMA, PGE2 can overcome the decrease induced by PMA of the CD3 expression and partially reduced the disappearance of the CD4 molecule. On the other hand PGE2 accelerated the loss of CD8 molecule expression. These events occurred only in CD4+ CD8+ immature thymocytes. An analogue of cAMP (dibutyryl cAMP) mimics the effect of PGE2, but not Br-cGMP. This differential regulation by PGE2 of the CD molecule expression on immature thymocytes may provide additional evidence on the role of PGE2 during the process of thymic differentiation.

Effect of PGE2 on thymocyte proliferation induced by Con A or IL-4 + PMA.

Prostaglandin E2 (PGE2) is known to inhibit peripheral T-lymphocyte and thymocyte proliferation activated by antigens, mitogens or anti-CD3 antibodies. In this study, we have investigated, the effect of PGE2 on thymocyte proliferation induced by the combination of IL-4 plus PMA. PGE2 inhibits the proliferation of thymocytes activated by ConA, whatever the culture period; in contrast PGE2 shifts the kinetics of thymocyte proliferation after stimulation by IL-4 plus PMA, but does not sustain the proliferation beyond day 3. This effect depends upon cell density, IL-4 concentration and on the time that PGE2 is added to the culture. By use of the cAMP inducer, forskolin, or a cAMP analog, db-cAMP, we observed the same results, PGE2 increases the proliferation of CD8+ corticoresistant thymocytes (CRT) activated by IL-4 plus PMA, but inhibits that of CD4+ CRT. These results suggest that PGE2 can regulate thymocyte proliferation differently according to the activation pathway and the thymic subpopulations.

Effect of arachidonic acid metabolites on CR1 expression by B-lymphocytes.

The effect of arachidonic acid metabolites on the expression of the receptor for the C3b/C4b fragment of complement (CR1) by human B-lymphocytes was investigated. Kinetic experiments to determine CR1 expression over time indicated that the maximal receptor number occurred at 2 h, followed by a return to baseline values. Addition of 10(-4) M puromycin to the cells suggested that the increase was due to the expression of an intracellular pool and not de novo synthesis of new receptor molecules. B-lymphocytes were incubated with arachidonic acid, 15-hydroxyeicosatetraenoic acid, leukotrienes B4 or C4 or prostaglandin E2 (PGE2). The quantity of membrane antigenic binding sites was determined before and after incubation. The lipoxygenase metabolites did not alter CR1 numbers. In contrast, PGE2 significantly decreased (P less than 0.05) the quantity of CR1 expressed. In kinetic experiments, PGE2 blocked the maximal expression of CR1 seen at 2 h, indicating that it prevents the appearance of an intracellular pool of receptor. These results show that CR1 number on B-lymphocytes can be altered by at least one arachidonic acid metabolite. This may offer a partial explanation for the inhibitory effects of PGE2 on B-cell proliferation and immunoglobulin secretion since CR1 is implicated in B-lymphocyte differentiation and specific antibody response.