The expression of COX2 protein induced by Lactobacillus rhamnosus GG, endotoxin and lipoteichoic acid in T84 epithelial cells.

AIMS: Lactobacillus rhamnosus GG (L. rhamnosus GG) possess immunomodulatory effects in the host. In the present study, the effect of L. rhamnosus GG on cyclooxygenase 2 (COX2) expression and its pharmacological control was investigated in human T84 colon epithelial cells. METHODS AND RESULTS: T84 cells were exposed to freeze-dried L. rhamnosus GG in vitro, and the expression of COX2 was detected by Western blot. CONCLUSIONS: L. rhamnosus GG induced COX2 expression in a time- and concentration-dependent manner in T84 cells. COX2 expression was inhibited by tyrosine kinase inhibitor genistein (100 micromol l(-1)), p38 mitogen-activated protein kinase (MAP kinase) inhibitor (SB203580; 1 micromol l(-1)) and dexamethasone (10 micromol l(-1)), whereas inhibitors of p42/44 MAP kinase (PD98095; 10 micromol l(-1)), protein kinase C (Ro 31-8220; 1 micromol l(-1)) and transcription factor nuclear factor kappa B (NF-kappaB) [pyrrolidinedithiocarbamate (PDTC) 100 micromol l(-1)] had practically no effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide novel information on the cellular mechanisms involved in the interaction between L. rhamnosus GG and colon epithelial cells.

Inhibition by nitric oxide-releasing compounds of E-selectin expression in and neutrophil adhesion to human endothelial cells.

The effects of two chemically unrelated nitric oxide (NO)-releasing compounds were studied on adhesion molecule expression in and neutrophil adhesion to human umbilical vein endothelial cells. Incubation of confluent monolayers of endothelial cells with increasing concentrations of lipopolysaccharide stimulated the adhesion of polymorphonuclear leukocytes to endothelial cells. Flow cytometric analysis showed that lipopolysaccharide treatment upregulated the expression of adhesion molecules E-selectin and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells. A novel NO-releasing compound GEA 3175 (1,2,3, 4-oxatriazolium, -3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt) inhibited lipopolysaccharide-induced adhesion being more potent than the earlier known NO donor S-nitroso-N-acetylpenicillamine. The increased E-selectin expression induced by lipopolysaccharide was significantly attenuated by the two NO donors tested whereas ICAM-1 expression remained unaltered. The present data show that NO donors inhibit E-selectin expression in and neutrophil adhesion to lipopolysaccharide-stimulated vascular endothelial cells. Thus, by inhibiting leukocyte adhesion NO donors may reduce leukocyte infiltration and leukocyte-mediated tissue injury in inflammation and ischemia-reperfusion injury.

Nitric oxide-releasing compounds inhibit neutrophil adhesion to endothelial cells.

In the present work, we demonstrated that chemically different nitric oxide (NO)-releasing compounds inhibit tumor necrosis factor alpha (TNF-alpha)-induced polymorphonuclear leukocyte adhesion to endothelial cells in vitro. Two mesoionic oxatriazole derivatives GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3(3, 4-dichlorophenyl)-chloride) and GEA 3175 (1,2,3,4-oxatriazolium, -3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt) were compared to the earlier-known NO donor SIN-1 (3-morpholino-sydnonimine). GEA 3162 (3-10 microM) and GEA 3175 (10-30 microM) inhibited human polymorphonuclear leukocyte adhesion to B(4) endothelial cells in a dose-dependent manner being more potent than SIN-1. In the present model, leukocytes rather than endothelial cells seemed to be the target of the effect of NO. Flow cytometric analysis showed that NO-releasing compounds did not alter TNF-alpha induced CD11/CD18 surface expression in polymorphonuclear leukocytes. The inhibitory action of NO-releasing compounds on adhesion paralleled with the increased synthesis of cGMP in polymorphonuclear leukocytes. Analogues of cGMP inhibited polymorphonuclear leukocyte adhesion indicating a role for cGMP in the action of NO donors. The results suggest that exogenous NO in the form of NO-releasing compounds inhibits polymorphonuclear leukocyte adhesion to endothelial cells, which may be implicated in the regulation of leukocyte migration and leukocyte-mediated tissue injury.

Inhibition by nitric oxide-releasing compounds of prostacyclin production in human endothelial cells.

1. The effects of two chemically unrelated nitric oxide (NO)-releasing compounds were studied on prostacyclin production in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs). The cells expressed cyclooxygenase-2 (COX-2) protein and produced prostacyclin by NS-398-sensitive manner suggesting that prostacyclin production derives principally by COX-2 pathway. 2. A novel NO-releasing oxatriazole derivative GEA 3175 (1-30 microm) inhibited LPS-induced production of prostacyclin in HUVECs in a dose-dependent manner being more potent than the earlier known NO-donor S-nitroso-N-acetylpenicillamine (SNAP). 3. The effects of the two NO-donors on prostacyclin synthesis were reversed when red blood cells were added into the culture indicating that the effects are due to NO released from the compounds. 4. Addition of exogenous arachidonic acid into the culture did not alter the inhibitory action of NO-donors suggesting that phospholipases are not the target of action of NO. 5. The NO-donors did not inhibit prostacyclin production in the presence of a selective COX-2 inhibitor NS-398. These data suggest that NO affects COX-2 pathway rather than has an overall effect on cyclooxygenases. 6. NO-releasing compounds did not alter the level of COX-2 protein expression in LPS-treated HUVECs as measured by Western blot analysis. 7. The results suggest that NO-donors inhibit the activity of COX-2 in human endothelial cells. A link between NO and the regulation of eicosanoid synthesis could represent an important mechanism in controlling vascular and inflammatory responses in pathophysiological states and during treatment with nitrovasodilators.

Nitric oxide-releasing oxatriazole derivatives inhibit human lymphocyte proliferation by a cyclic GMP-independent mechanism.

Two novel nitric oxide (NO)-releasing oxatriazole derivatives, GEA 3162 and GEA 3175, and an earlier known NO donor, S-nitroso-N-acetylpenicillamine (SNAP), inhibited cell proliferation and enhanced cGMP production in a concentration-dependent manner in human lymphocytes activated by lectin mitogen concanavalin A (ConA). The possible mediator role of cGMP in the antiproliferative action of NO donors was tested by pharmacological means. An inhibitor of guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, inhibited NO donor-induced cGMP production, whereas the antiproliferative action of NO donors remained unaltered. Phosphodiesterase inhibitors zaprinast and 3-isobutyl-1-methylxanthine potentiated and prolonged NO donor-induced increase in the concentrations of cGMP but did not enhance the antiproliferative action of NO donors. In addition, two analogs of cGMP, 8-bromo-cGMP and a more cell-permeable compound, 8-p-chlorophenylthio-cGMP, did not inhibit ConA-stimulated lymphocyte proliferation when used in concentrations of up to 300 microM. At millimolar concentrations, 8-bromo-cGMP had a moderate inhibitory action. These results suggest that nitric oxide-releasing oxatriazole derivatives inhibit proliferative responses in human lymphocytes by a cGMP-independent manner.

Inhibition of human lymphocyte proliferation by nitric oxide-releasing oxatriazole derivatives.

The effects of novel nitric oxide (NO)-releasing oxatriazole derivatives GEA 3162 and GEA 3175 were studied on cell proliferation and cGMP synthesis in human peripheral blood mononuclear cells stimulated with a lectin mitogen concanavalin A. GEA 3162 (1-30 microM) and GEA 3175 (3-30 microM) inhibited mononuclear cell proliferation in a dose-dependent manner being more potent than the earlier known NO-donor S-nitroso-N-acetylpenicillamine. The inhibitory action was more pronounced when submaximally stimulating concentrations of concanavalin A (0.1 and 1 microg/ml) were used and no inhibition was seen when concanavalin A concentrations were increased up to 10 microg/ml. The antiproliferative concentrations of GEA 3162, GEA 3175 and S-nitroso-N-acetylpenicillamine induced a rapid and transient increase in cGMP production in mononuclear cells cultured in the presence of concanavalin A. Both the antiproliferative action and the increased cGMP production were attenuated when red blood cells were added into the cultures indicating that NO is responsible for both of these actions. An analogue of cGMP, 8-bromo-cGMP (0.1-3 mM) reduced concanavalin A-induced proliferation in a dose-dependent manner suggesting that cGMP may be involved in the antiproliferative action of NO-donors. NO-releasing compounds have immunosuppressive actions which offer therapeutic possibilities and should be kept in mind as potential adverse events when these compounds are used in other indications.

3-Morpholino-sydnonimine-induced suppression of human neutrophil degranulation is not mediated by cyclic GMP, nitric oxide or peroxynitrite: inhibition of the increase in intracellular free calcium concentration by N-morpholino-iminoacetonitrile, a metabolite of 3-morpholino-sydnonimine.

This study was designed to clarify the mechanism of the inhibitory action of a nitric oxide (NO) donor 3-morpholino-sydnonimine (SIN-1) on human neutrophil degranulation. SIN-1 (100-1000 microM) inhibited degranulation (beta-glucuronidase release) in a concentration-dependent manner and concomitantly increased the levels of cGMP in human neutrophils in suspension. However, further studies suggested that neither NO nor increase in cGMP levels were mediating the inhibitory effect of SIN-1 on human neutrophil degranulation because 1) red blood cells or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl added as NO scavengers did not inhibit the effect; 2) inhibitors of cGMP synthesis (methylene blue) or phosphodiesterases (3-isobutyl-1-methylxanthine) did not produce changes in cell function correlating with the changes in cGMP. SIN-1 releases both nitric oxide and superoxide, which together form peroxynitrite. Chemically synthesized peroxynitrite (1-100 microM) did not inhibit, but at high concentrations (1000-2350 microM), it potentiated FMLP-induced beta-glucuronidase release from neutrophils. Thus formation of peroxynitrite from SIN-1 does not explain its inhibitory effects on neutrophil degranulation. The NO-deficient metabolite of SIN-1, SIN-1C (330-1000 microM) inhibited human neutrophil degranulation in a concentration-dependent manner similar to that of SIN-1 and reduced the increase in intracellular free calcium induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine. C88-3934 (330-1000 microM), another NO-deficient sydnonimine metabolite, also inhibited human neutrophil degranulation. In conclusion, the data shows that the NO-donor SIN-1 inhibits human neutrophil degranulation in a cGMP-, NO-, and peroxynitrite-independent manner, probably because of the formation of more stable active metabolites such as SIN-1C. The results demonstrate that studies on the role of NO and/or peroxynitrite carried out with SIN-1 and other NO-donors should be carefully re-evaluated as to whether the effects found are really attributable to NO or peroxynitrite and that in future studies, it will be crucial to carry out control experiments with the NO-deficient metabolites in any studies with sydnonimine NO-donors.

Inhibition by fenamates of calcium influx and proliferation of human lymphocytes.

1. Flufenamic and tolfenamic acids have recently been shown to inhibit receptor-mediated calcium influx in human neutrophils. The present work was designed to study the effects of these two nonsteroidal anti-inflammatory drugs on human peripheral blood lymphocyte activation. 2. Peripheral blood mononuclear cells (PBMNCs; containing 90% lymphocytes) were stimulated by mitogen concanavalin A (Con A) or by a combination of an inhibitor of microsomal Ca(2+)-adenosine triphosphatase thapsigargin (TG) and phorbol myristate acetate (PMA). The effects of the two fenamates on cell proliferation were compared with respective changes in calcium metabolism. 3. Flufenamic and tolfenamic acids (10-100 microM) inhibited both Con A and TG + PMA-induced [3H]-thymidine incorporation in a dose-dependent manner. At the same concentration range, the two fenamates inhibited the increase in intracellular free calcium concentration induced by Con A or TG + PMA. This effect was due to inhibition of calcium influx whereas calcium release from intracellular stores remained unaltered. 4. The inhibition of divalent cation influx was confirmed by showing that fenamates inhibited TG + PMA-induced Mn2+ influx. 5. The inhibitory effects of fenamates on PBMNC proliferation and Ca2+ influx were qualitatively similar with those of SK&F 96365, an earlier known inhibitor of receptor-mediated calcium entry. Ketoprofen, a chemically different prostaglandin synthetase inhibitor did not show similar suppressive effects on PBMNCs. 6. The data suggest that flufenamic and tolfenamic acids suppress proliferation of human peripheral blood lymphocytes by a mechanism which involves inhibition of Ca2+ influx and is not related to inhibition of prostanoid synthesis.