Expression and function of histamine receptors in human monocyte-derived dendritic cells Marco Idzko, MDa, Andrea la Sala, PhDb, Davide Ferrari, PhDc,

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Expression and function of histamine receptors in human monocyte-derived dendritic cells Marco Idzko, MDa, Andrea la Sala, PhDb, Davide Ferrari, PhDc, Elisabeth Panther, a, Yared Herouy, MDa, Stefan Dichmann, MDa, Maja Mockenhaupt, MDa, Francesco Di Virgilio, MDc,d, Giampiero Girolomoni, MDb, Johannes Norgauer, MDa Journal of Allergy and Clinical Immunology Volume 109, Issue 5, Pages 839-846 (May 2002) DOI: 10.1067/mai.2002.124044 Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 1 Human DCs express the mRNA for histamine receptors. DCs were prepared and matured as detailed in the Methods section. Experiments were repeated 5 times with identical results. Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 2 Histamine induces [Ca2+]i transients in immature DCs but not in mature DCs. Immature (A-C) and LPS-matured DCs (D) were loaded with Fura-2/AM and stimulated with different concentrations of histaminergic agonists. A, Histamine. B, Betahistine (H1 receptor agonist). C, R-α-MeH (H3 agonist). D, MIP-3β, but not histamine, induces Ca2+ transients in mature DCs. One representative of 7 similar experiments is shown. Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 3 Histamine induces actin polymerization in immature DCs but not in mature DCs. A, Cells were stimulated with grading doses of histamine. B, Betahistine and R-α-MeH, but not dimaprit (H2 receptor agonist), induce actin polymerization in immature DCs. The relative f-actin content was analyzed by means of flow cytometry after 25 seconds, as reported in the Methods section. Control experiments with RANTES (100 ng/mL) in immature DCs and with MIP-3β (100 ng/mL) in mature DCs showed relative f-actin content values of 2.12 ± 0.07 and 2.16 ± 0.08, respectively. Data are expressed as means ± SEMs (n = 5). Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 4 Histamine is chemotactic for immature DCs but for not mature DCs. Cells were exposed to the indicated histaminergic drugs at 37°C for 90 minutes in a Boyden chamber. The chemotactic index was calculated as reported in the Methods section. A, Histamine promotes the migration of immature DCs but not mature DCs. Control experiments with C5a in immature DCs and MIP-3β in mature DCs showed that both types of cells are able to migrate toward appropriate stimuli. B, The H1 and H3 receptor agonists, but not the H2 agonist, are chemotactic for immature DCs. C, The H1 and H3 receptor antagonists, diphenhydramine and thioperamide, reduce the chemotactic activity of histamine. Data are expressed as means ± SEMs (n = 5). **P < .001 and *P < .01 vs DCs treated with histamine alone. Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 5 Histamine, dimaprit, and R-α-MeH inhibit IL-12 release (A) and stimulate IL-10 secretion (B) from maturing DCs. Cells were incubated with LPS in the presence or absence of the indicated agonists. Supernatants were collected after 24 hours, and cytokines were measured by ELISA. One representative of 3 or 4 similar experiments is shown. Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 6 Histamine increases cAMP levels in mature DCs. A, Immature and mature DCs were treated with the indicated histamine concentrations. Forskolin (10−5 mol/L) was used as a positive control. B, Stimulation of mature DCs with dimaprit or R-α-MeH, but not with betahistine, also resulted in cAMP increase. The index was calculated as the ratio between stimulated cells and control medium. Data are means ± SEMs (n = 4). *P < .05 vs untreated DCs. Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 7 DCs matured in the presence of histamine are impaired in their capacity to initiate type 1 responses in vitro. Immature DCs were stimulated with and without 10−5 mol/L histamine in the presence or absence of LPS. After 24 hours, purified allogeneic CD45RA+ naive T lymphocytes were primed with generated DCs, and intracellular IFN-γ and IL-4 in T cells were analyzed by means of flow cytometry after 6 hours. Numbers indicate the percentages of positive cells in each quadrant. Data for 1 representative experiment are shown. Experiments were repeated 4 times with identical results, and the percentages of IFN-g - and IL-4-producing T cells stimulated by mature DCs were 60% ± 12% and 8% ± 5%, respectively (means ± SDs), whereas T cells activated by histamine-maturing DCs were 18% ± 5% IL-4+ and 20% ± 6% IFN-g+ (mean ± SD). Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions

Fig. 8 Biological function and signal transduction mechanisms of histamine in DCs. Journal of Allergy and Clinical Immunology 2002 109, 839-846DOI: (10.1067/mai.2002.124044) Copyright © 2002 Mosby, Inc. Terms and Conditions