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Histamine H4 receptor (H4R)-deficient mice (H4R−/−), H4R antagonist-treated 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 H4R−/− mice restored the EPR and LPR in H4R−/− 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 H4R−/− 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 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 H4R-expressing basophils to the nasal cavity and activation through FcεRI.
Allergic rhinitis (AR) is a common chronic disease, affecting more than 600 million people worldwide (1). The recurrent and chronic symptoms of allergic rhinitis, sneezing, itching, rhinorrhea, and nasal congestion, impair the quality of life in these patients and contribute to a major socio-economic burden. Similar to other allergic diseases, corticosteroids (topical) remain the most effective therapy in AR, despite major limitations (2). Only half of the patients gain adequate relief, while a large proportion suffers from persistent symptoms (Allergies in America: A landmark survey of nasal allergy sufferers (2006): Available online at www.mmcpub.com/scsaia/AdultSummary.pdf). New therapeutic targets in the treatment of AR are actively being investigated.
Histamine has long been recognized as an important mediator of allergic inflammation (3). To date, four subtypes of histamine receptors, H1, H2, H3, and H4 have been described (4, 5). Histamine H1 receptor antagonists (inverse agonists) have often been used as first-line therapy in AR, but they are only modestly effective at best (6). The histamine H4 receptor (H4R) has been identified most recently (7–11) and shown to be involved in the recruitment and activation of cells involved in allergic inflammatory responses, including eosinophils, T cells, dendritic cells, basophils, mast cells, and sensory nerve cells (12–15). In allergic rhinitis patients, the H4R was upregulated in nasal tissue (16). As a result, the H4R has been targeted in the treatment of allergic disease (17–21). For the most part, a primary focus in AR has been on mast cells. Mast cells share many features with basophils. Both cells express the high affinity IgE receptor (Fc epsilon receptor I, FcεRI) and secrete effector molecules such as histamine, platelet-activating factor (PAF), cysteinyl leukotrienes, and Th2 cytokines following activation by antigen-IgE crosslinking of FcεRI (22). Both play key roles in the development of allergic inflammation. Basophils, unlike mast cells which reside in the tissue, circulate in the blood and migrate to sites of inflammation. Although little is known about the direct role of basophils in allergic disease, several studies have revealed some unique characteristics that may be important to the initiation phase of the allergic response (23, 24). Limited studies have implicated basophils in the development of AR. Basophils have been demonstrated in nasal washes of patients with allergic rhinitis (25–27).
We established a method to induce nasal responsiveness in mice without influencing the lower airways, and demonstrated that increases in nasal resistance (RNA) were significantly correlated with decreases in respiratory frequency (RF), suggesting that RF is a valid, albeit indirect, indicator of nasal resistance or blockage in mice, which are obligate nasal breathers (28). In this experimental model of AR, we demonstrated that the combination of allergen, antigen-specific IgE, and FcεRI on mast cells resulted in the development of an early (EPR) or late phase (LPR) nasal response, the latter also dependent on IL-13 (28, 29). In the present study, we investigated the role of basophils in the development of the EPR and LPR in this model of AR. The results identified a critical role for the H4R on basophils in their recruitment to the nasal tissue.
Six week old female BALB/c mice were obtained from Harlan Laboratories (Indianapolis, IN) and H4 receptor-deficient (H4R−/−; BALB/c background) mice were initially provided by Dr. R. Thurmond, Johnson & Johnson Pharmaceutical Research and Development (San Diego, CA). FcεRI-deficient (FcεRI−/−; BALB/cByJ background) mice were originally provided by Drs. D. Dombrowicz and J. P. Kinet (Harvard Medical School, Boston, MA). IL-13-deficient (IL-13−/−) and IL-4−/−/IL-13−/− BALB/c mice were initially provided by Dr. D. Umetsu (Harvard Medical School, Boston, MA). All animals were maintained and bred in the animal facility at National Jewish Health (Denver, CO). Mice were housed under specific pathogen-free conditions and maintained on an OVA-free diet. All experimental animals used in this study were under a protocol approved by the Institutional Animal Care and Use Committee of National Jewish Health.
In this study, two models of AR were studied, one involving active sensitization and the other, passive sensitization. In the active sensitization approach, WT or H4R−/− mice were sensitized by intraperitoneal injection of 20 μg of OVA (Fisher Scientific, Pittsburgh, PA) emulsified in 2.25 mg of alum (Imject® Alum; Thermo Scientific Pierce Protein Research Products, Rockford, IL) in a total volume of 100 μl on days 0 and 14. From days 28–33, up to 6 daily nasal challenges with OVA (10% (w/v) in saline (15 μl for each nostril)) or saline alone as control were administered without anesthesia.
In the passive sensitization model, FcεRI−/− mice received OVA-specific IgE (2 μg/200 μl in HBSS, derived from OVA-specific IgE secreting hybridomas (30) or DNP-specific IgE (2 μg/200 μl in HBSS, Sigma-Aldrich) via the lateral tail vein on 2 consecutive days. Basophils from WT or H4R−/− mice were transferred 3 hrs after the last injection of IgE. Two hrs after basophil transfer, a single nasal challenge was performed in the same manner as described in the active sensitization model. Some groups of mice received histamine (0.2, 2, or 20 nmol/mouse) intranasally together with OVA.
Nasal responsiveness was assessed as described monitoring RF and RNA (28, 29, 31, 32). In the active sensitization model, RF values were assessed beginning immediately after the 4th OVA challenge using restrained single-chamber whole-body plethysmography (WBP; Buxco, Troy, NY). After the 4th OVA challenge, RF values were determined at 4–7, 9–12, 14–17, 19–22, 24–27, and 29–32 minutes (EPR). Twenty-four hrs after the 6th OVA challenge, RF and nasal cavity resistance (RNA) were determined (LPR). Baseline RF values were determined before the first challenge.
In the passive sensitization model, baseline RF values were obtained 2 hrs after basophil transfer. Following a single OVA challenge, EPR was monitored just after a single OVA or saline challenge with/without 20 nmol/mouse histamine or vehicle (15 μl for each nostril) and LPR (RF and RNA) was monitored 16 hrs after the EPR measurements. Measurements of RNA were carried out after RF measurements in the LPR as described.
A blunt 19-gauge needle was inserted into the nasopharynx through the pharynx and the nasal cavity was lavaged with 1.0 ml of ice-cold HBSS. Nasal cavity lavage fluids were then centrifuged at 480 × g at 4°C for 5 minutes, and the supernatants were collected and stored at −80°C until assayed.
IL-4, IL-5, and IL-13 levels in the nasal cavity lavage fluid were collected after the determination of RF and nasal cavity resistance for LPR and stored at −80°C until assayed. The cytokine levels were assessed by ELISA (eBioscience, San Diego, CA), according to the manufacturer’s directions. For the measurement of histamine levels in the active sensitization model, nasal cavity lavage fluid was collected 2 minutes after the 4th allergen challenge and stored at −80°C until assayed. Histamine levels were measured using an enzyme immunoassay kit (A05890, ALPCO Diagnostics, Salem, NH), as described by the manufacturer.
To determine the effect of basophil depletion, a basophil-depleting antibody, Ba103 (50 μg/mouse) which recognizes CD200R3 (23, 24, 33, 34) or isotype control rat IgG (50 μg/mouse) was injected into mice via the lateral tail vein one day before initiation of the challenges. Peripheral blood samples were obtained 4 or 8 days following treatment. Red blood cells in samples were lysed with Red Blood Lysing Buffer (Sigma-Aldrich, St. Louis, MO). Following blockade of FcγR with anti-mouse CD16/CD32 antibody, cells were stained with anti-mouse FcεRIα conjugated with PE (MAR-1; eBioscience) and anti-mouse CD117 (c-Kit) conjugated with APC (2B8; eBioscience). The labeled cells were analyzed by flow cytometry (Accuri C6, BD Biosciences).
The selective H4R antagonist, JNJ7777120, was purchased from Sigma Aldrich. JNJ7777120 (1, 5, 10 mg/kg) or vehicle control (0.5% methylcellulose-water) were gavaged 2.5 hrs prior to each OVA challenge on 6 consecutive days from days 28–33.
To mobilize basophils in bone marrow, 10 μg of mouse recombinant IL-3 (BD Biosciences, San Jose, CA) and 5 μg of anti-mouse IL-3 antibody (MP2-8F8; BD Biosciences) were mixed in 1.0 ml at room temperature for 1 minute and the IL-3/anti-IL-3 antibody complex solution was injected into WT or H4R−/− mice via the lateral tail vein (35). Four days after injection of IL-3/anti-IL-3 antibody, bone marrow cells were collected from femurs and tibias. After FcγR blockade with anti-mouse CD16/CD32 antibody (2.4G2; BD Biosciences), cells were labeled with anti-mouse CD49b conjugated with PE (DX5; eBioscience), anti-mouse FcεRIα conjugated with FITC (MAR-1; eBioscience), and anti-mouse CD117 (c-Kit) conjugated with APC (2B8; eBioscience). Labeled cells were sorted for CD117-negative, FcεRIα- and CD49b-double-positive populations using MoFlo XDP (Becton Coulter, Inc., Brea, CA). This provided a population of >95% basophils (Figure 4A, panel d). Purity of isolated basophils (FcεRIα-FITC- and CD49b-PE-double-positive but CD117-APC-negative populations) was confirmed using flow cytometry.
Expression of H4R on purified bone marrow basophils was examined by flow cytometry using anti-human H4R rabbit polyclonal antibody (MBL International Corporation, Woburn, MA). To identify the intracellular domain of the H4R, basophils were prefixed with 2% formalin followed by permeabilization with 0.1% saponin. After blocking of Fcγ receptors with anti-mouse CD16/CD32, anti-human H4R rabbit polyclonal antibody (MBL International Corporation, Woburn, MA) was used. These antibodies recognize the 1st cytoplasmic domain of human H4R which is >90% homologous to the mouse domain. Purified rabbit IgG (Invitrogen Corporation, Carlsbad, CA) was used as an isotype control. Then, goat anti-rabbit IgG conjugated with Alexa Fluor® 647 (Invitrogen Corporation) was added as the secondary antibody. The labeled cells were analyzed by flow cytometry (Accuri C6).
Purified basophils from WT or H4R−/− mice were suspended to 1×106 cells/ml in RPMI 1640 medium with 0.5% (w/v) bovine serum albumin (BSA). Six-hundred μl of the same culture medium containing 0, 0.01, 0.1, 1, 1, 10, or 100 μM of histamine were placed in the 24-well plates and 100 μl of suspended basophils were added onto the 5 μm pore size polycarbonate membrane Transwell® insert (6.5 mm size, Costar, Cambridge, MA). The plates were incubated for 2 hrs at 37°C, 5% CO2. Transwell inserts were removed gently and cells in the bottom chamber were resuspended in 100 μl of RPMI 1640 medium with 0.5% BSA and counted.
Purified basophils from WT or H4R−/− mice were suspended in RPMI 1640 medium containing 10% fetal calf serum and transferred into 96-well plate (1×105 cells in 100 μl/well). Basophils were incubated with histamine (1 μM) for 24 hrs. The dose of histamine was chosen based on maximum effects observed in chemotaxis assays. Basophils were also incubated with OVA-specific IgE (10 μg/ml) or DNP-specific IgE (10 μg/ml) followed by OVA (10 μg/ml) for 1 hr. After incubation, supernatants were collected and stored at −80°C until IL-4 and IL-13 measurements by ELISA.
In the active sensitization approach, basophils purified from H4R−/− or WT mice (0.6×106 cells/300 μl in HBSS) or HBSS alone as control were injected intravenously 2 hrs prior to the 4th OVA challenge in actively sensitized H4R−/− or WT mice. The numbers of basophils used in adoptive transfer experiments was determined from the literature (36–39) and preliminary experiments. In the passive sensitization model, basophils purified from H4R−/−, IL-13−/− or WT mice (0.6×106 cells/300 μl in HBSS) or HBSS alone were injected intravenously 2 hrs prior to a single OVA challenge in FcεRI−/− recipient mice.
Purified basophils from H4R−/− or WT mice (1×106 cells/100 μl in HBSS) were incubated with OVA-specific IgE (500 μg/ml) or DNP-specific IgE (500 μg/ml) for 30 minutes at room temperature. After washing with HBSS 3 times, basophils (0.6×106 cells in 300 μl of HBSS) or HBSS (vehicle control) were injected via the tail vein into naive FcεRI−/− mice. Two hrs after basophil transfer, a single nasal challenge was performed in the same manner as described in the active sensitization model. Some groups of mice received histamine (20 nmol/mouse) intranasally together with OVA followed by the measurements of EPR and LPR as described in the passive sensitization model.
To identify basophils in the nasal cavity, isolated basophils which were purified from either WT or H4R−/− mice and labeled with CellVue® Maroon (Ex max=647 nm, Em max=667 nm; Polysciences, Inc., Warrington, PA) were transferred into FcεRI−/− mice (0.6×106 cells/mouse) 3 hrs after passive sensitization with OVA-specific IgE or DNP-specific IgE and 2 hrs prior to a single OVA-vehicle, OVA-histamine, or histamine challenge. Two minutes after the challenge, the nasal cavity was lavaged with 1.0 ml of HBSS containing 1% formalin and collected into 1.5 ml tubes. Timing for the in vivo analyses following basophil transfer was determined based on earlier studies (36–39). Cells were resuspended in 50 μl of 0.5% (w/v) BSA-PBS followed by cell counting and flow cytometry (Accuri C6).
Values for all measurements are expressed as means±SEM. RF value changes were analyzed with repeated measures two-way ANOVA, followed by Bonferroni correction as a post-hoc test. All other values were analyzed with one-way ANOVA, followed by Bonferroni correction. Statistical analyses were performed using GraphPad Prism 5.0d (GraphPad Software Inc., La Jolla, CA). A P value <0.05 was considered statistically significant.
To identify the role of the H4R in the development of allergen-induced nasal responsiveness, we monitored RF and RNA in wild-type (WT) and H4R−/− mice. In the EPR, expressed as changes after the 4th ovalbumin (OVA) challenge, RF was significantly decreased in actively sensitized WT mice compared to saline challenged mice (Figure 1A). On the other hand, actively sensitized H4R−/− mice did not show a decrease in RF, similar to OVA sensitized but saline challenged WT or H4R−/− mice. In the LPR, actively sensitized WT mice demonstrated a significantly decreased RF whereas OVA sensitized and challenged H4R−/− mice did not show a significant change in RF (Figure 1B). In parallel, RNA in sensitized and OVA challenged WT mice was significantly higher than in saline challenged WT mice, whereas RNA in sensitized and OVA challenged H4R−/− mice was comparable to levels in sensitized and saline challenged WT or H4R−/− mice (Figure 1C). Analysis of cytokine levels in nasal cavity lavage fluid revealed that levels of IL-4, IL-5, and IL-13 were significantly increased in OVA sensitized and challenged WT mice, but not in H4R−/− mice (Figure 1D).
To confirm the mechanism underlying the failure of H4R−/− mice to develop nasal responses to allergen sensitization and challenge, we determined whether treatment with a selective H4R antagonist could mimic these findings in WT mice. As shown in Figure 2, H4R antagonist treatment of OVA sensitized WT mice during the OVA challenge period prevented the development of both the EPR and LPR (Figures 2A–C) in a dose-dependent manner. Cytokine analysis of nasal cavity lavage fluid showed that the increases in IL-4, IL-5, and IL-13 levels were also prevented by the inhibitor in a dose-dependent manner (Figure 2D).
To establish whether levels of histamine, a ligand of the H4R, were increased in this model, nasal cavity lavage fluids were analyzed soon after the 4th OVA challenge. As shown in Figure 3, histamine levels in nasal cavity lavage fluid were significantly increased 2 minutes following the 4th OVA challenge compared to the 4th saline challenge of OVA sensitized WT or FcεRI−/− mice. In contrast, following the 4th OVA challenge of FcεRI−/− mice which do not develop an EPR (28), nasal cavity histamine levels were extremely low. These data identify the importance of FcεRI-expressing resident mast cells as the major source of nasal cavity histamine (see above).
These results indicated a critical role for the H4R in the development of allergen-induced nasal responsiveness. In light of our previous demonstration that FcεRI was also essential in this setting (28), we focused on the role of the basophils, circulating cells known to express high levels of FcεRI. We first demonstrated that H4R was indeed expressed on basophils from WT mice (Figure 4B). To establish that the receptor was functional, chemotaxis assays were carried out using purified basophils from WT and H4R−/− mice. The number of basophils recovered from WT and H4R−/− mice were similar. Histamine induced WT basophil chemotaxis but not H4R−/− basophils, confirming the importance of the H4R in histamine-induced cell migration (Figure 4C). In addition, the capacity of basophils to produce cytokines was determined following stimulation with histamine or the combination of OVA and OVA-specific IgE. As shown in Figure 4D, the combination of OVA and OVA-specific IgE triggered the production of IL-4 and IL-13 from purified basophils, whereas histamine alone failed to do so. The levels of IL-4 and IL-13 following OVA and OVA-specific IgE stimulation of H4R−/− basophils were similar to levels from WT basophils (data not shown).
To determine whether basophils are involved in the development of allergen-induced EPR and LPR, basophils from OVA sensitized WT mice were depleted with Ba103 antibody prior to OVA challenge. Peripheral blood basophils were reduced by >80% up to 8 days following Ba103 antibody treatments (Figure 5A). The number of mast cells in nasal tissue was not altered in Ba103-treated mice (data not shown) confirming the results of a previous study (23). Following basophil depletion, the development of both the EPR (Figure 5B) and LPR (Figures 5C, 5D) were inhibited as were increases in Th2 cytokine levels in nasal cavity lavage fluid (Figure 5E).
To establish the critical requirement for H4R expression on basophils in the development of allergen-induced nasal responses, purified basophils from WT or H4R−/− mice were transferred to sensitized and challenged H4R−/− recipients prior to the 4th allergen challenge. Basophils from WT but not H4R−/− mice restored the development of the EPR and LPR, decreases in RNA, and increases in Th2 cytokine levels in nasal cavity lavage fluid (Figures 6A–D).
Together, these data identified the role of the H4R and basophils in the development of both the EPR and LPR. To more directly confirm the involvement of H4R-expressing basophils in these responses, FcεRI−/− recipients were passively sensitized with IgE to bypass the need for active sensitization and mast cells. As FcεRI−/− mice were not capable of releasing histamine into the nasal cavity (Figure 3), histamine was administered intranasally together with allergen in certain groups. The mice were passively sensitized with OVA-specific IgE or DNP-specific IgE and were recipients of basophils from WT, H4R−/−, IL-13−/−, or IL-4−/−/IL-13−/− mice. As shown in Figures 7A–C, only passively sensitized anti-OVA-IgE FcεRI−/− recipients of WT basophils and challenged with OVA and intranasal histamine developed both an EPR and LPR. In the absence of histamine or passive sensitization with the irrelevant anti-DNP-IgE, no EPR or LPR developed. Recipients of H4R−/− basophils failed to develop an EPR and LPR while recipients of IL-13−/− (or IL-4−/−/IL-13−/−, data not shown) basophils developed an EPR but not an LPR. In parallel, increases in nasal lavage fluid levels of IL-4 and IL-13 were only seen in the group which developed an EPR and LPR while recipients of IL-13−/− basophils which developed an EPR but not LPR demonstrated increases in IL-4 levels (Figure 7D). The response to histamine was dose-dependent; when mice were challenged with lower doses of histamine together with OVA, 2 nmol/mouse histamine induced modest nasal responses, but 0.2 nmol/mouse or lower doses did not elicit any nasal responses (data not shown).
To show the requirement for FcεRI on basophils in the development of nasal responses, purified basophils from WT or H4R−/− mice were cultured with OVA-specific IgE or DNP-specific IgE ex vivo prior to transfer into FcεRI−/− mice. Among the different groups of recipients, only the group that received OVA-specific, IgE-pretreated WT basophils followed by OVA-histamine challenge developed an EPR and LPR (Figure 9).
To establish that transferred basophils did indeed migrate into the target tissue, nasal cavity lavage fluid was collected 2 minutes after challenge following transfer of WT or H4R−/− basophils into the passively sensitized FcεRI−/− mice. Basophil numbers in nasal lavage fluid only increased following transfer of WT basophils after the OVA-histamine challenge (Figure 8B). Approximately 90% of the total cells were identified as basophils (Figure 8A). WT basophils were also detected in the nasal cavity lavage fluid of OVA-specific or DNP-specific IgE passively sensitized mice that were exposed to histamine even without OVA (Figure 8B), but they failed to develop any nasal responses. No basophils were detected in the absence of histamine or following transfer of H4R−/− basophils.
To identify which cell type in the nasal mucosa, a mast cell or basophil, was the initial source of histamine following activation through FcεRI, we assessed histamine levels in nasal cavity lavage fluid in OVA-specific IgE passively sensitized WT or FcεRI−/− mice. Histamine levels in passively sensitized WT mice were significantly increased on OVA challenge whereas passively sensitized FcεRI−/− mice, even those that received WT basophils, failed to increase the levels of histamine in the nasal cavity following OVA challenge (Figure 7E).
These data demonstrated that in the absence of mast cell activation (FcεRI−/− hosts), basophils expressing both FcεRI and the H4R were essential to the development of an EPR and LPR. Further, in the absence of mast cell activation (FcεRI−/− hosts), a source of histamine was also essential for the accumulation of basophils in the nasal cavity. Finally, to develop the LPR but not the EPR, the basophil release of IL-13 was also essential.
The symptoms of AR can be related to both the EPR and LPR with itching, sneezing, and rhinorrhea predominating in the EPR and congestion in the LPR. In order to better understand the underlying mechanisms, an experimental model of AR was developed in an attempt to mimic the human disease. A key to triggering nasal responsiveness without lower airway disease was intranasal administration of allergen without anesthesia and aspiration of allergen (28). In this approach, a distinct EPR and LPR could be elicited with changes in RF and RNA,, indicators of nasal obstruction, and the identification of individual pathways mediating these responses (28). RF was shown to correlate closely with RNA and a role for mast cells was demonstrated (29). In addition to the role of mast cells, the present study identified an essential role for basophils and their expression of both the H4R and FcεRI. In WT mice treated with an H4R antagonist administered during allergen challenge both the EPR and LPR were significantly reduced. To confirm these findings and exclude concerns about the specificity and potential off-target effects of the H4R antagonist, H4R−/− mice were also used. Allergen sensitized and challenged H4R−/− mice failed to develop both the EPR and LPR. These findings in receptor-deficient mice were confirmed In a different mouse model of AR, treatment with either an H4R antagonist, JNJ7777120, or H1R antagonist, ketotifen fumarate, prevented nasal symptoms such as sneezing and nasal rubbing perhaps through blockade of the H1R and H4R on nasal sensory nerves (31). Because of the high doses of the H1R antagonist used, the effects may reflect off-target activities (40). In addition to the effects on the EPR and LPR, antagonist treatment resulted in a significant reduction in Th2-type cytokine levels, IL-4, IL-5, or IL-13. Both IL-4 and IL-13 are central to OVA-specific antibody production and we previously demonstrated the importance of IL-13 in development of the LPR (32). H4R antagonism resulted in inhibition of T cell migration into the lung followed by decreased levels of Th2 cytokines in an experimental asthma model (41) and impairment of dendritic cell homing to lymph nodes in a model of atopic dermatitis (42). We previously demonstrated that FcεRI was also essential to the development of both the EPR and LPR (28). FcεRI is expressed primarily on mast cells and basophils and activation through the receptor induces degranulation, histamine release, and cytokine secretion in both cell types (43). Given the requirement for expression of both receptors, the findings that the H4R is effective in cell migration (4) and mast cells are primarily tissue resident cells, we hypothesized that basophils, which circulate, fulfill these requirements in the development of AR. To directly examine their role in AR, basophils were depleted using the depleting antibody, Ba103. Previously, and here, Ba103 was shown to deplete basophils for up to 8 days in spleen, bone marrow, and peripheral blood, without altering mast cell number in peritoneal lavage fluid (23). We also confirmed that Ba103 depleted basophils but not mast cells. Following Ba103 treatment and depletion of >80% basophils in the peripheral blood, allergen sensitized and challenged mice failed to develop an EPR or LPR and prevented the increases in Th2 cytokine levels in nasal cavity lavage fluid. Following activation by OVA and OVA-specific IgE, basophils produced both IL-4 and IL-13, whereas histamine itself did not induce cytokine production.
We confirmed H4R expression on basophils by flow cytometry analysis and increased histamine levels in nasal cavity lavage fluid. We then determined whether expression of this receptor was required for allergen-induced nasal responses. Basophils were isolated from WT or H4R−/− mice and adoptively transferred into allergen sensitized H4R−/− mice prior to the 4th allergen challenge. H4R−/− recipients which received WT but not H4R−/− basophils developed decreases in RF in the EPR and LPR, increases in RNA in the LPR, and increases in Th2 cytokine levels in the nasal cavity lavage fluid. These complimentary approaches established a critical role for basophils in the development of AR, and the transfer experiments identified the importance of H4R expression on these cells.
However, as mast cells can be activated through ligation of FcεRI as well as through H4R in chemotaxis and calcium mobilization assays (14), their role in the development of AR could not be discounted. We previously suggested the involvement of mast cells triggered through FcεRI in AR (28, 29). In order to determine the role of basophils and exclude the activation of mast cells through FcεRI, FcεRI−/− mice were used as recipients of basophils isolated from WT or H4R−/− mice. In addition, to avoid systemic sensitization with OVA/alum, recipients were passively sensitized with OVA-specific IgE prior to OVA challenge. Under these conditions only the transferred basophils but not host mast cells could be activated through the ligation of FcεRI. With these restrictions, only FcεRI−/− recipients that were passively sensitized with OVA-specific IgE and received WT but not H4R−/− basophils developed the full spectrum of allergen-induced nasal responses following challenge with OVA and exogenous histamine. Furthermore, ex vivo pretreatment of basophils with OVA-specific IgE also restored responsiveness in OVA-histamine challenged FcεRI−/− recipients. These results indicated that FcεRI was essential to the activation of basophils in AR. Together with our earlier data on the role of mast cells in AR (28, 29), the requirement for exogenous histamine implied that FcεRI was also important in allergen-specific IgE mast cell activation for the release of histamine as the initiator of the basophil-driven response in AR. Although mast cells can be activated through IgE-FcγRIII ligation (44), exogenous histamine was essential to the development of the nasal responses. Thus, nasal mast cell-derived histamine initiated the cascade by directing the migration of basophils through the H4R. In chemotaxis assays, only WT but not H4R−/− basophils responded to a histamine gradient. The response to histamine appeared to be restricted to chemotaxis, as addition of histamine to WT basophils did not result in degranulation or cytokine production. Confirming these findings, only WT but not H4R−/− basophils were demonstrated in the nasal cavity of OVA-IgE passively sensitized mice following OVA challenge together with histamine. Specificity of the response was shown as only OVA-specific IgE but not DNP-specific IgE triggered the EPR and LPR following WT basophil transfer and OVA-histamine administration in FcεRI−/− mice. The administration of histamine in the challenge phase was essential to the accumulation of basophils in the nasal lavage fluid but this was not sufficient to elicit a nasal response. The development of nasal responses was also dependent on activation of the basophils through FcεRI in that the absence of either OVA challenge or OVA-specific IgE passive sensitization, an EPR and LPR could not be detected. These data suggested that activated basophils were the source of the cytokines involved in AR, although we cannot rule out the contributions of other cell types. Basophils in the nose have been implicated in the pathogenesis of AR (45). Nasal allergen challenge in AR patients resulted in increased levels of FcεRI expression and IL-13 secretion by basophils (46). The number of basophils in the nasal mucosa of AR patients was shown to be increased significantly after segmental bronchial provocation, while the numbers of mast cells were decreased (47). In the experimental model of AR, we previously demonstrated that IL-13 played an essential role in the development of an LPR (32). As shown in the present study, both IL-13−/− and IL-4−/−/IL-13−/− basophils were capable of inducing an EPR but not an LPR in OVA-IgE, passively sensitized FcεRI−/− recipients following challenge with OVA and histamine.
Taken together, these findings identify the important role for both the H4R and FcεRI on basophils in AR and a sequential or biphasic cascade of events beginning with mast cell activation (through FcεRI) in the nasal tissue, the release of histamine followed by the recruitment of basophils to the nasal tissue (through the H4R), and subsequent activation (through FcεRI) of basophils in the nasal mucosa in an allergen-specific manner (Figure 10). This mast cell-histamine-H4R-basophil-FcεRI axis provides new opportunities for therapeutic intervention, overcoming the limitations of current treatment with H1R antagonists (inverse agonists) and intranasal corticosteroids.
Grant Support: This work was supported by NIH grants HL-36577 and AI-77609. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI or the NIH.
We thank Dr. Robin L Thurmond for providing the H4R−/− mice and Diana Nabighian for assistance in the preparation of this manuscript.