Leukotriene D4 induces gene expression in human monocytes through cysteinyl leukotriene type I receptor Grzegorz Woszczek, MD, PhD, Li-Yuan Chen, PhD,

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Presentation transcript:

Leukotriene D4 induces gene expression in human monocytes through cysteinyl leukotriene type I receptor Grzegorz Woszczek, MD, PhD, Li-Yuan Chen, PhD, Sahrudaya Nagineni, MS, Steven Kern, BS, Jennifer Barb, MS, Peter J. Munson, PhD, Carolea Logun, MS, Robert L. Danner, MD, James H. Shelhamer, MD Journal of Allergy and Clinical Immunology Volume 121, Issue 1, Pages 215-221.e1 (January 2008) DOI: 10.1016/j.jaci.2007.09.013 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 LTD4 induces EGR2 and FOSB genes in a time-dependent fashion. Monocytes were stimulated with LTD4 (100 nmol/L) for up to 4 hours, and mRNA expression was measured by means of TaqMan analysis. Data are presented as a fold change in comparison with vehicle (ethanol)–treated cells. Mean ± SD; n = 3. Journal of Allergy and Clinical Immunology 2008 121, 215-221.e1DOI: (10.1016/j.jaci.2007.09.013) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Calcium mobilization responses to CysLTs. Monocytes (A) and CysLTR1-transfected HEK293 cells (B) were prepared and calcium release was measured as indicated in the Methods section. Data are presented as means from 2 separate experiments performed in triplicate. Empty pcDNA-transfected HEK293 cells treated with different concentrations of LTD4 were used as controls. Journal of Allergy and Clinical Immunology 2008 121, 215-221.e1DOI: (10.1016/j.jaci.2007.09.013) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Characterization of calcium mobilization responses to LTD4 in monocytes. Elutriated monocytes were prepared and calcium release was measured as indicated in the Methods section. Cells were preincubated with inhibitors or vehicle control as indicated in the Results section and stimulated with LTD4 (100 nmol/L). Data from one of 3 experiments from 3 donors, each with similar results, are shown. Journal of Allergy and Clinical Immunology 2008 121, 215-221.e1DOI: (10.1016/j.jaci.2007.09.013) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 The effect of LTD4 on MAPK activation. Monocytes were stimulated with LTD4 (100 nmol/L) for different times, and phosphorylation of p44/42 and p38 was measured (A). Cells were preincubated with MK571 (100 nmol/L) for 10 minutes and stimulated with LTD4 (100 nmol/L) for 10 minutes (B). Data are representative of 3 independent experiments. Journal of Allergy and Clinical Immunology 2008 121, 215-221.e1DOI: (10.1016/j.jaci.2007.09.013) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Characterization of the pathway leading to LTD4-induced EGR2 and FOSB mRNA upregulation. Monocytes were preincubated with MK571 (MK; 100 nmol/L for 10 minutes), pertussis toxin (PTX; 100 ng/mL overnight), 2APB (50 μmol/L for 10 minutes), SB203580 (SB35; 5 μmol/L for 30 minutes), PD98059 (PD; 10 μmol/L for 30 minutes), BAPTA/AM (30 μmol/L for 30 minutes) (A and B) or with different concentrations of SB202190 (in micromoles per liter; 30 minutes) and U0126 (in micromoles per liter; 30 minutes) stimulated with LTD4 (100 nmol/L) for 30 minutes (C and D) and subjected to TaqMan analysis. Data are shown as a fold change in comparison with control vehicle-treated cells or expressed as the percentage of fold change induced by LTD4. Mean ± SD; n = 6 to 9. All inhibitors significantly inhibited LTD4-induced gene expression (P < .001 in comparison with LTD4 only–treated cells by using the Student t test). Journal of Allergy and Clinical Immunology 2008 121, 215-221.e1DOI: (10.1016/j.jaci.2007.09.013) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Chemotactic responses of monocytes to LTD4. A, Chemotactic activity of monocytes to different concentrations of LTD4 or CCL2 (5 nmol/L) as a positive control (Con) was measured as described in the Methods section. LTD4 induces chemotaxis of monocytes in a dose-dependent manner (∗P < .001, ANOVA). B, Cells were preincubated with MK571 (MK; 100 nmol/L for 10 minutes), pertussis toxin (PTX; 100 ng/mL overnight), SB203580 (SB35; 5 μmol/L for 30 minutes), SB202190 (SB21; 1 μmol/L for 30 minutes), PD98059 (PD; 10 μmol/L for 30 minutes), and U0126 (10 μmol/L for 30 minutes) and stimulated with LTD4 (100 nmol/L). Data from 3 different donors are presented as the migration index in comparison with vehicle control or percentage of migration index induced by LTD4 (mean ± SD). Journal of Allergy and Clinical Immunology 2008 121, 215-221.e1DOI: (10.1016/j.jaci.2007.09.013) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions