Suppression of the cutaneous immune response following topical application of the prostaglandin PGE2

UVB irradiation (290-320 nm) and topical applications of arachidonic acid (AA) in mice decrease the number of identifiable Langerhans cells and alter the cutaneous immune response. Application of contact allergens such as dinitrofluorobenzene (DNFB) to irradiated or AA-treated skin induces antigen-specific tolerance. Indomethacin (IM), a cyclooxygenase inhibitor, administered orally to mice prior to UVB irradiation or prior to the topical application of arachidonic acid, abrogates suppression of contact hypersensitivity (CHS) to DNFB. This suggests a byproduct of arachidonic acid generated through the cyclooxygenase pathway may be involved in the immune suppression. Topical application of various prostaglandins (PGE2, PGD2, PGF2 alpha, and CTXA2) did not cause alterations in the population density of the identifiable Ia+ dendritic Langerhans cells. PGE2, but no other tested agent, produced a suppression of the CHS response to DNFB. These observations suggests that of the various prostaglandins, PGE2 might be one of several biochemical signals which mediate the suppression of contact hypersensitivity reactions following ultraviolet radiation exposure. However, the mechanisms by which PGE2 produces its suppressive effects have not been identified.     

Rat pulmonary lipoxygenase: dioxygenase activity and role in xenobiotic metabolism.

1. Dioxygenase activity and the ability of pregnant rat lung lipoxygenase to oxidize xenobiotics were examined in vitro under a variety of experimental conditions. 2. More than 90% of the dioxygenase activity towards linoleic acid in the lung homogenate was found to be associated with the cytosolic fraction. The cytosolic enzyme exhibited pH optima at 6.5 and 9.5, the activity being two-fold greater at pH 9.5. To observe maximal dioxygenase activity (about 0.7 mumol of 13-hydroperoxylinoleic acid formed/min per mg protein) at pH 9.5, the presence of 6.0 mM linoleic acid was required. 3. Benzidine oxidation occurred at maximal rate of pH 6.5 when the reaction medium contained 1.0 mM benzidine and 13.5 mM linoleic acid. All eight xenobiotics tested were oxidized at significant rates by the lung cytosolic lipoxygenase. 4. Both dioxygenase activity and benzidine oxidation were inhibited by the inhibitors of lipoxygenase, viz. nordihydroguaiaretic acid, BHT, caffeic acid, esculetin, and gossypol, in a concentration-dependent manner. 5. The results suggest that oxidation of xenobiotics by lipoxygenase may be an important pathway of metabolism in the mammalian lung.     

The effect of prenatal exposure to the n-butylester of 2,4-dichlorophenoxyacetic acid (2,4-D) on the immune response in mice

Pregnant CD-1 mice were administered the n-butylester of 2,4-dichlorophenoxyacetic acid (2,4-D) by gastric intubation on day 11 of gestation at dosages ranging from 0 to 200 mg/kg (2,4-D content). The immune response in the female offspring was elevated at 6 weeks of age. The humoral immune response, antibody production against sheep red blood cells, was not altered by 2,4-D ester exposure during gestation. The mitogen responses of lymphocytes induced by concanavalin A, a T-lymphocyte mitogen, or by Escherichia coli lipopolysaccharide, a B-lymphocyte mitogen, were reduced in the highest exposure group (200 mg/kg), although the T-lymphocyte suppression was not statistically significant. A similar response pattern was observed in the background nonstimulated lymphocyte cultures, suggesting that the suppression was a generalized lymphocyte abnormality. Evaluation of the mitogen responses using stimulation indices to correct for the variable background responses demonstrated that 2,4-D produced no net suppressive effect in any of the treatment groups. Since in utero 2,4-D ester exposure produced no alterations in humoral immunity and only subtle effects on lymphocyte blastogenesis, it is unlikely to be of any immunotoxicological or immunoteratological significance. Further studies investigating commercial-grade 2,4-D formulations are necessary since these formulations contain other components that may potentially induce alterations in the immune system.     

The effect of platelet-activating factor and its antagonist--BN 52021--on immune reactions in vivo in mice and in vitro

The effect produced by the injection of platelet activation factor (PAF) and its antagonist BN 52021 on the intensity of humoral immune response in (CBA x C57BL)F1 mice was studied. PAF was found to stimulate the formation of antibodies to sheep red blood cells. In addition PAF stimulated the phagocytic activity of mouse peritoneal macrophages. The stimulation of immune response under the action of PAF may be attributed to an increase in the phagocytic activity of macrophages. The stimulating effect of PAF on immune response in vivo was abolished by the injection of BN 52021, the antagonist of PAF. At the same time the dose-dependent decrease of immune response was observed after the injection of BN 52021. Indomethacin, an inhibitor of prostaglandin synthesis, when administered to mice treated with BN 52021, abolished the BN 52021-induced suppression of humoral immune response. Mouse peritoneal macrophages, treated in vitro with BN 52021, were found to produce significantly more prostaglandin E than control macrophages. Thus, BN 52021 induced the suppression of humoral immune response in vivo; this suppression was probably due to the action of prostaglandin E2, a messenger of the second order. Besides, the PAF antagonist BN 52021 significantly decreased leukotriene B4 production by macrophages in vitro. BN 52021 may be supposed to switch over the synthesis and/or secretion of arachidonic acid from the lipoxygenase pathway to the cycloxygenase one.     

Dioxygenase and Co-oxidase activities of rat hepatic cytosolic lipoxygenase

The role of rat liver cytosolic lipoxygenase in the metabolism of benzidine was studied using linoleic acid as a cosubstrate. Under optimum assay conditions, cytosolic dioxygenase activity in the presence of 3.5 mM linoleic acid at pH 7.2 was 74.07 ± 1.43 nmoles/min/mg protein. Benzidine was oxidized at the rate of 3.18 ± 0.13 nmoles/min/mg cytosolic protein to benzidine diimine at pH 7.2 in the presence of 3.65 mM linoleic acid. Both dioxygenase and cooxidase reactions were inhibited by nordihydroguaiaretic acid in a concentration-dependent manner. Partially purified preparations of rat liver lipoxygenase, free of hemoglobin, exhibited a dioxygenase activity of 223.1 ± 65.9 nmoles/min/mg protein and cooxidase activity of 6.1 ± 0.5 nmoles/min/mg protein toward benzidine. These results suggest that hepatic lipoxygenase may play an important role in the metabolism of this hepatocarcinogen.     

Inhibition of cytotoxic T lymphocyte-mediated lysis by ETYA: effect independent of arachidonic acid metabolism

Cytotoxic T lymphocyte (CTL)-mediated lysis of target cells was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA) and other inhibitors of the lipoxygenase pathway at concentrations that inhibited arachidonic acid metabolism in mixed lymphocyte cultures. Inhibition was reversible and selective for the "lethal hit" stage in the CTL-target interaction. Studies to define CTL-specific arachidonic acid metabolites demonstrated that cloned CTL populations have little or no capacity to metabolize arachidonic acid. Therefore, inhibitor actions appear to be independent of the effects on CTL arachidonic acid metabolism. Alternative explanations for inhibitory effects are discussed.     

Inhibitors of arachidonic acid lipoxygenase impair the stimulation of inositol phospholipid hydrolysis by the T lymphocyte mitogen phytohaemagglutinin

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.     

A syngeneic MLR induced in vivo results in T-cell mediated immune suppression

The proliferation of murine T lymphocytes in response to syngeneic Ia bearing non-T cells (syngeneic mixed lymphocyte reaction, SMLR) has been shown to generate regulatory T cells in vitro. An in vivo regulatory role has therefore been proposed for the SMLR. To study this role more directly, we examined the effects of repeated iv injection of mice with activated syngeneic B cells. Three such weekly injections induced a suppression of the plaque forming cell response to a subsequent injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH). The suppression was transient and could not be maintained by additional injections of activated syngeneic B cells. The suppression was transferable to syngeneic recipients with splenic lymphocytes. Continued weekly iv injections of LPS induced blasts, as well as weekly intraperitoneal injections, caused enhancement rather than inhibition of the response to iv injected TNP-KLH. The enhancement was prevented by injection of anti-L3T4. Spleen cells from mice which had received three iv injections of activated syngeneic cells suppressed an in vitro secondary response to TNP-KLH by normal immune spleen cells. The cells responsible for the immune suppression were Thy 1.2+. The results indicate that repeated exposure to activated B cells causes activation of suppressor pathways but does not bring about a chronic state of immune suppression.     

Reactive oxygen production associated with arachidonic acid metabolism by peritoneal macrophages

The nature of the calcium-dependent chemiluminescence observed in peritoneal macrophages after exposure to the calcium ionophore A23187 or during the phagocytosis of zymosan has been investigated. Eicosatetraynoic acid, an inhibitor of the lipoxygenase and cyclooxygenase pathways of arachidonic acid metabolism, inhibited the calcium-dependent chemiluminescence whereas indomethacin, a selective inhibitor of the cyclooxygenase pathway, did not. Arachidonic acid induced chemiluminescence only in phagocytosing cells, whilst 15-HPETE, an intermediate of the lipoxygenase pathway, generated a similar, transient chemiluminescent response in either unstimulated or phagocytosing cells. The results suggest that the lipoxygenase pathway may be a significant source of the reactive species of oxygen that give rise to chemiluminescence. Prostaglandin E₁ inhibited the chemiluminescence induced by zymosan and A23187, but did not affect that generated in response to 15-HPETE or arachidonic acid, suggesting that the inhibition is directed at a step either connected with or occurring prior to the release of free arachidonic acid by the cells.     

In vitro study of frog adrenal function--IX. Evidence against the involvement of lipoxygenase metabolites in the control of steroid production

The possible role of arachidonic acid metabolites of the lipoxygenase pathway in the regulation of steroidogenesis was studied in vitro using perifused frog interrenal (adrenal) glands. Graded doses of arachidonic acid (10(-6)-10(-4)M) increased the production of corticosterone and aldosterone in a dose-dependent manner. In the presence of indomethacin (5 X 10(-6)M), the effect of arachidonic acid on steroid secretion was totally abolished. Nordihydroguaiaretic acid (NDGA: 10(-6)M), a lipoxygenase inhibitor, did not alter the spontaneous secretion of corticosteroids and did not impair the stimulatory effect of arachidonic acid. In the presence of NDGA, both ACTH and angiotensin II were still able to stimulate corticosteroid production. Our data support the view that arachidonic acid metabolites play an important role in the regulation of amphibian steroidogenesis. Moreover, the results show that the lipoxygenase pathway is not involved in the spontaneous secretion of corticosteroids and in angiotensin II- or ACTH-induced steroidogenesis.     

Coordinate actions of arachidonic acid and protein kinase C in gonadotropin-releasing hormone-stimulated secretion of luteinizing hormone

The relative contributions of arachidonic acid and protein kinase C during GnRH-stimulated LH release were investigated in cultured rat anterior pituitary cells. Maximal or near-maximal concentrations of arachidonic acid or the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, were less effective than a maximal dose of GnRH in stimulating LH release. However, the effect of a combination of arachidonic acid and phorbol ester was equivalent with that of GnRH. The protein kinase C inhibitor, retinal, significantly reduced GnRH- and phorbol-induced, but not arachidonic acid-stimulated, LH release. The lipoxygenase inhibitors, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, partially inhibited GnRH- and arachidonic acid-stimulated, but not phorbol-induced, LH secretion. Simultaneous addition of retinal and either lipoxygenase inhibitor completely abolished LH responses elicited by GnRH, as well as by combined treatment with arachidonic acid and the phorbol ester. These results suggest that hormone release is mediated by phospholipid-dependent mechanisms that are coordinated during the stimulation of LH secretion by GnRH.     

Effect of inhibitors of arachidonic acid metabolism on mitogenesis in human lymphocytes: possible role of thromboxanes and products of the lipoxygenase pathway.

Although it is already known that prostaglandins inhibit lymphocyte responses to mitogens the role of other products of arachidonic acid (AA) metabolism has not previously been investigated. Various inhibitors of AA metabolism were studied for their effects on mitogenesis in human lymphocytes, including imidazole, benzylimidazole, N-0164, L-8027, 5, 8, 11, 14 eicosatetraynoic acid, nordihydroguaiaretic acid, indomethacin, and aspirin. Selective or partially selective inhibitors of thromboxane synthesis, such as imidazole, benzylimidazole, N-0164, and L-8027 inhibited the mitogenic response at concentrations that also substantially affect thromboxane B2 synthesis in platelet-free lymphocyte preparations. Since indomethacin failed to reverse the inhibition by imidazole or N-0164, it is probably due to decreased thromboxane synthesis per se rather than secondary increases in prostaglandin synthesis. Eicosatetraynoic acid and nordihydroguaiaretic acid were more effective inhibitors of mitogenesis than of thromboxane synthesis. Since these agents also affect the lipoxygenase pathway, it is possible that part of their action is at this level. Thus, in addition to the inhibitory effects of prostaglandins on mitogenesis, other products of AA metabolism may promote the response.     

Characterization of immunomodulatory properties and accessory cell function of small intestinal epithelial cells.

We have studied the immunomodulatory properties of epithelial cells from the small intestine on T cell immune function in vitro. Proliferation of lymph node cells stimulated either with antigen or with mitogen was inhibited by epithelial cells in a dose-dependent fashion. The epithelial cell-mediated suppression of lymphocyte proliferation was blocked by indomethacin, a cyclooxygenase pathway inhibitor, demonstrating that the suppressive effect of epithelial cells was related to prostaglandin secretion. Furthermore, the action of epithelial cell-secreted prostaglandin on lymphocytes was related to its effect on IL-2 as the suppressive effect of epithelial cells was abrogated by the addition of exogenous IL-2. As previously reported, epithelial cells constitutively express MHC class II and we found them able to present antigen in a class II-restricted fashion when their suppressive effects were blocked by indomethacin. Furthermore, epithelial cells activated by LPS secrete an IL-1 like molecule in a fashion analogous to other antigen-presenting cells. These results demonstrate that epithelial cells can both enhance and suppress in vitro T cell immune responses and further characterize the mechanisms by which intestinal epithelial cells may function in gut-associated immune responses.     

Assessment of a role for phospholipase A2 and arachidonic acid metabolism in human lymphocyte natural cytotoxicity

The reagents quinacrine, hydrocortisone, and dexamethasone have been assumed to affect phospholipase A2 (PA2) when they reduce natural killer (NK) activity. However, these reagents did not reduce lymphocyte incorporation of [14C]arachidonate, which implies that they are not acting as PA2 inhibitors in this lymphocyte system. However, p-bromophenacyl bromide (BPB), which is an active site inhibitor of PA2, irreversibly abrogated NK activity of pretreated lymphocytes, disrupted target cell binding, and reduced [14C]arachidonic acid incorporation by 70-80% as compared to controls. Other observations contrary to expectations for PA2 inhibitors were: (1) quinacrine inhibited NK lysis when lymphocytes were pretreated and (2) the glucocorticoids only inhibited NK activity when continuously present in the assay. Furthermore, NK inhibition by hydrocortisone did not require protein synthesis. The lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosotetraynoic acid (ETYA), and hydroxyphenylretinamide, and not cycloxygenase inhibitors, reduced NK activity. These data suggest that arachidonate must be metabolized through the 5-lipoxygenase pathway in order to function in NK.     

On the role of arachidonic acid metabolites in mast-cell mediated mitogenesis in the rat

We investigated whether the mitogenic response induced by local mast-cell secretion in the rat mesentery was affected by suppression of phospholipase A2, lipoxygenase, or cyclooxygenase in arachidonic acid metabolism. Enzyme inhibitor was given in a single intravenous dose 5 min before intraperitoneal injection of the mast-cell secretagogue 48/80. Mepacrine, a phospholipase A2 inhibitor, suppressed the generation of both leukotrienes (SRS) and prostaglandins (PG), whereas the lipoxygenase inhibitor BW 755C reduced the generation of SRS, and the cyclooxygenase inhibitor indomethacin significantly suppressed the generation of PG. None of the enzyme inhibitors affected the basal mesenteric histamine content or histamine release in the mesentery after exposure to 48/80, and none of them affected mast-cell-mediated mitogenesis in the mesentery as judged by specific DNA activity and mitosis counting. The stimulation of DNA synthesis and mitosis initiated by secreting mast cells is apparently not mediated or modulated by synthesis of leukotrienes, prostaglandins, or other known arachidonic acid metabolites.     

The effect of BW775C on respiratory distress in the rat induced by arachidonic acid

BW775C, an inhibitor of the lipoxygenase and cyclo-oxygenase pathways, inhibits the respiratory distress induced by arachidonic acid in rats. The degree of respiratory distress was measured in terms of respiratory rate using electrodes implanted at each side of the thorax. Indomethacin, an inhibitor of the cyclooxygenase pathway, failed to influence the respiratory distress induced by arachidonic acid. The results implicate the lipoxygenase pathway, i.e. the leukotrienes synthesis inhibition, in the respiratory distress induced by arachidonic acid.     

Diacylglycerol lipase pathway is a minor source of released arachidonic acid in thrombin-stimulated human platelets

It has been postulated that the diacylglycerol lipase pathway is a predominant source of the free arachidonic acid which is released from phospholipids upon the exposure of human platelets to thrombin. The amount of released arachidonic acid and other fatty acids in thrombin-stimulated platelets was determined in the presence of BW755C, the cyclooxygenase/lipoxygenase inhibitor, and in relation to phosphatidylinositol degradation and phosphatidic acid formation. A stearic acid:arachidonic acid molar ratio approaching unity would be expected in the free fatty acid fraction if the latter pathway were a major source of released arachidonic acid. Our results indicate that the diacylglycerol lipase pathway contributes a maximum of 3-4 nmol of arachidonic acid/2 X 10(9) platelets or 12-15% of the total arachidonic acid released (25.8 nmol/2 X 10(9) platelets) upon exposure to thrombin (2 units/ml) for 4 min. Trifluoperazine inhibited most of the thrombin-dependent free arachidonic acid release but only 15% of the absolute loss of arachidonic acid from phosphatidylinositol. Therefore, we conclude that the diacylglycerol lipase pathway represents only a minor source of the free arachidonic acid that is released upon thrombin stimulation of human platelets.     

The role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. I. Induction of regulatory macrophages in normal mice by the in vivo administration of rGM-CSF

Using a dimethylbenzanthracene-induced immunogenic nonmetastatic murine mammary adenocarcinoma in BALB/c mice, our previous work has shown that splenocytes from tumor bearers have reduced responses to both mitogens and Ag including tumor-associated Ag. NK and cytotoxic T cell activities are also reduced in splenocytes of tumor bearers. Mac-1+2+ macrophages induced in mammary tumor bearers are capable of down-regulating lymphocyte responses to mitogens and tumor-associated Ag by cell to cell contact interaction and increased PGE2 production. We have found that the tumor constitutively releases a granulocyte-macrophage (GM)-CSF-like factor in vivo and in vitro, which may be responsible for the systemic increase in cells of the macrophage lineage in tumor-bearing mice. A tumor cell line established from the in vivo tumor expresses and releases GM-CSF as shown by Northern and Western blot analyses. Daily i.p. injections for 3 wk of 10,000 U of rGM-CSF into normal mice induces hemopoietic and immunologic alterations similar to those observed in tumor bearers. Mac-1+ and/or Mac-2+ macrophages can also be detected in the spleens and bone marrow of the mice treated with rGM-CSF. Additionally, splenocytes from rGM-CSF-treated mice have reduced responses to mitogens and their peritoneal exudate cells can cause in vitro down-regulation of proliferative responses of lymphocytes from normal mice. The suppression can be partially reversed by the addition of indomethacin to the cultures suggesting that PGE2 may contribute to the effect. rGM-CSF enhances the in vitro release of PGE2 by the spleen, bone marrow, and peritoneal cells of normal mice. These data indicate that the high levels of GM-CSF constitutively produced by the tumor may be responsible for the hemopoietic changes and immunologic alterations observed in tumor-bearing mice.     

In vitro locomotion of allosensitized T lymphocyte clones in response to metabolites of arachidonic acid is subset specific

The effects of the arachidonic acid metabolites prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) on the in vitro random migration of cloned murine T lymphocytes (derived from limiting dilution analysis of a C57BL/6 anti-DBA/2 mixed leukocyte culture) were examined. Experiments were also performed to study the effects of the cyclooxygenase inhibitor indomethacin on both random lymphocyte migration and lymphocyte migration in the presence of PGE2. The responses of cloned lymphocytes to PGE2 and LTB4 were compared with those of unsensitized lymph node lymphocytes. PGE2 at 100 ng/ml significantly inhibited (p less than 0.001) the in vitro migration of helper clones of T lymphocytes, but had no effect on random migration of cytotoxic T cells or helper independent cytotoxic (HIT) cloned cells. In contrast, LTB4 significantly (p less than 0.001) enhanced the random locomotion of helper, cytotoxic, and "HIT" cloned cells at 0.1 and 0.3 ng/ml. The effects of both PGE2 and LTB4 were found to be completely reversible by cell washing. Indomethacin (10(-7) M) did not alter random migration of any of the clones, and in particular, did not affect the inhibition of helper lymphocyte migration induced by PGE2. Unsensitized bulk lymph node lymphocyte migration was not affected by either PGE2 or LTB4. The results suggest that modulation of lymphocyte locomotor function by environmental stimuli may depend on cellular activation, and the locomotor responses of activated lymphocytes to arachidonic acid metabolites may be subset specific.     

The effects of products and inhibitors of arachidonic acid metabolism on the hamster sperm acrosome reaction

The mammalian sperm acrosome reaction (AR) is a fusion and fenestration of sperm head membranes which is essential for fertilization. Our earlier work demonstrated that arachidonic acid could stimulate the AR 15 min after addition to hamster sperm capacitated by incubation for 4.5 h. The present study was undertaken to determine whether inhibitors of arachidonic acid metabolism could affect the stimulation of the AR by arachidonic acid and whether products of its metabolism could stimulate the AR. Phenidone or nordihydroguaiaretic acid, inhibitors of both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, and docosahexaenoic acid, a cyclo-oxygenase pathway inhibitor, inhibited the AR induced by arachidonic acid. PGE2, a product of the cyclo-oxygenase pathway of arachidonic acid metabolism and 5- or 12-hydroxyeicosatetraenoic acid (HETEs) products of the lipoxygenase pathway, stimulated the AR when added to sperm capacitated by incubation for 4.5 h. Prostaglandins not derived from arachidonic were also tested: PGE1 stimulated the AR, but PGF1 alpha and PGA2 did not. We suggest that arachidonic acid metabolites produced by the sperm and by the female reproductive tract are important for the mammalian sperm AR.