Modulation of the population density of identifiable epidermal Langerhans cells associated with enhancement or suppression of cutaneous immune reactivity

The epidermis on the backs or ears of DBA/2 mice treated for 7 days with a 20% concentration of monobenzyl ether of hydroquinone (MBEH) had a significantly greater population density of ATPase- and Ia-positive cells compared with control mice treated with diluent. There was no decrease or increase in ATPase- or Ia-positive cells at sites distal from the treated tissue. This increase in population density of Langerhans cells was associated with a significant increase in functional afferent immune reactivity measured by allergic contact hypersensitivity. We also found evidence for enhanced efferent immune reactivity. Animals treated on the ears for 7 days with MBEH were sensitized to DNFB on untreated back. MBEH treated ears with more Ia-positive Langerhans cells demonstrated a threefold greater increase in swelling after the DNFB challenge than the control mice. Results of other studies suggest that the afferent and efferent enhanced immune reactivity produced by MBEH are local effects. We postulated that MBEH produced its effects by activating the oxidation of arachidonic acid (AA) to prostaglandins. To test this, we applied AA to mouse skin. AA has a biphasic effect on epidermal Langerhans cells: in low doses it increases their number; in high amounts it decreases the number of identifiable cells with either the Ia or the ATPase technique. An increased population density of identifiable epidermal Langerhans cells induced with AA was correlated with an increase in afferent and efferent immune reactivity. In contrast, reduction of Langerhans cells with larger amounts of AA suppress the afferent and efferent limb of the immune response. DNFB applied to skin with decreased Langerhans cell density from AA induced a state that mimics immune tolerance. The findings are significant because we report the only method to either increase or decrease the population density of Langerhans cells: and to modulate up or down the afferent or efferent limbs of the cutaneous immune response. Our results also suggest that the Langerhans cell may be involved in the efferent limb of the immune efferent response. These effects may be modulated in part by products of AA metabolism.     

Arachidonic acid metabolism in guinea pig Langerhans cells: studies on cyclooxygenase and lipoxygenase pathways

Epidermal Langerhans cells are macrophage-like la+ leukocytes that are critically involved in cutaneous immune reactions. Because macrophages exert their immunoregulatory activity in part by generation of oxygenated arachidonic acid metabolites, we systematically studied arachidonic acid transformations by purified guinea pig Langerhans cells and compared them with mixed epidermal cells and Langerhans cell-depleted keratinocytes. Products formed from arachidonic acid by cell homogenates were measured after thin-layer or reverse-phase high-pressure liquid chromatographic separation. In addition, leukotriene B4 and C4 formation was assessed in supernatants of Ca ionophore A23187-challenged intact cells by radioimmunoassay. Mixed epidermal cells converted arachidonic acid predominantly via cyclooxygenase and 12-lipoxygenase pathways. The main products were prostaglandin D2 (PGD2) and 12-hydroxyeicosatetraenoic acid (12-Hete), although significant amounts of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were formed as well. PGD2 synthesis was dependent on the presence of reduced glutathione. The product spectrum formed by Langerhans cell-depleted keratinocytes was virtually indistinguishable from mixed epidermal cells. In contrast, Langerhans cells showed a markedly different metabolism of arachidonic acid. They exhibited an exceedingly high PGD2-generating capacity, whereas only minor amounts of 12-HETE and very low amounts of other prostaglandins were synthesized. The PGD2/12-HETE ratio was 1.22 for mixed epidermal cells and 4.37 for Langerhans cells. Leukotriene production from exogenous or endogenous arachidonic acid could not be demonstrated by either radioenzymatic or radioimmunologic detection methods. We conclude that guinea pig Langerhans cells transform arachidonic acid predominantly to PGD2, which might mediate significant immunoregulatory, inflammatory, and antitumoral activity in the skin.     

Regulation of ultraviolet radiation induced cutaneous photoimmunosuppression by Toll-like receptor-4

UVB radiation is a potent immunosuppressive agent that inhibits cell-mediated immune responses. The mechanisms by which UVB radiation influences cell-mediated immune responses have been the subject of extensive investigation. However, the role of innate immunity on photoimmunological processes has received little attention. The purpose of this study was to determine whether Toll-like receptor-4 (TLR4) contributed to UV-induced suppression of contact hypersensitivity (CHS) responses. TLR4^−/− and wild type C57BL/6 (TLR4^+/+) mice were subjected to a local UVB immunosuppression regimen consisting of 100 mJ/cm² UVB radiation followed by sensitization with the hapten DNFB. Wild type TLR4^+/+ mice exhibited significant suppression of contact hypersensitivity response, whereas TLR4^−/− developed significantly less suppression. The suppression in wild type TLR4^+/+ mice could be adoptively transferred to naïve syngeneic recipients. Moreover, there were significantly fewer Foxp3 expressing CD4⁺CD25⁺ regulatory T-cells in the draining lymph nodes of UV-irradiated TLR4^−/− mice than TLR4^+/+ mice. When cytokine levels were compared in these two strains after UVB exposure, T-cells from TLR4^+/+ mice produced higher levels of IL-10 and TGF-β and lower levels of IFN-γ and IL-17. Strategies to inhibit TLR4 may allow us to develop immunopreventive and immunotherapeutic approaches for management of UVB induced cutaneous immunosuppression.     

Studies on the role of antigen-presenting cells in the systemic suppression of contact hypersensitivity by UVB radiation

Exposure of mice to UVB radiation produces a highly selective, systemic immunosuppression associated with the appearance of suppressor T lymphocytes. Suppression of delayed hypersensitivity to hapten-coupled syngeneic cells has been shown to result from an altered distribution of antigen-presenting cells. The purpose of this study was to determine whether an alteration in the activity of antigen-presenting cells could account for the systemic suppression of contact hypersensitivity (CHS) by UVB radiation. Fluorescein isothiocyanate (FITC) was used for contact sensitization because it uses different antigen-presenting cells than does oxazolone to induce CHS. Our previous studies demonstrated that CHS to oxazolone was suppressed by UVB irradiation. In these studies, we show that exposure of mice to UVB radiation before epicutaneous application of FITC onto unirradiated skin markedly decreased the CHS response to FITC painted on unexposed ears. Cyclophosphamide-sensitive suppressor T cells were detectable in the spleens of mice exhibiting decreased CHS. The antigen-presenting activity of cells in lymph nodes draining the site of epicutaneous sensitization (DLN cells) was assessed by injecting them into the hind footpads of syngeneic recipients and measuring the CHS response to FITC 6 days later. Viable DLN cells from UVB-irradiated, FITC-sensitized mice were equal to those from unirradiated, FITC-sensitized mice in their ability to induce CHS in normal recipients. No sensitization resulted when killed DLN cells were used for immunization, indicating that sensitization was not caused by reprocessing of antigen by host cells. We conclude that impairment of the CHS reaction in UVB-irradiated mice does not appear to be blocked at an initial step of antigen uptake, processing, or presentation, but must be impaired at some other step in the immunologic pathway.     

Eicosanoids mediate microaggregation and nodulation responses to bacterial infections in black cutworms,Agrotis ipsilon,and true armyworms,Pseudaletia unipuncta

Nodulation is the first, and quantitatively predominant, cellular defense reaction to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in true armyworms, Pseudaletia unipuncta, and black cutworms, Agrotis ipsilon, immediately prior to intrahemocoelic injections with heat-killed preparations of the bacterium, Serratia marcescens, severely impaired the nodulation response. Five eicosanoid biosynthesis inhibitors, including dexamethasone (a phospholipase A(2) inhibitor), indomethacin, ibuprofen (cyclooxygenase inhibitors), phenidone (dual lipoxygenase/cyclooxygenase inhibitor) and eicosatetraynoic acid (an arachidonic acid analog that inhibits all arachidonic acid metabolism) severely reduced nodulation in infected insects. The dexamethasone effects were reversed by treating true armyworms with arachidonic acid immediately after infection. In addition to these pharmacological findings, we demonstrate that an eicosanoid biosynthesis system is present in these insects. Arachidonic acid is present in fat body phospholipids at about 0.4% of total phospholipid fatty acids. Fat body expressed a phospholipase A(2) that can hydrolyze arachidonic acid from the sn-2 position of cellular phospholipids. Fat body preparations were competent to biosynthesize prostaglandins, of which PGE(2) was the major product. These findings support the hypothesis that eicosanoids mediate cellular immune reactions in insects.     

Genetic basis of ultraviolet-B effects on contact hypersensitivity

The genetic basis of the effects of ultraviolet B(UVB) radiation on the induction of contact hypersensitivity (CH) to dinitrofluorobenzene (DNFB) has been explored in genetically defined mice. It was found that acute, low-dose UVB radiation produced profound depletion of epidermal Langerhans cells (LC) at UVB-treated sites in all strains of mice tested. However, when DNFB was applied to UVB radiation sites, unresponsiveness developed in some strains of mice, but vigorous contact hypersensitivity was induced in others. The UVB-susceptible phenotype proved dominant or codominant in F₁ hybrids derived from parental strains of the susceptible and UVB-resistant phenotypes. Experiments conducted in one set of F₁ hybrids derived from two UVB-susceptible parental strains displayed UVB resistance, suggesting gene complementation, and showed that more than one genetic locus was involved. Segregant backcross populations, analyzed for the capacity to develop CH after UVB treatment and skin painting with DNFB, revealed that at least two, and probably three, independent genetic loci participate in determining UVB resistance. Results of experiments with H-2 congenic and recombinant mice derived from the B10 background implicated class I genes of the major histocompatibility complex as relevant genetic factors. These results indicate that there is a dissociation between the effects of UVB radiation on epidermal Langerhans cells and the capacity of a cutaneous surface to support the induction of contact hypersensitivity. The data indicate that the induction of CH to haptens is dependent on normal numbers of functional LC at the skin painting site only in some strains of mice. The data imply that in the so-called UVB-resistant strains of mice, alternative (non-Langerhans cell-dependent) mechanisms allow for the induction of CH. Several independent genetic loci, one of which appears to be H-2, govern this UVB-related effect.     

An ELISA method to measure inhibition of the COX enzymes

The cyclooxygenase (COX) reaction can be monitored by measurement of oxygen consumption, peroxidase co-substrate oxidation or prostaglandin (PG) detection. This protocol describes a procedure measuring cyclooxygenase activity by quantifying PGE2 produced by enzymatic conversion of arachidonic acid, in the presence or absence of potential inhibitors. This high-throughput method has the advantage that it directly measures cyclooxygenase activity and requires little enzyme. The first part of the assay consists of incubating arachidonic acid, cyclooxygenase and the test samples to generate prostaglandins. The second part uses an ELISA method to quantify the amount of PGE2 produced by the enzymatic reaction. The isolation of COX-1 and COX-2 enzymes is also described. This protocol can be completed in approximately 23 h, including 16-h and 4-h incubation phases. This does not include enzyme preparation (3 h for COX-1 and 24 h for COX-2) or preparation of ELISA plates (23 h, including incubation).     

IL-10 contributes to the inhibition of contact hypersensitivity in mice treated with photodynamic therapy

We have explored the effect of photodynamic therapy (PDT) with verteporfin on the induction and expression of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB) in normal mice and IL-10-deficient mice. Our results indicate that DNFB sensitized mice given PDT with verteporfin and whole body red light irradiation exhibited a significant reduction in CHS compared with control animals. Administration of rIL-12 reversed the effect(s) of PDT as did treatment of mice with anti-IL-10-neutralizing Ab. Knockout mice deficient in IL-10 were found to be resistant to the inhibitory effects of PDT. In vitro proliferative responses using spleen cells from DNFB-sensitized and PDT-treated mice showed a significantly lower response to DNBS as compared with cells from DNFB-sensitized mice or DNFB and PDT-treated IL-10-deficient mice. Finally, naive mice exposed to PDT exhibited an increase in skin IL-10 levels, which peaked between 72 and 120 h post-PDT. Together these data support the role of IL-10 as a key modulator in the inhibition of the CHS response by whole body PDT.     

Gender differences in UV-induced inflammation and immunosuppression in mice reveal male unresponsiveness to UVA radiation

Immunosuppression attributed mainly to the UVB (290-320 nm) waveband is a prerequisite for skin cancer development in mice and humans. The contribution of UVA (320-400 nm) is controversial, but in mice UVA irradiation has been found to antagonise immunosuppression by UVB. In other studies of photoimmune regulation, protection mediated via oestrogen receptor-β signalling was identified as a normal endogenous defence in mice, and was shown to depend on UVA irradiation. A gender bias in photoimmune responsiveness was thus suggested, and is tested in this study by comparing the UV-induced inflammatory and immune responses in male and female hairless mice. We report that male mice, which show greater skin thickness than females, developed a less intense but slower resolving sunburn inflammatory oedema, correlated with reduced epidermal expression of pro-inflammatory IL-6 than females following solar simulated UV (SSUV, 290-400 nm) exposure. On the other hand, the contact hypersensitivity reaction (CHS) was more severely suppressed by SSUV in males, correlated with increased epidermal expression of immunosuppressive IL-10. Exposure to the UVB waveband alone, or to cis-urocanic acid, suppressed CHS equally in males and females. However, whereas UVA irradiation induced immunoprotection against either UVB or cis-urocanic acid in females, this protection was significantly reduced or abrogated in males. The results indicate that males are compromised by a relative unresponsiveness to the photoimmune protective effects of UVA, alone or as a component of SSUV. This could explain the known gender bias in skin cancer development in both mice and humans.     

Ultraviolet irradiation of mice reduces the competency of bone marrow-derived CD11c+ cells via an indomethacin-inhibitable pathway

Direct UV irradiation of dendritic cells and Langerhans cells reduces their Ag presenting ability. However, the effects of UV on CD11c(+) cells located distally to the point of irradiation are poorly understood. Three days after UV irradiation (8 kJ/m(2)) of BALB/c mice, bone marrow cells were isolated and cultured for 7 d with IL-4 and GM-CSF for the propagation of CD11c(+) cells. Bone marrow-derived CD11c(+) cells from UV-irradiated or nonirradiated mice were loaded with dinitrobenzene sulfonic acid and injected into the ear pinnas of naive BALB/c mice. After 7 d, the ears were painted with 2,4-dinitro-1-fluorobenzene and the ear swelling determined 24 h later. A reduced contact hypersensitivity response was found in mice injected with CD11c(+) cells from the UV-irradiated animals compared with those injected with cells from the nonirradiated animals. Further, a long-lasting suppression of the memory response to 2,4-dinitro-1-fluorobenzene was created. This suppressed response corresponded to increased IL-10 and PGE(2) secretion by freshly isolated bone marrow cells from UV-irradiated mice, and to increased myelopoiesis. The reduction in competence of bone marrow-derived CD11c(+) cells from UV-irradiated mice was not due to delayed maturation, as it was maintained upon LPS exposure prior to CD11c(+) cell purification. The UV-induced effect was reversed by the administration of indomethacin to mice prior to UV irradiation and could be reproduced by s.c. PGE(2). These results show that UV irradiation of mice can affect the function of bone marrow-derived CD11c(+) cells via a mechanism inhibitable by indomethacin; this pathway is likely to contribute to systemic UV-induced immunosuppression.     

Effects of prostaglandins on intraocular pressure recovery rate in rabbits

The actions of a number of prostaglandins (PGs) were studied in unanesthetized rabbits using an intraocular pressure (IOP) recovery-rate method. In topical doses of 0.1 to 10 micrograms, these compounds accelerated the rate at which IOP returned to control levels after an infusion of hypertonic saline. In general, PGE1 appeared more potent than the other PGs at these doses. Arachidonic acid also increased the IOP recovery rate. The effect of arachidonic acid was completely blocked by the cyclooxygenase inhibitor indomethacin. Recovery rate responses to arachidonic acid were increased further after pretreatment with the lipoxygenase inhibitor phenidone. When administered alone, phenidone itself accelerated IOP recovery; this action was also blocked by indomethacin. The IOP recovery rate method appears to be a useful tool for studying ocular effects of PGs and other products or inhibitors of arachidonic acid metabolism.     

CD4+ and CD8+ T cell priming for contact hypersensitivity occurs independently of CD40-CD154 interactions

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-gamma-producing CD8(+) T cells, whereas CD4(+) T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8(+) and CD4(+) T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154(-/-) animals. DNFB sensitization of CD154(-/-) mice primed IFN-gamma-producing CD8(+) T cells and IL-4-producing CD4(+) T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8(+), but not CD4(+), T cell development and the CHS response to challenge. F(ab')(2) of MR1 failed to inhibit CD8(+) T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8(+) T cell development and CHS responses in mice depleted of CD4(+) T cells or in CD4(-/-) mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4(+) T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-gamma-producing CD8(+) T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8(+) T cell development where anti-CD154 mAb acts indirectly through CD4(+) T cells to impair the ability of hpLC to prime CD8(+) T cells.     

Cyclooxygenase enzymes and prostaglandins in reproductive tract physiology and pathology

Prostaglandins, thromboxanes (TX) and leukotrienes, collectively referred to as eicosanoids, are cyclooxygenase (COX) metabolites of arachidonic acid (AA). Prostaglandins, have been recognised for many years as key molecules in regulating reproductive tract physiology and pathology. Numerous recent studies in in vitro model systems and knockout mouse models have demonstrated specific functional roles for the respective cyclooxygenase enzymes, prostaglandins and prostanoid receptors. Here we review the findings obtained in several of these studies with emphasis on the roles played by cyclooxygenase enzymes and prostaglandins, specifically prostaglandin E2 (PGE2) and F2alpha in reproductive tract physiology and pathology.     

Is prostaglandin E2 involved in the pathogenesis of fever? Effects of interleukin-1 on the release of prostaglandins.

Interleukin-1 (IL-1) induces the formation of PGE2 from monocytes, fibroblasts, muscle cells, and brain tissue by increasing the intracellular concentrations of CA2+; this cation, in turn, activates a phospholipase which cleaves arachidonic acid from either diacylglycerol or a membrane phospholipid. In addition, IL-1 increases the synthesis of cyclooxygenase, as evidenced by the increased conversion of arachidonic acid into prostaglandins after fibroblasts are pre-incubated with IL-1. Evidence is also presented that fever is caused by interleukin-1-induced prostaglandin E2.     

Time-course evaluation and treatment of skin inflammatory immune response after ultraviolet B irradiation

Skin exposure to high doses of ultraviolet B (UVB) radiation generates a severe inflammatory skin response. In the present study we aim to investigate, using in vitro and in vivo models, the time-course of the inflammatory skin immune response after an acute exposure to UVB irradiation, as well as its modulation by a topical non-steroidal anti-inflammatory drug (NSAID) treatment, naproxen. PGE2 production and TNF-alpha levels increase in a post-irradiation time-dependent manner both in vivo and in vitro. This production pattern is also reflected in the iNOS expression levels in vivo and in the IL-6 levels in vitro. Changes observed in these mediators are correlated with histological alterations and dermal infiltration after the acute UVB irradiation. Naproxen treatment notably reduces PGE2 production and iNOS expression, reflecting the COX-NOS crosstalk already reported, although it causes an important increment in TNF-alpha synthesis in the epidermis of irradiated mice. Taken together, our data indicates that the epidermis is severely damaged by UVB radiation but then it is able to fully recover, and that the immune response is modulated by the NSAID treatment, since it is able to reduce the levels of some mediators as well as it can increase others.     

The influence of mono- and divalent cations on the cardiac metabolism of arachidonic acid

Our previous study indicated that, in the isolated rabbit heart, perfusion with Ca2+ free Krebs Henseleit buffer (KHB) results in increased conversion of exogenous arachidonic acid to PGE2 and 6-keto-PGF1 alpha, probably as the result of increased availability of substrate to cyclooxygenase. Since perfusion with Ca2+ free buffer is known to cause alterations in the cardiac content of various mono- and divalent cations, the present study was performed to determine: a) The relationship between the conversion of exogenous arachidonic acid to prostaglandins and cardiac content of Na+, K+, Ca2+ and Mg2+; and b) Whether enhanced arachidonic acid conversion to prostaglandins during Ca2+ free perfusion is due to reduced incorporation of this fatty acid into tissue lipids. Perfusion of the rabbit heart with Ca2+ free buffer produced a significant reduction in the tissue content of Na+, K+, Ca2+ and Mg2+. However, the production of 6-keto-PGF1 alpha from exogenous arachidonic acid was linearly correlated with tissue Mg2+. These observations, together with our finding that perfusion with Ca2+ free KHB reduced the incorporation of [3H] arachidonic acid into tissue lipids, suggests that Ca2+ free perfusion may, by reducing the activity of arachidonyl CoA synthetase (a Mg2+ dependent enzyme), decrease the acylation of arachidonic acid into lipids, thus increasing the availability of arachidonic acid to cyclooxygenase.     

Mechanism of action of arachidonic acid in the isolated perfused rat heart

The purpose of this study was to elucidate the mechanism of action of arachidonic acid in the isolated rat heart perfused with Krebs solution at a constant flow. Administration of arachidonic acid, 3.3-33 nmol, into the heart caused a small transient increase followed by a pronounced decrease in coronary perfusion pressure and increased myocardial tension, heart rate, and the output of prostaglandins (6-keto-PGF1 alpha, PGE2, and PGF2 alpha). Administration of structurally similar fatty acids, dihomo-gamma-linolenic acid, and 8,14,17-eicosatrienoic acid, produced vasoconstriction and decreased myocardial tension without affecting heart rate or the output of prostaglandins. Infusion of PGI2, PGF2 alpha, or PGE2 produced coronary vasodilation and increased myocardial tension, whereas PGF2 alpha increased heart rate, an effect which was not prevented by propranolol. Indomethacin blocked the effect of arachidonic acid on myocardial tension and heart rate, but only reduced the duration of coronary vasodilation. The initial component of arachidonic acid induced coronary vasodilation which was unaffected by indomethacin and also remained unaltered during the infusion of three structurally dissimilar lipoxygenase inhibitors, eicosatetraynoic acid, nordihydroguaiaretic acid, and 1-phenyl-3-pyrazolidone. Indomethacin did not alter the effects of the exogenously administered prostaglandins on perfusion pressure or myocardial tension; however, it blocked the effect of PGF2 alpha on heart rate. The effect of arachidonic acid or PGF2 alpha to increase heart rate was not blocked by thromboxane synthetase inhibitors, imidazole, or OKY-1581. We conclude that the cardiac effects of arachidonic acid are mediated primarily through its conversion to cyclooxygenase products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indomethacin worsens and a leukotriene biosynthesis inhibitor accelerates mucosal healing in rat colitis.

The implication of leukotrienes as mediators of inflammation and recent evidence that prostaglandin analogues provide a beneficial effect during experimental colitis led to the speculation that (i) leukotrienes may be injurious and (ii) prostaglandins may be protective to colonic mucosa. Using a 2% acetic acid induced rat colitis model, we administered specific cyclooxygenase (indomethacin) and leukotriene biosynthesis inhibitors (MK-886) to examine the effect of endogenous prostaglandins and leukotrienes on colonic macroscopic injury, mucosal inflammation as measured by myeloperoxidase activity, net in vivo intestinal fluid absorption, and colonic PGE2 and LTB4 levels as measured by in vivo rectal dialysis. Indomethacin treatment prior to induction of colitis reduced endogenous mucosal PGE2 levels and exacerbated macroscopic ulceration and net fluid absorption. Addition of the exogenous PGE1 analogue misoprostol to the indomethacin-exacerbated colitis completely healed colonic macroscopic ulceration and inflammation but only partially improved fluid absorptive injury. The specific leukotriene biosynthesis inhibitor MK-886 administered prior to induction of colitis healed macroscopic ulceration and inflammation but not fluid absorptive injury. This mucosal reparative effect of MK-886 occurred at a dose that reduced colonic LTB4 synthesis while concomitantly enhancing PGE2 levels. Combining MK-886 with misoprostol treatment improved not only macroscopic ulceration and inflammation but also provided a synergistic effect that maintained net colonic fluid absorption at noncolitic control levels. These studies suggest that, during the induction of experimental colitis, endogenous prostaglandins play a pivotal role in providing a mucosal healing effect, and that leukotriene biosynthesis inhibitor may manifest part of its beneficial effect by shifting arachidonic acid metabolism towards production of prostaglandins.     

Pathways of arachidonic acid metabolism in human amnion cells at term

We have compared the metabolism of (3H) arachidonic acid by monolayers of human amnion, cells obtained prior to or following labor at term. Radiolabel was either added exogenously or previously incorporated into cellular phospholipid pools to compare metabolism of arachidonic acid from different substrate sources. Cells obtained both prior to and following labor synthesized metabolites co-chromatographing on HPLC with di- and mono-HETEs and also a metabolite with polarity corresponding to a epoxyeicosatrienoic acid. Both types of cells were able to synthesize PGE2 when (3H) arachidonic acid was added exogenously. However, only those cells obtained following labor synthesized PGE2 from (3H) arachidonic acid incorporated into intracellular pools. These findings suggest that the cyclooxygenase and PGE2 isomerase enzymes are present in amnion prior to delivery but that exogenous arachidonic acid would be required for PGE2 synthesis at that time as the enzymes do not appear to be linked to a source of endogenous arachidonic acid. At the time of parturition, there may be a switching on of an enzyme system to generate arachidonic acid from intracellular pools specifically for PGE2 synthesis or alternatively coupling of such a system to a cyclooxygenase-PGE2 isomerase system resulting in PGE2 synthesis. These findings raise intriguing new possibilities for the regulation of eicosanoid synthesis in amnion which may include membrane topography, substrate pool-enzyme linking and regulation of specific phospholipase enzymes.     

Prostaglandin E2 increases growth and motility of colorectal carcinoma cells

Chronic use of nonsteroidal anti-inflammatory drugs results in a significant reduction of risk and mortality from colorectal cancer in humans. All of the mechanism(s) by which nonsteroidal anti-inflammatory drugs exert their protective effects are not completely understood, but they are known to inhibit cyclooxygenase activity. The cyclooxygenase enzymes catalyze a key reaction in the conversion of arachidonic acid to prostaglandins, such as prostaglandin E(2) (PGE(2)). Here we demonstrate that PGE(2) treatment of LS-174 human colorectal carcinoma cells leads to increased motility and changes in cell shape. The prostaglandin EP(4) receptor signaling pathway appears to play a role in transducing signals which regulate these effects. PGE(2) treatment results in an activation of phosphatidylinositol 3-kinase/protein kinase B pathway that is required for the PGE(2)-induced changes in carcinoma cell motility and colony morphology. Our results suggest that PGE(2) might enhance the invasive potential of colorectal carcinoma cells via activation of major intracellular signal transduction pathways not previously reported to be regulated by prostaglandins.