- Email: [email protected]
191 Inhibitory effect of rupatadine on TNF-alpha release from human monocytes and mast cell lime HMC-1
562
Abstracts
J ALLERGY CLIN IMMUNOL JANUARY
188
189
Dexamethasone Decreases the Levels of Surface FceRI Exptession in Mouse Mast Cells M Yamaguchi*, M likura*, M Mi.yamaSU*, H Nagase*, K Hirai*. Y Furumotof. C Rat. R Teshima$. SJ Gal&, K Yamamoto* *University of Tokyo, Tokyo, Japan tJuntendo University, Tokyo, Japan *National Institute of Health Sciences, Tokyo, Japan &Stanford University, Stanford, CA Surface FceRJ expression in mast cells is strongly upregulated by factors including IgE. which leads toenhanced mast cell function to IgEmediated stimulation. Modulation of mast cell surface FceRl expression may be thus potentially important for therapeutic approach to allergic diseases. In this study, we tested whether glucocorticoids directly act on mast cells and affect the levels of their surface FceRI expression. Mouse peritoneal mast cells (PMCs), bone marrow-derived cultured mast cells (BMCMCs), or mast cell line, CLMCK57.1 (C57) cells were incubated with dexamethasone (DEX) or other steroids. Surface FceRI expression was analyzed by flow cytometry using mouse IgE and FITC-labelled anti-IgE antibody. DEX at 10 - 100 nM decreased the levels of surface FceRI expression in mouse PMCs, BMCMCs, and C57 cells. DEX partly suppressed IgE-induced increase in mast cell surface Fc&RI levels. The decrease in levels of surface FcERI by DEX was also observed in BMCMCs whose FcERI had been maximally upregulated by IgE. The inhibitory effect of DEX on surface FcERI expression was evident after 4 to 6 hours of incubation of BMCMCs or C57 cells with DEX. Other glucocorticoids such as hydrocortisone and methylprednisolone, but not estradiol, progesterone, or testosterone, reduced surface F&U expression in mast cells. DEX did not affect mast cell surface FqRII/III levels assessed using 2.462 mAb. In conclusion, glucocorticoids decrease the surface FcERI levels in mast cells incubated in both the presence and absence of IgE. The inhibitory effect of glucocorticoids on mast cell surface FcERI expression may be one of useful anti-allergic actions of the drugs. Comparison of the Priming Effect of Two Polymorphic Forms of IL-3 on Leukotriene Production A Schweiger. M Baldini, IC Lehman, DA Stem, FD Martinez, M Halonen Respiratory Sciences Center, University of Arizona, Tucson, AZ A polymorphism in the coding region of the IL-3 gene, located on chromosome 5q (1) leads to a change in the amino-acid sequence in the protein in residue 27 (proline to serine). These two different forms of rhIL-3 are available from commercial sources (Biosource”’ (proline) and R&DTM and GibcoTM (serine)). We sought to determine if these two different forms of IL-3 varied in function. IL-3 primes basophils for the production of sulfidoleukotrienes upon stimulation with anti-IgE. We obtained blood from 8 healthy adult volunteers and performed a dextran separation. The resulting mixed cell populations were primed with the two forms of IL-3 for various periods and then stimulated with anti-IgE. Production of combined LTC,, LTD, and LTE, (pg/ml) was measured by ELISA (Buehlmann, Switzerland). The values after 24 hrs of priming are shown,
Leukotrienes
(pglml)
mean
fold (SD)
Rh-IL3
IO-12 10-10 1O-s
Proline 0.99 (0.21) 1.76 (1.16) 2.74 (1.27)
increase
over
control
(n=8) Serine 1.29 (0.44) 2.90 (1.52) 3.21 (1.21)
We conclude that the binding of two naturally occuring hIL-3 to the IL-3 receptor differs in affinity or efftcacy. tJeong MC, Navani A, Oksenberg JR. (1998) Molecular lar Probes, 12:49-53.
p-Value 0.023 0.016 0.008 forms
of
& Cellu-
2000
199
Olopatadine Inhibits TNFa Release From Human Conjunctival Mast Cells Ellen 8. Cook. James L. Stahl, Neal b? Barney, Frank M. Graziano University of Wisconsin-Madison TNFa release from human conjunctival mast cells likely plays a crucial role in allergic ocular inflammation via increasing ICAM- 1 on epithelial cells and triggering other proinflammatory events. This immediate and prolonged release of TNFa in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined.Olopatadine (PatanolB) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine. tryptase, and PGD2 release in vitro and promote decreased HI receptor binding activity in vitro and functional Hl receptor antagonism in viva. The objective of these studies was to investigate the effect of olopatadine on TNFa release from purified human conjunctival mast cells challenged with anti-IgE antibody. Human conjunctival mast cells were purified (>95Yo) from cadaveric tissues using a procedure combining enzymatic digestion and Pertoll@ gradient centrifugation. These purified mast cells were incubated with olopatadine for 30 min then challenged with anti-IgE antibody for 90 min. Supernatants were analyzed for TNFa. Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFu release in a concentration dependent manner (optimum concentration for TNFa release was IO ug/ml anti-IgE antibody). Pre-incubation with olopatadine resulted in a dose dependent decrease in anti-IgE antibody mediated TNFa release (IC50 = 13. I PM). A concentration of 3mM olopatadine reduced TNFa release to the level of unchallenged controls. Olopatadine inhibited anti-IgE antibody mediated release of TNFa from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.
191
Inhibitory Effect of Rupatadine on TNF-alpha Release From Human Monocytes and Mast Cell Line HMC-1 M MerIm*. I Ramis*, D Balsa*, M Queraltf, P Brazist. A Plrigdemontt *Uriach Research Center and *Department of Pharmacology, Veterinary, UAB. Barcelona, Spain Mast cells are not only major contributors to the early response in allergic processes but also are able to synthesize a variety of cytokines which play a key role in the development of the late phase reactions. Among these cytokines, TNF-alpha is of particular importance in causing allergic inflammation. We compared the effects of rupatadine (RUP), a new antihistamine and antiallergic drug, with those of loratadine (LOR) and cetirizine (CET) on TNF-alpha release. Human mast cells HMC-1 were cultured in Iscove’s medium with 10% fetal calf serum and stimulated with 0.3 pM A23 187 and 80 nM tetradecanoyl phorbol acetate. After incubation in the presence or absence of test compounds (0.1-30 PM, 3 h, 37°C). TNF-alpha release was measured by bioassay. RUP, LOR. and CET inhibited TNF-alpha release, with ICso values of 2.0 f 0.9.2.1 rt 1.1 and 7.7 + 2.6 PM, respectively. Inhibition was further confirmed in mononuclear cells isolated from human donors and stimulated with bacterial lipopolysaccharide (026:B6 10 pg/ml, 19 h. 37°C). RUP and CET (10 pM) practically abrogated TNF-alpha release, whereas LOR showed 57% inhibition. In conclusion, RUP and also antihistamines LOR and CET are able to inhibit TNF-alpha release from human mast cells and monocytes. This effect could be beneficial in the late phase reactions present in allergic inflammation.
Abstracts
J ALLERGY CLIN IMMUNOL JANUARY
188
189
Dexamethasone Decreases the Levels of Surface FceRI Exptession in Mouse Mast Cells M Yamaguchi*, M likura*, M Mi.yamaSU*, H Nagase*, K Hirai*. Y Furumotof. C Rat. R Teshima$. SJ Gal&, K Yamamoto* *University of Tokyo, Tokyo, Japan tJuntendo University, Tokyo, Japan *National Institute of Health Sciences, Tokyo, Japan &Stanford University, Stanford, CA Surface FceRJ expression in mast cells is strongly upregulated by factors including IgE. which leads toenhanced mast cell function to IgEmediated stimulation. Modulation of mast cell surface FceRl expression may be thus potentially important for therapeutic approach to allergic diseases. In this study, we tested whether glucocorticoids directly act on mast cells and affect the levels of their surface FceRI expression. Mouse peritoneal mast cells (PMCs), bone marrow-derived cultured mast cells (BMCMCs), or mast cell line, CLMCK57.1 (C57) cells were incubated with dexamethasone (DEX) or other steroids. Surface FceRI expression was analyzed by flow cytometry using mouse IgE and FITC-labelled anti-IgE antibody. DEX at 10 - 100 nM decreased the levels of surface FceRI expression in mouse PMCs, BMCMCs, and C57 cells. DEX partly suppressed IgE-induced increase in mast cell surface Fc&RI levels. The decrease in levels of surface FcERI by DEX was also observed in BMCMCs whose FcERI had been maximally upregulated by IgE. The inhibitory effect of DEX on surface FcERI expression was evident after 4 to 6 hours of incubation of BMCMCs or C57 cells with DEX. Other glucocorticoids such as hydrocortisone and methylprednisolone, but not estradiol, progesterone, or testosterone, reduced surface F&U expression in mast cells. DEX did not affect mast cell surface FqRII/III levels assessed using 2.462 mAb. In conclusion, glucocorticoids decrease the surface FcERI levels in mast cells incubated in both the presence and absence of IgE. The inhibitory effect of glucocorticoids on mast cell surface FcERI expression may be one of useful anti-allergic actions of the drugs. Comparison of the Priming Effect of Two Polymorphic Forms of IL-3 on Leukotriene Production A Schweiger. M Baldini, IC Lehman, DA Stem, FD Martinez, M Halonen Respiratory Sciences Center, University of Arizona, Tucson, AZ A polymorphism in the coding region of the IL-3 gene, located on chromosome 5q (1) leads to a change in the amino-acid sequence in the protein in residue 27 (proline to serine). These two different forms of rhIL-3 are available from commercial sources (Biosource”’ (proline) and R&DTM and GibcoTM (serine)). We sought to determine if these two different forms of IL-3 varied in function. IL-3 primes basophils for the production of sulfidoleukotrienes upon stimulation with anti-IgE. We obtained blood from 8 healthy adult volunteers and performed a dextran separation. The resulting mixed cell populations were primed with the two forms of IL-3 for various periods and then stimulated with anti-IgE. Production of combined LTC,, LTD, and LTE, (pg/ml) was measured by ELISA (Buehlmann, Switzerland). The values after 24 hrs of priming are shown,
Leukotrienes
(pglml)
mean
fold (SD)
Rh-IL3
IO-12 10-10 1O-s
Proline 0.99 (0.21) 1.76 (1.16) 2.74 (1.27)
increase
over
control
(n=8) Serine 1.29 (0.44) 2.90 (1.52) 3.21 (1.21)
We conclude that the binding of two naturally occuring hIL-3 to the IL-3 receptor differs in affinity or efftcacy. tJeong MC, Navani A, Oksenberg JR. (1998) Molecular lar Probes, 12:49-53.
p-Value 0.023 0.016 0.008 forms
of
& Cellu-
2000
199
Olopatadine Inhibits TNFa Release From Human Conjunctival Mast Cells Ellen 8. Cook. James L. Stahl, Neal b? Barney, Frank M. Graziano University of Wisconsin-Madison TNFa release from human conjunctival mast cells likely plays a crucial role in allergic ocular inflammation via increasing ICAM- 1 on epithelial cells and triggering other proinflammatory events. This immediate and prolonged release of TNFa in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined.Olopatadine (PatanolB) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine. tryptase, and PGD2 release in vitro and promote decreased HI receptor binding activity in vitro and functional Hl receptor antagonism in viva. The objective of these studies was to investigate the effect of olopatadine on TNFa release from purified human conjunctival mast cells challenged with anti-IgE antibody. Human conjunctival mast cells were purified (>95Yo) from cadaveric tissues using a procedure combining enzymatic digestion and Pertoll@ gradient centrifugation. These purified mast cells were incubated with olopatadine for 30 min then challenged with anti-IgE antibody for 90 min. Supernatants were analyzed for TNFa. Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFu release in a concentration dependent manner (optimum concentration for TNFa release was IO ug/ml anti-IgE antibody). Pre-incubation with olopatadine resulted in a dose dependent decrease in anti-IgE antibody mediated TNFa release (IC50 = 13. I PM). A concentration of 3mM olopatadine reduced TNFa release to the level of unchallenged controls. Olopatadine inhibited anti-IgE antibody mediated release of TNFa from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.
191
Inhibitory Effect of Rupatadine on TNF-alpha Release From Human Monocytes and Mast Cell Line HMC-1 M MerIm*. I Ramis*, D Balsa*, M Queraltf, P Brazist. A Plrigdemontt *Uriach Research Center and *Department of Pharmacology, Veterinary, UAB. Barcelona, Spain Mast cells are not only major contributors to the early response in allergic processes but also are able to synthesize a variety of cytokines which play a key role in the development of the late phase reactions. Among these cytokines, TNF-alpha is of particular importance in causing allergic inflammation. We compared the effects of rupatadine (RUP), a new antihistamine and antiallergic drug, with those of loratadine (LOR) and cetirizine (CET) on TNF-alpha release. Human mast cells HMC-1 were cultured in Iscove’s medium with 10% fetal calf serum and stimulated with 0.3 pM A23 187 and 80 nM tetradecanoyl phorbol acetate. After incubation in the presence or absence of test compounds (0.1-30 PM, 3 h, 37°C). TNF-alpha release was measured by bioassay. RUP, LOR. and CET inhibited TNF-alpha release, with ICso values of 2.0 f 0.9.2.1 rt 1.1 and 7.7 + 2.6 PM, respectively. Inhibition was further confirmed in mononuclear cells isolated from human donors and stimulated with bacterial lipopolysaccharide (026:B6 10 pg/ml, 19 h. 37°C). RUP and CET (10 pM) practically abrogated TNF-alpha release, whereas LOR showed 57% inhibition. In conclusion, RUP and also antihistamines LOR and CET are able to inhibit TNF-alpha release from human mast cells and monocytes. This effect could be beneficial in the late phase reactions present in allergic inflammation.