Outi Kosonen

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 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.

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 μM) 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.

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.

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.

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 Ca2+‐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 μm) 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.

Effects of Nimesulide, Acetylsalicylic Acid, Ibuprofen and Nabumetone on Cyclooxygenase-1- and Cyclooxygenase-2-Mediated Prostanoid Production in Healthy Volunteers ex vivo

: The beneficial actions of non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with inhibition of cyclooxygenase-2 (COX-2), whereas some of their adverse effects are associated mainly with inhibition of COX-1. Selective COX-2 inhibitors reduce the risk of gastrointestinal adverse events, but increase the risk of thromboembolic events pointing to importance of optimal COX-1/COX-2 inhibition in drug safety. We compared the effects of acetylsalicylic acid, ibuprofen, nabumetone and nimesulide on COX-1 and COX-2 pathways in healthy volunteers in an ex vivo set-up using single oral doses commonly used to treat acute pain. In a randomized, double-blind four-phase cross-over study, 15 healthy volunteers were given orally a single dose of either acetylsalicylic acid 500 mg, ibuprofen 400 mg, nabumetone 1 g or nimesulide 100 mg. Blood samples were drawn before and 1, 3, 6, 24 and 48 hr after the drug for the assessment of COX-1 and COX-2 activity. COX-1 activity was measured as thromboxane(2) production during blood clotting and COX-2 activity as endotoxin-induced prostaglandin E(2) synthesis in blood leucocytes. The data show that after a single oral dose these four NSAIDs have different profiles of action on COX-1 and COX-2. As expected, acetylsalicylic acid appeared to be COX-1-selective and ibuprofen effectively inhibited both COX-1 and COX-2. Nabumetone showed only a slight inhibitory effect on COX-1 and COX-2. Nimesulide caused almost complete suppression of COX-2 activity and a partial reduction of COX-1 activity. This confirms the relative COX-2 selectivity of nimesulide.

Nitric oxide-releasing compounds inhibit the production of interleukin-2, -4 and -10 in activated human lymphocytes.

In the present study, we investigated the effects of nitric oxide donors, GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3(3,4-dichlorophenyl)-chloride), GEA 3175 (1,2,3,4-oxatriazolium,3-(3-chloro-2-methylphenyl)-5-[[(4-methylphenyl) sulfonyl]amino]-, hydroxide inner salt) and S-nitroso-N-acetylpenicillamine (SNAP), on the production of Th1 [interleukin (IL)-2] and Th2 (IL-4 and IL-10) type cytokines in activated human lymphocytes. Lymphocytes were stimulated with concanavalin A or a combination of thapsigargin and phorbol myristate acetate in the absence or in the presence of nitric oxide donors. Concanavalin A induced expression of IL-2 mRNA and production of IL-2, and the combination of thapsigargin and phorbol myristate acetate induced expression of IL-4 and IL-10 mRNAs and production of IL-4 and IL-10. These effects were inhibited by the nitric oxide donors in a dose-dependent manner, GEA 3162 and GEA 3175 being more potent than SNAP on a molar basis. The results show that nitric oxide donors have immunomodulatory properties in both Th1- and Th2-derived responses.