J. Goossens

Effects of antioxidants on cyclooxygenase and lipoxygenase activities in intact human platelets : Comparison with indomethacin and ETYA

Five antioxidative agents (BW755C, 1-naphtol, NDGA, propylgallate and quercetin) were compared with indomethacin and ETYA for their effects on (14C) arachidonic acid metabolism by cyclooxygenase (CO) and lipoxygenase (LPO) enzymes in intact human platelets. All tested compounds inhibited CO activity in a concentration-dependent manner. LPO activity was suppressed by NDGA, propylgallate, quercetin and ETYA but strongly enhanced by BW755C, 1-naphtol and indomethacin. Whereas NDGA and ETYA showed almost equi-potent inhibitory effects towards both fatty acid oxygenases, propylgallate and quercetin were found to be respectively 6.5 and 4 times better inhibitors of LPO than of CO activities. These data indicate that antioxidants affect arachidonic acid metabolism in intact human platelets in different ways: BW755C and 1-naphtol exerted the same activity as indomethacin, a selective CO blocker, whereas NDGA, propylgallate and quercetin behaved as ETYA, a dual CO-LPO inhibitor. Considering their inhibition selectivity, propylgallate and quercetin may serve as prototypes for more specific blockers of LPO activity.

The mitogenic activity of OKT3 and anti-Leu 4 monoclonal antibodies: a comparative study.

The mitogenic properties of OKT3 (IgG2a) and anti-Leu 4 (IgG1), two monoclonal antibodies directed at the same surface antigen of human T cells, have been studied. Both antibodies, at subnanomolar concentrations, induced thymidine incorporation of comparable magnitude into human peripheral blood mononuclear cell cultures. Their mitogenicity reached peak values 3 days after stimulation, was totally inhibitable by monoclonal OKT11A antibody, and was strictly dependent on the presence of monocytes as auxiliary cells. Whereas mononuclear cells from all blood donors tested were proliferative to OKT3, cells from 12 of 30 donors (40%) were unresponsive to anti-Leu 4. The incidence of this unresponsiveness was significantly higher for cells from female than male donors, showing 60 and 20% nonresponders, respectively. Anti-Leu 4 unresponsiveness was found to be due to the inability of autologous monocytes to exert their auxiliary function; thus, lymphocytes from anti-Leu 4 nonresponders became mitogenically inducible by anti-Leu 4 after addition of monocytes from anti-Leu 4 responders, whereas lymphocytes from anti-Leu 4 responders failed to respond to anti-Leu 4 after supplementation of monocytes from anti-Leu 4 nonresponders. Considering the different immunoglobulin subtype of OKT3 (IgG2a) and anti-Leu 4 (IgG1), our results indirectly suggest that the auxiliary function of monocytes is mediated by their Fc receptors. Unresponsiveness to anti-Leu 4 could then be explained by the absence, paucity, or functional deficiency of monocytic Fc receptors for IgG1.

Mitogenic actions of orthoclone OKT3 on human peripheral blood lymphocytes: Effects of monocytes and serum components

The Orthoclone monoclonal antihuman T lymphocyte antibody, OKT3, induced maximal DNA, RNA and protein synthesis in peripheral mononuclear blood cells (PMBC) at concentrations as low as 10 ng ml-1. This pronounced mitogenic activity was highly dependent on the presence of monocytes: removal of these cells from PMBC suspensions by complement (C)-dependent lysis with the antimonocyte antibody OKM1, completely abrogated the proliferative responsiveness of the remaining lymphocytes. The addition of adherent cells to OKM1-treated PMBC demonstrated the strict monocyte requirement for the mitogenic activity of OKT3. Mitogenic responses to OKT3 were most marked when PMBC were cultured in media containing heat-inactivated fetal calf serum (FCS) but they were considerably weaker in cultures supplemented with heat-inactivated human serum (HS). Moreover, aggregated human IgG and its Fc fragments (but not monomeric IgG and its Fab fragments) inhibited the mitogenicity of OKT3: their inhibition could be explained by stimulation of monocytes, resulting in increased prostaglandin E release, since (a) prostaglandin E2 itself strongly suppressed OKT3 activity and (b) indomethacin blocked the inhibitory effects of aggregated HuIgG. The present data demonstrate that OKT3 shows a particular pattern of mitogenicity: the strict monocyte requirement, the inhibitory effects of HS, aggregated human IgG and prostaglandin E2 were not observed for the phytomitogen PHA.

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.

Liarozole fumarate inhibits the metabolism of 4-keto-all-trans-retinoic acid

The metabolism of 4-keto-all-trans-retinoic-acid (4-keto-RA), a biologically active oxygenated metabolite of all-trans-retinoic (RA), has been examined. In vitro, incubation of [14C]4-keto-RA with hamster liver microsomes in the presence of NADPH produced two major radioactive metabolites which were more polar than the parent compound. Following isolation, appropriate derivatization and analysis by GC-MS, these compounds were tentatively identified as 2-hydroxy- and 3-hydroxy-4-ketoretinoic acid. Formation of both hydroxy-keto derivatives was suppressed by the imidazole-containing P450 inhibitor liarozole fumarate (IC50, 1.3 microM). In vitro, an i.v. injection of 4-keto-RA (20 micrograms) into rats was followed by rapid disappearance of the retinoid from plasma with a half-life of 7 min. Pretreatment with liarozole fumarate (40 mg/kg, -60 min) reduced the elimination rate of 4-keto-RA: it prolonged the plasma half-life of the retinoid to 12 min, without affecting its distribution volume. These results indicate the important role of the P450 enzyme system in the metabolism of 4-keto-RA both in vitro and in vivo. The inhibitory effect of liarozole fumarate on this metabolic process may contribute to the reported retinoid-mimetic activity of this drug.

Arabinogalactan- and dextran-induced ear inflammation in mice: differential inhibition by H1-antihistamines, 5-HT-serotonin antagonists and lipoxygenase blockers.

Intravenous injection of arabinogalactan or dextran together with pontamine sky-blue dye into mice increased vascular permeability and led to marked blueing of the ears. Arabinogalactan caused a rapidly progressing ear blueing (maximal coloration 20–30 min after injection). This response was suppressed by pretreating the animals with the histamine H1-antihistamines levocabastine and loratadine. In contrast, dextran induced a slowly evolving ear inflammation (maximal coloration 60–90 min after injection), which was blocked by the 5-HT-serotonin antagonists cinanserin, metergoline and ritanserin. Furthermore, the dextran reaction was inhibited by the lipoxygenase (LO)/cyclooxygenase (CO) inhibitors BW540C, BW755C and phenidone and by the specific 5-LO inhibitor AA-861. Both arabinogalactan and dextran responses were inhibited by aprotinin, a kallikrein inhibitor, and the mixed H1/5-HT antagonists astemizole and azatadine. The inflammogenic activity of the polysaccharides was not affected by administration of the CO inhibitors indomethacin and suprofen, the thromboxane synthetase inhibitor dazoxiben, the H2-antihistamines cimetidine and ranitidine, the anticholinergics isopropamide or the PAF-antagonist L-652,731.These data indicate the existence of distinctive endogenous molecules that mediate the pinnal extravasation reaction to both polysaccharides: histamine for arabinogalactan, serotonin and lipoxygenase-derived arachidonic acid metabolites for dextran.

Pharmacological profile of JNJ-27141491 [(S)-3-[3,4-difluorophenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester], as a noncompetitive and orally active antagonist of the human chemokine receptor CCR2.

The interaction between CC chemokine receptor 2 (CCR2) with monocyte chemoattractant proteins, such as MCP-1, regulates the activation and recruitment of inflammatory leukocytes. In this study, we characterized (S)-3-[3,4-difluoro-phenyl)-propyl]-5-isoxazol-5-yl-2-thioxo-2,3-dihydro-1H-imidazole-4-carboxyl acid methyl ester (JNJ-27141491) as a noncompetitive and orally active functional antagonist of human (h)CCR2. JNJ-27141491 strongly suppressed hCCR2-mediated in vitro functions, such as MCP-1-induced guanosine 5′-O-(3-[35S]thio)triphosphate binding; MCP-1, -3, and -4-induced Ca2+ mobilization; and leukocyte chemotaxis toward MCP-1 (IC50 = 7–97 nM), whereas it had little or no effect on the function of other chemokine receptors tested. The inhibition of CCR2 function was both insurmountable and reversible, consistent with a noncompetitive mode of action. JNJ-27141491 blocked the binding of 125I-MCP-1 to human monocytes (IC50 = 0.4 μM), but it failed to affect MCP-1 binding to mouse, rat, and dog cells (IC50 > 10 μM). Therefore, transgenic mice, in which the mouse (m)CCR2 gene was replaced by the human counterpart, were generated for in vivo testing. In these mice, oral administration of JNJ-27141491 dose-dependently [5–40 mg/kg q.d. (once daily) or b.i.d.] inhibited monocyte and neutrophil recruitment to the alveolar space 48 h after intratracheal mMCP-1/lipopolysaccharide instillation. Furthermore, treatment with JNJ-27141491 (20 mg/kg q.d.) significantly delayed the onset and temporarily reduced neurological signs in an experimental autoimmune encephalomyelitis model of multiple sclerosis. Taken together, these results identify JNJ-27141491 as a noncompetitive, functional antagonist of hCCR2, capable of exerting oral anti-inflammatory activity in transgenic hCCR2-expressing mice.

Cytoprotective effects of disodium cromoglycate on rat stomach mucosa

1 The cytoprotective effects of the anti‐asthmatic drug, disodium cromoglycate (DSCG), on gastric mucosal necrosis induced by ethanol in rats were studied. Subcutaneous, but not oral, DSCG prevented the formation of gastric lesions and this effect was dose‐dependent between 1.25 and 40 mg kg−1, with an ED50 value of 6.8 mg kg−1. Maximal cytoprotection occurred 15–30 min after DSCG treatment. 2 Histological examination revealed that DSCG effectively protected the gastric mucosa against ethanol‐induced vascular congestion, haemorrhage, epithelial desquamation and mucosal oedema. 3 Enhanced production of endogenous prostaglandins, which are known cytoprotective compounds, could not explain the mucosal protection. At a dose of 40 mg kg−1, DSCG did not change prostaglandin E2 or 6‐keto‐prostaglandin F1α concentrations in gastric mucosal tissue, although its cytoprotective activity was partially inhibited by prior treatment of the animals with indomethacin.

E-rosette formation at 37 °C: Analysis with monoclonal OKT antibodies

Abstract When cultured in the presence of PHA, a proportion of human peripheral blood mononuclear cells acquires the capacity to form E rosettes with sheep erythrocytes that are resistant to incubation at 37 °C. The nature of this 37 °C stable E-rosette formation was investigated using a panel of monoclonal OKT antibodies directed to human T-lymphocyte surface antigens. OKT11A antibody, at a concentration of 0.2–0.4 μg/ml, markedly blocked 37 °C E rosetting. OKT1, OKT3, OKT4, OKT6, and OKT8 antibodies, when tested at 10 μg/ml, show no such inhibiting activity. Quantitative studies with 125 I-labeled OKT11A indicated that the antibody interacted strongly with both 37 °C E-rosetting and nonrosetting cells, the association constant being 1.6–2.0 × 10 9 M −1 . However, on the average, a threefold higher concentration of OKT11A receptor sites was found on 37 °C E-rosette-forming cells (14.8 × 10 4 sites/cell) than on nonrosetting cells (4.8 × 10 4 sites/cell). Our data suggest that 37 °C E-rosette formation is governed by a lymphocyte surface determinant recognized by OKT11A antibody. “Overexpression” of OKT11A antigenic sites on a proportion of PHA-stimulated lymphocytes may explain their capacity to form 37 °C stable E-rosettes.

The effects of antioxidants on the stimulation of mouse thymocytes by concanavalin A.

Abstract The antioxidative compounds, 2-mercaptoethanol (2-ME), butylated hydroxyanisole (BHA), diphenylphenylenediamine (DPPD) and ethoxyquinureide substantially enhanced the DNA synthetic response of mouse thymocytes to concanavalin A (Con A). RNA and protein was not or only slightly enhanced. With the exception of 2-ME, maximal potentiating effect was obtained when antioxidants were added at the initiation of the cultured period. Supplementation of antioxidant pretreated fetal calf serum (FCS) to Con A stimulated thymocytes results in the same elevated DNA responses. These data indicate a characteristic pattern of costimulatory activity of these antioxidants. It is suggested that their activity is mediated by an activated FCS component.