H4 histamine receptor mediates optimal migration of mast cell precursors to CXCL12 Veronique Godot, PhD, Michel Arock, MD, Gilles Garcia, MD, Francis.

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H4 histamine receptor mediates optimal migration of mast cell precursors to CXCL12 Veronique Godot, PhD, Michel Arock, MD, Gilles Garcia, MD, Francis Capel, Carine Flys, Michel Dy, MD, Dominique Emilie, MD, Marc Humbert, MD Journal of Allergy and Clinical Immunology Volume 120, Issue 4, Pages 827-834 (October 2007) DOI: 10.1016/j.jaci.2007.05.046 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Histamine modulates CXCL12 activity on human PrMCs. A, Number of migrated HMC1 cells toward histamine alone or in association with CXCL12 (n = 10). B, Migration of PrMCs to CXCL12, SCF, and LTB4 with or without histamine (n = 5). Migrations with histamine alone were 0.78% ± 0.21% cells. C, Checkerboard analysis of HMC1 cell migration (n = 5). ∗P < .05. Journal of Allergy and Clinical Immunology 2007 120, 827-834DOI: (10.1016/j.jaci.2007.05.046) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Signaling through the H4 receptor (H4R) enhances CXCL12-driven migration. A, Western blot analysis of histamine receptor expression in MCs. B, Migration of HMC1 cells to CXCL12 and histamine after incubation with 10−5 mol/L or 10−6 mol/L pyrilamine, cimetidine, thioperamide, or JNJ (left); inhibition of histamine receptor expression by specific siRNA (right). Results are expressed as percentage of histamine activity on CXCL12-driven migration (n = 4). C, Migration of HMC1 cells toward CXCL12 and clobenpropit (Clobpt; n = 7). ∗P < .05. Journal of Allergy and Clinical Immunology 2007 120, 827-834DOI: (10.1016/j.jaci.2007.05.046) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Culture supernatants from IgE-activated mature MCs modulate CXCL12 activity on HMC1 cells. A, Number of migrated HMC1 cells to CXCL12 combined with supernatants (1:100 dilution) obtained from cultures of FcɛRI-activated MCs or their controls (IgE-sensitized MCs without anti-IgE, IgG control cross-linking; n = 6). B, The same experiment as described above was done with HMC1 cells previously incubated with thioperamide or control vehicle. ∗P < .05. Journal of Allergy and Clinical Immunology 2007 120, 827-834DOI: (10.1016/j.jaci.2007.05.046) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Histamine/CXCL12 synergy is specific of the MC population. Migration of human CD4+ T cells (A) or monocytes (B) to CXCL12 with or without histamine. Results are expressed as the number of migrated cells (n = 3). Journal of Allergy and Clinical Immunology 2007 120, 827-834DOI: (10.1016/j.jaci.2007.05.046) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Effects of histamine on HMC1 cytoskeletal reorganization induced by CXCL12. Levels of polymerized actin (F-actin) were evaluated as a final readout of HMC1 cytoskeletal reorganization. A, Percentage of F-actin in response to CXCL12, histamine, or their combination (n = 5). B, Responses to CXCL12 after incubation (30 minutes) with histamine (10−6 mol/L) or medium (n = 5). ∗P < .05. Journal of Allergy and Clinical Immunology 2007 120, 827-834DOI: (10.1016/j.jaci.2007.05.046) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 CXCR4 expression on the cell surface is not modulated by histamine. A, RT-PCR detection of CXCR4 and CXCR7 in MCs. B, Flow cytometric analysis (empty histograms, anti-CXCR4 mAb; gray histograms, control mAb) of CXCR4 expression in HMC1 cells incubated for 30 minutes with histamine. C, CXCR4 recycling in HMC1 cells incubated for 10 and 90 minutes with CXCL12 alone or with histamine. Results are representative of 3 experiments. Journal of Allergy and Clinical Immunology 2007 120, 827-834DOI: (10.1016/j.jaci.2007.05.046) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions