Rifampin inhibits prostaglandin E2 production and arachidonic acid release in human alveolar epithelial cells

Rifampin, a potent antimicrobial agent, is a major drug in the treatment of tuberculosis. There is evidence that rifampin also serves as an immunomodulator. Based on findings that arachidonic acid and its metabolites are involved in the pathogeneses of Mycobacterium tuberculosis infections, we investigated whether rifampin affects prostaglandin E(2) (PGE(2)) production in human alveolar epithelial cells stimulated with interleukin-1beta. Rifampin caused a dose-dependent inhibition of PGE(2) production. At doses of 100, 50, and 25 microg/ml, it inhibited PGE(2) production by 75%, 59%, and 45%, respectively (P < 0.001). Regarding the mechanism involved, rifampin caused a time- and dose-dependent inhibition of arachidonic acid release from the alveolar cells. At doses of 100, 50, 25, and 10 mug/ml, it significantly inhibited the release of arachidonic acid by 93%, 64%, 58%, and 35%, respectively (P < 0.001). Rifampin did not affect the phosphorylation of cytosolic phospholipase A(2) or the expression of cyclooxygenase-2. The inhibition of PGE(2), and presumably other arachidonic acid products, probably contributes to the efficacy of rifampin in the treatment of tuberculosis and may explain some of its adverse effects.     

Autoregulatory vasodilation enhances renal prostaglandin E2 and associated renin release during arachidonic acid infusion in dogs

To examine whether autoregulatory dilation of preglomerular vessels enhances prostaglandin (PG)E2 and renin release during arachidonic acid infusion, the ureter was occluded or the renal artery constricted in anesthetized dogs. Intrarenal arachidonic acid infusion (40 micrograms X kg-1 X min-1) increased PGE2 release by 41 +/- 17 pmol/min at control pressures and by 149 +/- 60 pmol/min during ureteral occlusion. Arachidonic acid infusion (160 micrograms X kg-1 X min-1) increased PGE2 release by 149 +/- 60 pmol/min at control pressures, by 505 +/- 211 pmol/min during ureteral occlusion and by 581 +/- 201 pmol/min during renal arterial constriction. Thus, PGE2 release during arachidonic acid infusion was trebled by autoregulatory dilation. Arachidonic acid infusion (160 micrograms X kg-1 X min-1) raised renin release by 6 +/- 2 micrograms of angiotensin I per min at control pressures, by 25 +/- 9 micrograms of angiotensin I per min during renal arterial constriction and during ureteral occlusion by 16 +/- 4 micrograms of angiotensin I per min, which was not significantly higher than induced by the lower rate of infusion. Arachidonic acid infusion (160 micrograms X kg-1 X min-1) raised renal blood flow by 54 +/- 5% at control pressures but exerted no vasoactive effect during ureteral occlusion and renal arterial constriction. We conclude that autoregulatory dilation enhances the stimulatory effects of arachidonic acid on renal PG synthesis. Both increased intrarenal PG concentration and autoregulatory dilation may contribute to enhancement of renin release. The stimulatory effects of arachidonic acid on PG synthesis and renin release are independent of the vasoactive effects of arachidonic acid.     

Sphingosine synergistically stimulates tumor necrosis factor alpha- induced prostaglandin E2 production in human fibroblasts

Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor alpha (TNF alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF alpha signal transduction. Because TNF alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF alpha-induced PGE2 production. We found that sphingosine enhanced TNF alpha-induced PGE2 production by as much as 18-fold over TNF alpha alone. Sphingosine appeared to stimulate TNF alpha-induced PGE2 production independent of TNF alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF alpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF alpha on phospholipase A2 activity. It appears that sphingosine affects TNF alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.     

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.     

Cachectin/tumor necrosis factor stimulates collagenase and prostaglandin E2 production by human synovial cells and dermal fibroblasts

Cachectin/TNF (tumor necrosis factor), an endotoxin-induced murine macrophage hormone implicated in the pathogenesis of cachexia and shock, has been found capable of stimulating collagenase and prostaglandin E2 (PGE2) production by isolated human synovial cells and dermal fibroblasts. This bioactivity associated with cachectin is comparable to that observed with the monokine interleukin 1 (IL-1), previously suggested as the major mediator of proteolysis. The ability of cachectin/TNF to stimulate collagenase and PGE2 production suggests that it may play a role in tissue destruction and remodelling, as these processes occur in inflammatory diseases.     

Effect of platelet-activating factor and serum-treated zymosan on prostaglandin E2 synthesis, arachidonic acid release, and contraction of cultured rat mesangial cells

The interaction of inflammatory cells and glomerular prostaglandins (PG) may be important during glomerulonephritis. We therefore examined the influence of platelet-activating factor (PAF), (a mediator of inflammation released from leukocytes) and of phagocytosis of zymosan on arachidonic acid metabolism and on cell contractility in rat glomerular mesangial cells in culture. PAF increased PGE2 synthesis (determined by radioimmunoassay) within minutes (threshold: 10(-10)M; maximal effect: 10(-7)M). Serum-treated zymosan also stimulated PGE2, but with a slower onset. In cells prelabeled with [14C]arachidonic acid both PAF and serum-treated zymosan released 14C from phospholipids and increased free [14C]arachidonate. The ratio of 14C-release to PGE2 was, however, different with PAF and serum-treated zymosan, indicating different phospholipid pools. Under phase-contrast microscopy, PAF caused contraction of mesangial cells with a dose-response and time-course parallel to that for PGE2 synthesis. Serum-treated zymosan caused no contraction. The PAF-induced contraction was enhanced by PG synthesis inhibition and was attenuated by addition of PGE2, indicating a feedback mechanism. The mesangial contraction by PAF may be important in favoring deposition of immune complexes, while the PGE2 synthesis stimulated by PAF and by phagocytosis of zymosan may counteract the deleterious effects of PAF during induction of glomerulonephritis.     

Effect of homocysteine on arachidonic acid release in human platelets

BACKGROUND: It has been suggested that homocysteine is implicated in the risk of atherosclerosis and thrombosis. The pathogenic mechanism has not been clarified, but oxygen-free species produced by the homocysteine metabolism and auto-oxidation could have a role. DESIGN: We have studied the effect of homocysteine on arachidonic acid release in human platelets. Two important products of arachidonic acid metabolism - thromboxane B2 (TXB2) and reactive oxygen species (ROS) - have been assayed. RESULTS: Results indicate that homocysteine induces arachidonic acid release that is partially inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA). Platelet incubation with homocysteine significantly increases basal levels of TXB2 and ROS. The effect is time- and dose-dependent. The TXB2 formation is strictly correlated with the arachidonic acid release. Moreover, ROS accumulation is largely inhibited by ETYA and partially reduced by diphenyleneiodonium (DPI), suggesting the involvement both of enzymes metabolising arachidonic acid (cyclooxygenase, lipooxygenase, cytochrome P450 monooxygenase) and of NAD(P)H oxidase. CONCLUSION: Homocysteine induces oxidative stress in human platelets in vitro. The unbalance in platelet redox-state and the increased TXB2 formation may generate hyperactivation, contributing to a thrombogenic state leading to cardiovascular diseases.     

Preincubation of Candida albicans strains with amphotericin B reduces tumor necrosis factor alpha and interleukin-6 release by human monocytes.

The release of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) by human monocytes stimulated with whole heat-killed Candida albicans CA3 (a clinical isolate) and CA2 (a germ tube-negative mutant) either treated or not treated with amphotericin B was investigated. The optimal release of the cytokines was observed at 24 h of incubation of the yeasts with the monocytes for both TNF-alpha and IL-6. The levels ranged from 10,500 to 19,000 U/ml for TNF-alpha and from 350 to 460 pg/ml for IL-6. Germ tube-negative mutant CA2 induced the release of TNF-alpha at levels significantly (P < 0.05) lower than those induced by clinical isolate CA3, while no major differences were observed between the two strains with regard to their capacity to induce the release of IL-6. In all instances, preincubation of the yeasts with a sublethal concentration of amphotericin significantly reduced cytokine production. These results suggest that drug-induced alterations of fungal outer structures may affect the interactions between the yeasts and the monocytes, resulting in a reduced level of secretion of cytokines.     

Endothelium-dependent and independent responses to prostaglandin H2 and arachidonic acid in isolated dog cerebral arteries

The addition of prostaglandin (PG) H2 produced a transient contraction followed by a relaxation in helical strips of dog cerebral arteries partially contracted with PGF2 alpha or K+. The contraction was abolished by removal of endothelium, and the relaxation was potentiated. Relaxation induced by PGI2 was not influenced by endothelium denudation. The PGH2-induced contraction in strips with intact endothelium was not influenced by OKY-046, a thromboxane A2 synthesis inhibitor, but was abolished by treatment with ONO3708, an antagonist of vasoconstrictor PGs, whereas the relaxation was inhibited by tranylcypromine or diphloretin phosphate, a nonselective PG antagonist. Contraction induced by arachidonic acid (AA) was reversed to relaxation by removal of endothelium or treatment with ONO3708. Treatment with indomethacin attenuated the AA-induced contraction in the intact strips and also the relaxation in the strips treated with ONO3708 or denuded of endothelium. It may be concluded that vasoconstrictor PGs are synthesized from PGH2 or AA mainly in endothelium, and the production of PGI2 from PGH2 is not dependent on endothelium. Thromboxane A2 in concentrations sufficient to elicit significant contractions does not appear to be liberated from the cerebroarterial wall stimulated by PGH2.     

Human recombinant interleukin 1 stimulates collagenase and prostaglandin E2 production by human synovial cells.

The pathogenesis and progression of rheumatoid arthritis involves the production of biologically active lymphokines and monokines. Of these, interleukin 1 (IL-1) has been somewhat of a controversial molecule because it seems to evoke various biological responses in several different tissues. In these studies we demonstrate that three biological properties of human monocyte-derived IL-1 (T-lymphocyte activation and human synovial cell prostaglandin E2 and collagenase production) co-purify. The complementary DNA for the prominent pI 7 form of human IL-1 was expressed, purified, and tested. Any controversy now appears resolved since homogeneous recombinant human IL-1 stimulates prostaglandin E2 and collagenase from human synovial cells as well as activates T cells in vitro.     

Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50's of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.     

Prostaglandin E2 and [14C]arachidonic acid release by carbachol in the isolated canine parietal cell

Parietal cells have the capacity to synthesize prostaglandins (PGs) and these PGs have a significant effect on parietal cell acid secretion. We examined the stimuli responsible for the control of PG production by parietal cells. Two approaches were utilized. One consisted of measuring PGE2 concentration in the incubation media containing dispersed canine parietal cells stimulated with increasing concentrations of carbachol, histamine plus a phosphodiesterase inhibitor and pentagastrin. PGE2 was measured by radioimmunoassay. The other consisted of measuring the release of radioactive arachidonic acid by parietal cells prelabeled with [4C]arachidonic acid to increasing concentrations of the secretagogues. Both techniques gave very similar results. Only carbachol stimulated the release of PGE2 as well as [14C]arachidonic acid into the incubation media. The increase in PGE2 release was from a base line of 44.3 +/- 10.8 pg/ml to 51.0 +/- 11.7, 55.2 +/- 11.1 and 69.3 +/- 14.3 pg/ml at carbachol concentrations of 10(-6), 10(-5) and 10(-4) M, respectively. In prelabeled cells, carbachol stimulated 1072 +/- 141 and 1264 +/- 155 cpm/ml above basal at concentrations of 10(-5) and 10(-4) M, respectively. Neither histamine nor pentagastrin stimulated PGE2 or [14C]arachidonic acid release significantly at any concentration. The effect of carbachol on the release of [14C]arachidonic acid was blocked by atropine and the exclusion of calcium from the incubation media. Our data suggest that the cholinergic tone to the stomach with subsequent interaction of acetylcholine with muscarinic receptors determines the amount of PG production by the parietal cells. Prostaglandins may then modulate acid secretion by limiting the combined, potentiated effects of the secretagogues.     

Turnover of a prostaglandin precursor, arachidonic acid, in rheumatoid arthritis.

The plasma concentration and the turnover of free arachidonic and oleic acids were determined in patients with rheumatoid arthritis and in control subjects. The plasma level of free arachidonic acid, but not of oleic acid, was significantly decreased in male rheumatoid patients. Female patients did not differ from healthy subjects in this respect. Following treatment with indomethacin, a significant increase in the plasma concentration of free arachidonic acid was observed in rheumatoid patients of both sexes. It is suggested that the findings in the rheumatoid patients reflect a partial exhaustion of the pool of arachidonic acid serving as precursor in the synthesis of prostaglandin.     

Human monocytes and gingival fibroblasts release tumor necrosis factor-α, interleukin-1α and interleukin-6 in response to particulate and soluble fractions ofPrevotella melaninogenica andFusobacterium nucleatum

Summary In this study we provide evidence that structural and soluble components of periodontopathogenic bacteria, such asPrevotella melaninogenica andFusobacterium nucleatum, induce the release of cytokines in vitro known to cause in vivo necrotic inflammatory phenomena and bone resorption (tumor necrosis factor-α, interleukin-1α and interleukin-6). Human monocytes and gingival fibroblasts were cultivated in vitro in the presence of both particulate and soluble bacterial fractions. A dose-dependent production of tumor necrosis factor-α by monocytes and gingival fibroblasts was observed in the presence of fractions ofP. melaninogenica andF. nucleatum. Interleukin-1α was produced in approximately the same quantities in the presence of soluble fractions of eitherP. melaninogenica orF. nucleatum, but in greater quantities in response to particulate fractions ofP. melaninogenica. Monocytes release larger amounts of interleukin-1α (about 3000 pg/ml) than gingival fibroblasts (about 1500 pg/ml). Interleukin-6 was released in greater quantities by monocytes in the presence of the pellet fraction ofP. melaninogenica (about 5.5 ng/ml), but gingival fibroblasts released larger amounts of interleukin-6, especially in the presence of particulate and soluble components ofF. nucleatum (about 12 ng/ml). The ability to induce the release of these cytokines notably increases the pathogenic potential of the bacteria involved in the damage of periodontal tissue.     

Human granulocyte-macrophage colony-stimulating factor induces expression of the tumor necrosis factor gene by the U937 cell line and by normal human monocytes.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts profound effects on the proliferation, differentiation, and effector function of myeloid lineage cells. In contrast to its growth-promoting effects on normal myeloid progenitor cells, we found that GM-CSF unexpectedly inhibited the colony growth of U937 cells in agar culture. Furthermore, medium conditioned by recombinant GM-CSF(rGM-CSF)-treated U937 cells was found to exert an inhibitory effect on subsequent U937 colony growth that was partially due to the presence of tumor necrosis factor (TNF). By Northern blot analysis, rGM-CSF was shown to induce expression of the TNF gene in U937 cells and in T-lymphocyte-depleted, monocyte-enriched peripheral blood mononuclear cells. Furthermore, rGM-CSF was observed to significantly enhance TNF secretion by monocytes stimulated with endotoxin and phorbol myristate acetate (PMA). These data suggest that some of the biological effects of GM-CSF may be amplified through the release of monokines such as TNF.