Sequential engagement of FcεRI on Mast Cells and Basophil Histamine H(4) Receptor and FcεRI in Allergic Rhinitis

Abstract

Histamine H(4) receptor (H(4)R)-deficient mice (H(4)R(-/-)), H(4)R antagonist-treated wild-type (WT) mice, and WT mice depleted of basophils failed to develop early (EPR) or late phase (LPR) nasal responses following allergen sensitization and challenge. Basophil transfer from WT but not H(4)R(-/-) mice restored the EPR and LPR in H(4)R(-/-) mice. Following passive sensitization with OVA-specific IgE, FcεRI(-/-) recipients of WT basophils plus OVA and histamine developed an EPR and LPR. OVA-IgE passively sensitized FcεRI(-/-) recipients of H(4)R(-/-) basophils and OVA and histamine challenge failed to develop an EPR or LPR, and basophils were not detected in nasal tissue. In contrast, recipients of basophils from IL-13(-/-) and IL-4(-/-)/IL-13(-/-) mice developed an EPR but not an LPR. These results demonstrate the development of allergic rhinitis proceeded in two distinct stages: histamine release from FcεRI-activated mast cells, followed by histamine-mediated recruitment of H(4)R-expressing basophils to the nasal cavity and activation through FcεRI.

Figure 1

Early phase (EPR) and late phase (LPR) nasal responses in OVA sensitized and challenged WT and H₄R^−/− mice. (A) EPR was monitored as changes in respiratory frequency (RF) over a period of 32 minutes after the 4^th OVA challenge. (B) LPR was detected as a change in RF 24 hrs after the 6^th OVA challenge. (C) Nasal cavity resistance (R_NA) in the LPR and (D) cytokine levels in nasal cavity lavage fluid. OVA/saline; sensitized to OVA but challenged with saline. OVA/OVA; sensitized and challenged with OVA. Shown are the means±SEM, n=9–14 in each group. **P<0.01 compared to WT OVA/saline group. ##P<0.01 compared to WT OVA/OVA group.

Figure 2

Effects of H₄R antagonist JNJ7777120 treatment on OVA-induced nasal responses in the EPR and LPR of WT mice. The inhibitor (1, 5, or 10 mg/kg) was administered 2 hrs prior to each challenge. Changes in (A) EPR, (B) LPR, (C) R_NA, (D) cytokine levels in nasal cavity lavage fluid. Shown are the means±SEM, n=9–14 in each group. *P<0.05, **P<0.01, compared to OVA/saline vehicle group, #P<0.05, ##P<0.01 compared to OVA/OVA vehicle group.

Figure 3

Histamine levels in nasal cavity lavage fluid 2 minutes following the 4^th saline or 4^th OVA challenge in OVA sensitized WT or FcεRI^−/− mice. Shown are the means±SEM, n=9–14 in each group. **P<0.01, compared to OVA/saline vehicle group, ##P<0.01 compared to OVA/OVA vehicle group.

Figure 4

(A) Gating strategy for purification of basophils from bone marrow cells. Bone marrow cells from mice treated with IL-3 and anti-IL-3 antibody were stained with anti-mouse CD49b-PE, anti-mouse FcεRIα-FITC and anti-mouse CD117 (c-Kit)-APC after FcγR blockade with anti-mouse CD16/CD32 antibody. Basophils were purified by sorting (MoFlo XDP, Becton Coulter, Inc., Brea, CA). (a) Bone marrow cells were gated on FSC and SSC (region R1; 74.3% of total cells) and (b) region R1 was gated on CD117-APC-negative and FcεRIα-FITC-positive cells (region 2), and then cells were collected (c) FcεRIα-FITC- and CD49b-PE-double positive populations (region R3). After sorting of FcεRIα- and CD49b-double positive but CD117-negative populations, post-sorting validation was performed (d; FcεRIα-FITC and CD49b-PE, 99.7% purity). (B) H₄R expression on WT basophils. CD117-negative, FcεRIα- and CD49b-double-positive populations were stained with anti-H₄R rabbit antibodies followed by goat anti-rabbit IgG antibody. (C) In vitro basophil chemotaxis assay to histamine. The chemotactic effects of 0, 0.01, 0.1, 1, 10, and 100 μM histamine on basophils from WT or H₄R^−/− mice were examined using 24-well plate and Transwell^® inserts and counting cells that migrated through to the bottom. Shown are the means±SEM, n=5 in each group. *P<0.05, **P<0.01 compared to the 0 μM histamine and H₄R^−/− basophils, ##P<0.01, compared to 1 μM histamine. (D) In vitro basophil cytokine production. Purified basophils (1×10⁵ cells/well) were incubated with/without 10 μg/ml OVA and/or 100 μg/ml OVA-specific or DNP-specific IgE for 24 h. Some groups of cells were incubated with 1 μM histamine for 24 h. Shown are the means±SEM, n=6 in each group. **P<0.01 compared to medium group.

Figure 5

(A) Effects of basophil depletion following Ba103 antibody administration. Ba103 Ab (50 μg/mouse) was administrated to WT mice and depletion was confirmed counting CD117-negative, FcεRIα-positive cells, (a) Ba103 antibody, (b) Isotype control antibody on day 4 and on day 8, (c) Ba103 antibody, (d) Isotype control antibody. (a, b) Peripheral blood basophils were depleted following Ba103 antibody treatment by 82–85% on day 4, (c, d) peripheral blood basophils were depleted by Ba103 antibody by 81–86% on day 8. The effects of basophil depletion on OVA-induced nasal responses in WT mice. Basophil depletion antibody Ba103 or isotype control antibody was administered one day prior to initiation of 4 or 6 consecutive days of allergen challenge. (B) EPR, (C) LPR, (D) R_NA, and (E) cytokine levels in nasal cavity lavage fluid. Shown are the means±SEM, n=6 in each group. ##P<0.01 compared to the OVA/OVA isotype control antibody-treated group. **P<0.01 compared to OVA/saline group.

Figure 6

Transfer of WT but not H₄R^−/− basophils restores nasal responsiveness in sensitized and challenged H₄R^−/− recipients. Basophils isolated from WT or H₄R^−/− donors were transferred 2 hrs prior to the 4^th OVA challenge. Changes in (A) EPR, (B) LPR, (C) R_NA, and (D) cytokine levels in nasal cavity lavage fluid. Shown are the means±SEM, n=6–9 in each group. ##P<0.01 compared to H₄R^−/− recipients of H₄R^−/− basophils.

Figure 7

The combination of WT basophils, OVA-IgE passive sensitization, and OVA challenge with histamine restores nasal responsiveness in FcεRI^−/− recipients accompanied by increased basophil numbers in the nasal cavity. FcεRI^−/− recipient mice were passively sensitized with OVA-specific IgE or DNP-specific IgE followed by a single OVA or saline challenge with/without histamine. Basophils from WT, H₄R^−/−, or IL-13^−/− donors were transferred into recipients 2 hrs prior to the single challenge. (A) EPR, (B) LPR (16 hrs after the single challenge), (C) R_NA, and (D) cytokine levels in nasal cavity lavage fluid. Nasal lavage fluid was collected 16 hrs after the single challenge. (E) Histamine levels in nasal cavity lavage fluid from OVA-specific IgE passively sensitized WT and OVA-specific IgE passively sensitized FcεRI^−/− mice with/without WT basophil transfer. Nasal cavity lavage fluid was collected 2 minutes after the single OVA challenge. Shown are the means±SEM, n=6–9 in each group. ##P<0.01 compared to all other groups.

Figure 8

Detection of transferred basophils in nasal cavity lavage fluid. (A) Identification of transferred basophils in nasal cavity lavage fluid. Sorted basophils (CD117-APC-negative, FcεRIα-FITC- and CD49b-PE-double positive populations) were labeled with CellVue^® Maroon and then transferred into passively sensitized FcεRI^−/−recipient mice. Nasal cavity lavage fluids were collected in 1 ml HBSS and suspended in 1% formalin, 2 minutes after a single challenge. Labeled, live cells in nasal cavity lavage fluids were first gated on FSC/SSC (dot plot, panels a–e). Within the gated cells, numbers of labeled cells (transferred basophils) were identified (FL4). (panels a, f): Nasal cavity lavage fluid from FcεRI^−/− recipients treated with vehicle only following passive sensitization with OVA-specific IgE and OVA-histamine challenge; (panels b, g): FcεRI^−/− recipients of WT basophils following passive sensitization with OVA-specific IgE and OVA challenge; (panels c, h): FcεRI^−/− recipients of H₄R^−/− basophils following sensitization with OVA-specific IgE and OVA-histamine challenge; (panels d, i): FcεRI^−/−recipients of WT basophils following sensitization with OVA-specific IgE and OVA-histamine challenge; or (panels e, j): FcεRI^−/− recipients of WT basophils following sensitization with OVA-specific IgE and challenge with histamine alone. (B) Basophil numbers in nasal lavage fluid, which was collected 2 minutes after a single OVA or saline challenge of passively sensitized FcεRI^−/− mice. Basophil numbers were increased in recipients of WT but not H₄R^−/− basophils and following histamine (20 nmol/mouse) challenge. Shown are the means±SEM, n=6 in each group. ##P<0.01 compared to mice which did not receive basophils, or received H₄R^−/− basophils, or recipients which received OVA-vehicle challenge.

Figure 9

Transfer of OVA-specific IgE but not DNP-specific IgE pretreated WT basophils restores nasal responsiveness in non-sensitized but OVA-histamine challenged FcεRI^−/− recipient mice in both EPR and LPR. Isolated basophils from WT or H₄R^−/− mice were transferred into non-sensitized FcεRI^−/− mice 2 hrs prior to a single challenge of OVA or saline with/without 20 nmol/mouse histamine. (a) EPR, (b) LPR (16 hrs after the EPR), (c) R_NA. ##P<0.01 compared to all other groups.

Figure 10

Model for development of EPR and LPR in AR. Step 1; activation of mast cells (MC) in the nasal tissue following crosslinking of FcεRI resulting in the release of histamine. Step 2; histamine binds to H₄R on basophils (Ba). Step 3; leading to migration of basophils to the nasal tissue. Step 4; activation of basophils in the nasal tissue following cross-linking of FcεRI resulting in early phase (EPR) mediator release and late phase (LPR) cytokine production.