Glucocorticoids inhibit chemokine generation by human eosinophils

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Presentation transcript:

Glucocorticoids inhibit chemokine generation by human eosinophils Misato Miyamasu, PhDa, Yoshikata Misaki, MD, PhDb, Shinyu Izumi, MDa, Toshiaki Takaishi, MD, PhDa, Yutaka Morita, MD, PhDa, Haruji Nakamura, PhDc, Kouji Matsushima, MD, PhDd, Tadashi Kasahara, PhDe, Koichi Hirai, MD, PhDa Journal of Allergy and Clinical Immunology Volume 101, Issue 1, Pages 75-83 (January 1998) DOI: 10.1016/S0091-6749(98)70196-4 Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 1 Effect of DEX on released or cell-associated IL-8 of C5a-stimulated eosinophils. Eosinophils (n = 6) were incubated with DEX or control vehicle for 5 minutes. Cells were then stimulated with C5a (10–7 mol/L) plus CB (5 μg/ml) for 18 hours. Supernatants (open columns) were harvested by centrifugation and cell lysates (closed columns) were obtained by the addition of 0.5% Nonidet P-40 to the pellets. Immunoreactive IL-8 was measured by ELISA. Bars represent SEM. EtOH, Ethanol. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 2 Effect of DEX on IL-8 or MCP-1 production by eosinophils. Eosinophils were incubated with DEX for 5 minutes and then stimulated with C5a (filled circles, 10–7 mol/L) plus CB (5 μg/ml), FMLP (filled squares, 10–6mol/L) plus CB or ionomycin (open circles, 1 μmol/L) for 18 hours. Immunoreactive IL-8 (A) or MCP-1 (B) in cultures were assayed by ELISA. Value is expressed as a percentage of control production initiated by each stimulus in absence of DEX. Control IL-8 production by C5a, FMLP, or ionomycin was 2553 ± 1304 pg, 1229 ± 220 pg, and 3504 ± 1972 pg/106 eosinophils, respectively. Control MCP-1 production induced by C5a, FMLP, or ionomycin was 789 ± 237 pg, 523 ± 90 pg and 1858 ± 820 pg/106 eosinophils, respectively. Values in parentheses indicate number of experiments. IOM, Ionomycin. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 3 Effects of delayed addition of DEX on C5a-induced eosinophil chemokine generation. Eosinophils (n = 4) were treated with DEX (10–7 mol/L) for the indicated times before or after the addition of C5a (10–7 mol/L) plus CB (5 μg/ml). After 18 hours of stimulation, IL-8 (A) and MCP-1 (B) were assayed in cultures by ELISA. Values are expressed as percentage of control production (1307 ± 435 pg and 1516 ± 568 pg/106 eosinophils for IL-8 and MCP-1, respectively) induced by C5a in the absence of DEX. NS, Not significant. *p < 0.05, **p < 0.01. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 4 Effect of various steroids on C5a-induced chemokine generation by eosinophils. Eosinophils were incubated with hydrocortisone (open circles), methylprednisolone (filled circles), testosterone (filled triangles), estradiol (open triangles), or progesterone (filled squares) for 5 minutes. Cells were then stimulated with C5a (10–7 mol/L) plus CB (5 μg/ml) for 18 hours and IL-8 (A) or MCP-1 (B) in cultures were assayed by ELISA. Value is expressed as percentage of control production induced by C5a in absence of steroids. Values in parentheses indicate number of experiments. HC, Hydrocortisone; MP, methylprednisolone; Test, testosterone; Est, estradiol; Prog, progesterone. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 5 Effect of DEX on C5a-induced chemokine generation by IL-5-primed eosinophils. Eosinophils were preincubated with (open circles) or without (filled circles) 10 nmol/L IL-5 for 30 minutes, after which the cells were incubated with DEX for 5 minutes. Cells were then stimulated with C5a (10–7 mol/L) plus CB (5 μg/ml) for 18 hours. IL-8 (A, n = 3) or MCP-1 (B, n = 3) in cultures were assayed. IL-8 production induced by C5a from nonprimed and primed eosinophils was 625 ± 287 pg and 2438 ± 811 pg/106 eosinophils, respectively. MCP-1 production induced by C5a from nonprimed and primed eosinophils was 415 ± 85 pg and 2186 ± 872 pg/106 eosinophils, respectively. Values are expressed as percentage of control production from nonprimed and non-drug-treated eosinophils stimulated with C5a plus CB. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 6 Inhibition of C5a- or ionomycin-induced IL-8 mRNA accumulation by DEX. Eosinophils were preincubated with or without DEX for 5 minutes and then stimulated with C5a (open circles, 10–7 mol/L) plus CB (5 μg/ml) or ionomycin (filled circles, 1 μmol/L) for 2 hours. The RNA was reverse-transcribed to cDNA. A, A 40-cycle PCR was performed, and products were electrophoresed on agarose gel followed by staining with ethidium bromide. Representative experiment is shown. B, IL-8 mRNA level was determined by competitive PCR as described in Methods. Value is expressed as percentage of control (i.e., non-drug-treated eosinophils stimulated with the corresponding stimulus). Blank value (i.e., IL-8 mRNA level of eosinophils cultured with CB) is also shown (filled squares). Each point represents the mean ± SEM of three different experiments. IOM, Ionomycin. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions

FIG. 7 Inhibition of C5a- or ionomycin-induced MCP-1 mRNA accumulation by DEX. Eosinophils were preincubated with or without DEX for 5 minutes and then stimulated with C5a (filled circles, 10–7 mol/L, n = 4) plus CB (5 μg/ml) or ionomycin (filled squares, 1 μmol/L, n = 3) for 2 hours. RNA was reverse-transcribed to cDNA and amplified with primers for β-actin or human MCP-1. A, 30-cycle PCR was performed, and products were electrophoresed on agarose gel followed by staining with ethidium bromide. Representative experiment is shown. B, Ratios of MCP-1 to β-actin were determined by ELISA as described in Methods. Value is expressed as percentage of control (i.e., non-drug-treated eosinophils stimulated with the corresponding stimulus). Open symbols indicate value of eosinophils exposed to control vehicle (blank). IOM, Ionomycin. Journal of Allergy and Clinical Immunology 1998 101, 75-83DOI: (10.1016/S0091-6749(98)70196-4) Copyright © 1998 Mosby, Inc. Terms and Conditions