Mary Carol Conroy

Selective regulation of human neutrophil functions by the cell activation inhibitor CI-959.

The cell activation inhibitor CI‐959 [5‐methoxy‐3‐(1‐methylethoxy)‐N‐1H‐tetrazol‐5‐ylbenzo[b]thiophene‐2‐carboxamide, monosodium salt] was evaluated for its effects on human neutrophil functions. CI‐959 inhibited spontaneous migration and chemotaxis toward N‐formyl–methionyl‐l‐leucyl‐l‐phenylalanine (fMLP) with 50% inhibition (IC50) values of 3.6 and 3.1 μM, respectively. CI‐959 also inhibited superoxide anion generation in response to C5a, fMLP, serum‐opsonized zymosan (SOZ), concanavalin A (Con A), and calcium ionophore A23187 with IC50 values of 2.5, 4.7, 14.5, 5.4, and 14.8 μM, respectively. In comparison, CI‐959 inhibited myeloperoxidase release in response to C5a, fMLP, SOZ, and Con A with IC50 values of 11.6, 16.1, 7.5, and <1.0 μM, respectively, while inhibiting the response to A23187 by only 5.5% at 100 μM. At concentrations up to 100 μM, CI‐959 had no effect on the respiratory burst or degranulation in response to L‐α‐1,2‐dioctanoylglycerol (DiC8) or phorbol 12‐myristate 13‐acetate (PMA). In addition, the compound inhibited leukotriene B4 release stimulated by fMLP and SOZ (IC50 values 4.0 and 2.5 μM, respectively), while having less activity against the A23187‐stimulated response (IC50>100 μM). These results demonstrate that CI‐959 inhibits cellular responses to stimuli that mobilize intracellular calcium. For cellular responses to ionophore‐mediated calcium influx, only oxygen radical production was inhibited by CI‐959. CI‐959 was further evaluated for its effects on neutrophil stimulus‐response coupling. At 100 μM, CI‐959 had no effect on human neutrophil phospholipase C or protein kinase C. CI‐959 inhibited fMLP‐stimulated intracellular calcium mobilization and calcium influx with IC50 values of 16.7 and 3.1 μM, respectively, and exhibited less potent calmodulin antagonist activity (IC50 = 90.5 μM). These results indicate that CI‐959 may exert its stimulus‐ and response‐specific inhibitory effects on neutrophil functions, in part, through inhibition of calcium‐regulated signalling mechanisms. J. Leukoc. Biol. 55: 443–451; 1994.

Specific inhibition of phorbol ester and DiC8-induced histamine release from human basophils by the protein kinase C inhibitors, H-7 and H-9

The release of histamine and other inflammatory mediators from human basophils is triggered by numerous stimuli, including chemical, physical and receptor-mediated activators. Several mechanisms of cell activation including protein kinase C activation have been proposed to operate in these cells. We used phorbol ester and DiC8 to induce histamine release from human basophils and the protein kinase C inhibitors H-7 and H-9 to inhibit this release. Both DiC8 and TPA induced histamine release were inhibited by H-7 (ID 50 = 37 mcM) and H-9 (IC 50 = 20 mcM). However, anti-IgE, fmlp and A23187-induced histamine release were unaffected. In contrast, the calmodulin antagonists W-7 and perphenazine effectively inhibited histamine release by all five stimuli. Therefore, different biochemical pathways appear to be critical for basophil activation depending on the nature of the stimulus used.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922: mechanism of inhibitory activity

3,7-Dimethoxy-4-phenyl-N-1H tetrazol-5-yl-4H-furo[3,2-b]-indole-2- carboxamide, L-arginate (CI-922) is a potent inhibitory of human neutrophil functions in response to a variety of stimuli. In this report, the effects of CI-922 on specific processes involved in stimulus-response coupling are evaluated. CI-922 does not inhibit human neutrophil phospholipase C or protein kinase C activities. CI-922 is shown to inhibit calmodulin-dependent enzyme activation. The calmodulin antagonist activity is confirmed by calmodulin-Sepharose affinity chromatography. These results suggest that CI-922 inhibits neutrophil activation by preventing the activation of calmodulin-dependent enzymes, implying a critical role for such enzymes in stimulus-response coupling.

Inhibition of interleukin-2 production and lymphocyte responsiveness by the cell activation inhibitor, CI-959

CI-959 (5-methoxy-3-(1-methylethoxy)-N-1H-tetrazol-5-yl-benzo[b]-thiophene-2-carboxamide), an antiallergy compound, blocked release of IL-2 from Con A stimulated rat splenocytes and human lymphocytes with respective IC50s of 19.1 and 23.1 μM. Inhibition of IL-2 production required the presence of CI-959 in culture medium for the first 9 hr. CI-959 also inhibited Con A-stimulated rat and human lymphocyte proliferation with IC50s of 4.7 and 5.4 μM, respectively. Inhibition of the Con A proliferative response could not be overcome by exogenous recombinant human IL-2 (300 units/ml) in either the rat or human systems. Although potent in the human mixed lymphocyte reaction (IC50=3.5 μM), CI-959 was less effective in blocking the PHA response (IC50=43.9 μM), and had minimal effect on the release of IL-1 and TNFα from LPS-stimulated human monocytes. These findings suggest that CI-959 selectively inhibits some lymphocyte functions, as opposed to monocyte functions, and that among these is the production of IL-2.

Cytoprotective Effects of CI-959 in the Rat Gastric Mucosa: Modulation of Leukocyte Adhesion

BACKGROUND & AIMS CI-959 is an anti-inflammatory agent that inhibits neutrophil adhesion, respiratory burst, and mast cell histamine release in vitro. In view of the emerging role of neutrophils in gastric erosive damage, the goals of this study were to assess the gastric cytoprotective effects of CI-959 and identify the mechanism responsible for this action. METHODS Cytoprotective effects in the rat nonsteroidal anti-inflammatory drug and ethanol erosion models were assessed using image analysis. The in vivo effects of CI-959 on gastric acid secretion, arachidonic acid metabolism, and intracellular sulfhydryl and leukocyte adhesion were also examined. RESULTS CI-959 protected prophylactically against the erosive damage induced by aspirin, indomethacin, and ethanol with 50% effective doses (ED50s) of 0.05, 1.0, and 0.07 mg/kg administered orally, respectively. When administered after indomethacin or ethanol, CI-959 had no effect on the healing of erosive damage. CI-959 did not alter gastric acid secretion, arachidonic acid metabolism, or intracellular sulfhydryl levels. In vivo, CI-959 blocked leukocyte adhesion in intravital microscopy studies using indomethacin (ED50, < 5 mg/kg orally) or platelet-activating factor (50% inhibiting concentration, approximately 10 mumol/L) as the adhesion stimulus. CONCLUSIONS The most likely mechanism responsible for the cytoprotective effects of CI-595 is its inhibitory effects on leukocyte trafficking and/or adhesion.

Differential inhibition of histamine release from human basophils induced by antigen, anti-IgE or N-formyl-L-methionyl-L-leucyl-L-phenylalanine.

We report differential effects of various compounds on inhibition of histamine release from washed leukocytes stimulated through IgE (using antigen or anti-IgE antibody) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors. Inhibition of IgE-induced release was seen for several compounds listed in order of potency (IC50 in microM): CI-922 (3,7-dimethoxy-4-phenyl-N-(1-H-tetrazol-5-yl)-4-H-furo-[3, 2-b]-indole-2-carboxamide, 1-arginate) less than NDGA less than BW755c less than proxicromil less than isamoxole less than phenidone. Other compounds including FPL 55712, meclofenamate, and indomethacin were inactive or enhanced IgE-mediated release. When FMLP was used to stimulate histamine release the order of potency (IC50) changed: meclofenamate = FPL 55712 = proxicromil less than CI-922 less than NDGA = BW755c less than indomethacin less than isamoxole = phenidone. The inhibition of FMLP-induced histamine release by FPL 55712, meclofenamate and indomethacin in the absence of inhibition (or even enhancement) of histamine release by the IgE mechanism suggests that different pathways of activation and/or control are critical in the release process when different stimuli are used to activate basophils.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-949.

The allergic mediator release inhibitor CI‐949 [5‐methoxy‐3‐(1‐methylethoxy)‐1‐phenyl‐N‐1H‐tetwol‐5‐yl‐1H‐indole‐2‐carboxamide, L‐arginine salt] was evaluated for its effects on human neutrophil functions. CI‐949 (100 μM) inhibited spontaneous migration and chemotaxis toward f‐met‐leu‐phe (FMLP) by 49.1% and 45.8%, respectively. At the same concentration, CI‐949 inhibited the phagocytosis of serum‐opsonized zymosan (SOZ) by 39.0%. CI‐949 inhibited leukotriene B4 and thromboxane B2 release in response to SOZ with IC50S of 2.0 μM and 3.3 μM, while inhibiting the response to FMLP with IC50s of 1.7 and 2.0 μM. CI‐949 also inhibited myeloperoxidase release from primary lysosomal granules in response to the following stimuli with the respective IC50s (μM): C5a (40.3); FMLP (34.4): SOZ (21.4); concanavalin A (Con A) (3.9); and calcium ionophore A23187 (91.2). In contrast, CI‐949 inhibited lysozyme release from secondary granules in response to SOZ and Con A with IC50S of 99.3 and 56.1 μM, while inhibiting the response to C5a, FMLP, and A23187 by 41.2%, 52.4%, and 10.0%, respectively, at 100 μM. CI‐949 (100 μM) had no inhibitory effect against lysozyme release in response to L‐α‐1,2 dioctanoylglycerol (DiC8), or phorbol 12‐myristate 13‐acetate (PMA). CI‐949 inhibited superoxide anion generation stimulated by FMLP and Con A with IC50S of 33.9 and 25.8 μM, while inhibiting the response to C5a, SOZ, and A23187 by 36.6%, 24.8%, and 14.1% and having no effect on the response to DIC8 or PMA at 100 μM. These results demonstrate preferential inhibition of arachidonic acid metabolism and degranulation of primary lysosomal granules by CI‐949 with selectivity for stimuli which promote intracellular calcium mobilization or calcium influx.

Differential regulation of the activation of human eosinophils, macrophages, and neutrophils: Effect of the allergic mediator release inhibitor CI-949

The allergic mediator release inhibitor CI-949 [5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H-indole-2-carboxamide,l-arginine salt] was evaluated for its effect on the activation of human eosinophils, macrophages, and neutrophils by the phagocytic stimulus serum-opsonized zymosan (SOZ). CI-949 inhibited the SOZ-stimulated respiratory burst of eosinophils, measured as the generation of superoxide anion, with an IC50 of 22.8 μM. At concentrations up to 100μM, CI-949 had no inhibitory effect against superoxide anion generation by human macrophages, while inhibiting the response of human neutrophils by only 24.8 percent. CI-949 exhibited a different profile of inhibitory activity against lysosomal enzyme release by these cells. At 100 μM, CI-949 had no inhibitory effect against release of eosinophil peroxidase while inhibiting release of the macrophage lysosomal enzyme N-acetyl-β-d-glucosaminidase by only 11.7 percent. In contrast, CI-949 inhibited the release of the neutrophil primary granule enzyme myeloperoxidase with an IC50 of 21.4μM, while inhibiting release of lysozyme from secondary granules with an IC50 of 99.3μM. These results demonstrate that oxygen radical generation and lysosomal enzyme release by human eosinophils, macrophages, and neutrophils are differentially regulated by CI-949. These results suggest that these inflammatory cells may have distinct stimulus-response coupling mechanisms.

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922: differential inhibition of responses to a variety of stimuli.

The allergic mediator release inhibitor 3,7‐dimethoxy‐4‐phenyl‐N‐1H‐tetrazol‐5‐yl‐4H‐furo[3,2‐b]indole‐2‐carboxamide, L‐arginate (CI‐922) is a potent inhibitor of human neutrophil functions in vitro. Over a concentration range from 1 to 100 μmol CI‐922 inhibits the chemotactic response of neutrophils to the synthetic chemotaxin N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP). CI‐922 also inhibits respiratory and secretory responses of neutrophils in response to agents that stimulate phospholipase C‐dependent phosphoinositide hydrolysis to generate the second messengers inositol 1,4,5, trisphos‐phate and 1,2 diacylglycerol, including: the plasma membrane receptor‐specific ligands FMLP and C5a; serum‐opsonized zymosan; concanavalin A; and the guanine nucleotide regulatory protein‐specific stimulus guanosine‐5′‐0‐(3‐thiotriphosphate) (GTPγS). CI‐922 also inhibits neutrophil functions stimulated by the calcium ionophore A23187. In contrast, CI‐922 does not inhibit neutrophil responses to protein kinase C‐specific stimuli such as phorbol 12‐myristate 13‐acetate (PMA) or L‐α‐1,2 dioctanoylglycerol (DiC8). CI‐922 also fails to inhibit the synergistic activation of the respiratory burst by suboptimal concentrations of PMA and calcium ionophore A23187. The observation that CI‐922 inhibits neutrophil responses to a variety of soluble and particulate stimuli, excluding protein kinase C‐specific stimuli, allows us to postulate the site of action of the compound. We propose that CI‐922 inhibits neutrophil activation at a site distal to signal transduction through the guanine nucleotide regulatory protein required for second messenger generation but proximal to phosphorylation reactions mediated by protein kinase C and calmodulin‐dependent protein kinases.

CI-959, a New, Potential Antiallergic Drug, Inhibits Mediator Release from Lung and Contractions of Human Airways in vitro

Many symptoms of the immediate allergic response can be attributed to the synthesis/release and subsequent actions of histamine and metabolic products of arachidonic acid oxidation. CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazole-5-yl-benzo(b) thiophene-2-carboxamide], a new, potential antiallergic drug, inhibited the release of histamine, immunoreactive sulfidopeptide leukotrienes C4, D4 and E4 and immunoreactive thromboxane B2 from immunologically activated guinea-pig and human lung cells in vitro. The IC50s of CI-959 using guinea-pig lung were: histamine, 0.8 +/- 1.4 microM; leukotriene, 0.7 +/- 1.6 microM and thromboxane, 9.6 +/- 3.3 microM. Using human lung the IC50s were: 2.3 +/- 1.3 microM for histamine; 0.3 +/- 5.1 microM for leukotriene, and 0.3 +/- 2.6 microM for thromboxane. CI-959 caused a concentration-dependent inhibition of anti-IgE-induced contractions of human bronchial muscle. Mean percent inhibitions were 45, 65 and 96 at 1, 3 and 10 microM, respectively. Cromolyn, 10 microM, inhibited bronchial contractions only 42%. The ability of CI-959 to inhibit these immunologically induced contractions indicates that the release of all mediators responsible for bronchoconstriction was effectively inhibited. These data suggest that CI-959 may be effective in preventing the development of symptoms directly related to inflammatory mediator release in a variety of allergic and inflammatory states.