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Physiologic assessment of allergic rhinitis in mice: Role of the high-affinity IgE receptor (FcɛRI)
Physiologic assessment of allergic rhinitis in mice: Role of the high-affinity IgE receptor (FceRI) Satoko Miyahara, MD, PhD, Nobuaki Miyahara, MD, PhD, Katsuyuki Takeda, MD, PhD, Anthony Joetham, BS, and Erwin W. Gelfand, MD Denver, Colo Mechanisms of asthma and allergic inflammation
Background: There have been few reports using animal models to study the development of allergic rhinitis. Characterization of such a model in mice would be advantageous given the availability of reagents and gene-manipulated strains. Objective: We sought to develop a murine model of allergic rhinitis in the absence of lower airway changes. Methods: After sensitization and challenge, both wild-type and FceRI-deficient mice were studied for their ability to develop early- and late-phase nasal responses. In the invasive approach, direct measurements of nasal airway resistance (RNA) were obtained; in the noninvasive approach using whole-body plethysmography, respiratory frequency and expiratory and inspiratory times were monitored. In both approaches, nasal responses were determined either acutely after challenge (early phase) or 24 hours after challenge (late phase). Results: After challenge of sensitized mice, RNA significantly increased. In parallel, respiratory frequency significantly decreased and was highly correlated with the increases in RNA. Sensitized wild-type mice had an early-phase nasal response and persistent nasal blockage (late-phase response) after allergen challenge. In contrast, sensitized and challenged FceRI a-chain–deficient mice did not have an early-phase nasal reaction and exhibited reduced nasal blockage and lower IL-13 levels in nasal tissue homogenates. Conclusions: These data indicate that FceRI is essential to development of an early-phase nasal response and contributes to the development of the late-phase nasal response. These invasive and noninvasive approaches provide new opportunities to evaluate the mechanisms underlying the development of nasal responses to allergen and to assess various therapeutic interventions. (J Allergy Clin Immunol 2005;116:1020-7.) Key words: Rodent, rhinitis, hyperresponsiveness, allergy
Allergic rhinitis (AR), the most common form of atopic disease, has an estimated prevalence ranging from 5% to 22%,1 with enormous associated costs for treatment.2 The typical symptoms of AR in human subjects are well known, mainly sneezing and nasal blockage.3,4 The 3
From the Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center. Supported by National Institutes of Health grants HL-36577, HL-61005, and AI-42246 and US Environmental Protection Agency grant R835702. Received for publication January 18, 2005; revised August 8, 2005; accepted for publication August 10, 2005. Reprint requests: Erwin W. Gelfand, MD, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80206. E-mail: gelfande@ njc.org. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.08.020
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Abbreviations used AR: Allergic rhinitis MCh: Methacholine OVA: Ovalbumin RF: Respiratory frequency RNA: Nasal airway resistance WBP: Whole-body plethysmography WT: Wild-type
major causes of the nasal blockage are thought to be dilitation of capacitance vessels in the nasal septum and turbinates, edematous swelling of nasal membranes, and the direct result of secretions.5-8 The mechanisms underlying the development of bronchial asthma have been analyzed, measuring lower airway function. By using murine strains with targeted mutations of an array of relevant genes, the role of a number of factors contributing to the pathogenesis of lower airway hyperresponsiveness in asthma has been reported.9-13 In contrast, there have been relatively few reports on the development of nasal hyperresponsiveness in AR using an animal model.14,15 The physiologic assessment of AR in mice, especially monitoring early-phase responses and nasal blockage, have not been well established. Nasal reactivity in AR has been shown to occur in 2 phases: early-phase and late-phase responses. Earlyphase responses occur within minutes of exposure to the allergen and tend to produce sneezing, itching, and clear rhinorrhea; the late-phase response reaction occurs 6 to 24 hours after local allergen challenge of subjects with atopic rhinitis and is characterized by congestion, fatigue, malaise, and irritability.2 Persistent tissue edema and eosinophils, mast cells, TH2-type lymphocytes, and macrophages are thought to be involved.2 However, the detailed mechanisms underlying the development of these nasal responses have not been well defined. Given the complexity of the potential contributors to the development of AR and the difficulty in dissecting the mechanisms in human subjects, an animal model of AR is essential, especially one that can address physiologic changes in this model. Furthermore, a model that was limited to changes in the upper airways without lower airway changes was believed to be important. To this end, we used both invasive and noninvasive methods to monitor early and late nasal responses accompanied by
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TABLE I. Antibody responses and nasal eosinophils after 6 nasal challenges OVA-specific: Sensitization
Challenge
None None OVA None OVA
None OVA OVA OVA OVA
<10 27 6 13 674 6 50 à <10 664 6 93§
PBS/PBS WT PBS/OVA6 WT OVA/OVA6 WT PBS/OVA6 FceRI2/2 OVA/OVA6 FceRI2/2
IgG1 (EU/mL)
Total IgE (ng/mL)
<10 646 6 204* 2552 6 72 à 273 6 174 2255 6 31§
862 23 6 5* 221 6 13 à <10 308 6 28§
Eosinophils in nasal septum (/mm2)
16 325 1493 186 1102
6 6 6 6 6
13 136 212 à 56 255§
OVA-specific antibody and total Ig levels in the serum and tissue infiltration with major basic protein–positive eosinophils in nose tissue after 6 nasal challenges are shown. Mice were sensitized and challenged as described in the Methods section. Serum Ig and antibody levels were assessed 24 hours after the sixth nasal challenge in untreated mice (PBS/PBS), mice that were challenged only (PBS/OVA6), and sensitized and challenged mice (OVA/OVA6). The number of eosinophils is shown per square millimeter of nasal septal membrane, excluding cartilage (n 5 8 in each group). Means 6 SEM are shown. *P < .05 and P < .01 compared with untreated group (PBS/PBS). àP < .01 compared with the group that was only challenged (PBS/OVA6). §P < .01 compared with the group that was only challenged (PBS/OVA6) in FceRI2/2 mice.
increased nasal airway resistance (RNA) in mice and in the absence of lower airway changes. We demonstrate the development of allergen-induced increases in RNA and show that decreases in respiratory frequency (RF) were highly correlated with increases in RNA. We monitored early- and late-phase nasal responses in wild-type (WT) and FceRI-deficient (FceRI2/2) mice and showed that sensitized and challenged FceRI2/2 mice did not have an early-phase nasal response to allergen, as well as having a significantly lower late-phase response.
Measurement of respiratory parameters with whole-body plethysmography Twenty-four hours after the last antigen challenge, RF, expiratory time, and inspiratory time were measured in unrestrained conscious animals by using single-chamber whole-body plethysmography (WBP; Buxco, Troy, NY). For measurement of respiratory parameters during the early-phase reaction, mice received OVA (20 mL of 25 mg/mL) through the nostril after measurement of baseline values and were then placed back into the box. RF, inspiratory time, and expiratory time were measured at 4, 10, 15, 20, 25, 30, and 60 minutes.
METHODS
Statistical analysis
See the Methods section in the Online Repository in the online version of this article at www.jacionline.org for additional material.
All results were expressed as the mean 6 SEM. ANOVA was used to determine the levels of difference between all groups. Pairs of groups were compared by using the unpaired 2-tailed Student t test. The time course change in the same groups was compared by using the paired t test. The P value for significance was set at .05.
OVA sensitization and nasal challenge Mice were assigned to either the control or treatment groups on the basis of the following: (1) the ovalbumin (OVA)/OVA group received OVA sensitization followed by OVA intranasal challenge every 24 hours; (2) the PBS/OVA group received OVA intranasally daily without intraperitoneal sensitization; and (3) the PBS/PBS group received intraperitoneal PBS followed by PBS intranasal instillation. OVA/OVA3 indicates that these mice were sensitized to OVA and received 3 nasal challenges. Mice were sensitized by means of intraperitoneal injection of 20 mg of OVA (Grade V; Sigma Chemical, St Louis, Mo) emulsified in 2.25 mg of alum (AlumImuject; Pierce, Rockford, Ill) in a total volume of 100 mL on days 0 and 14. This was followed by daily challenge by instillation of OVA diluted in PBS (20 mL of 25 mg/mL) in the nostril without anesthesia from day 28 onward. The solutions were shown to contain less than 1 EU of LPS per milligram of protein.
Measurement of RNA For more information, see Fig E1 in the Online Repository in the online version of this article at www.jacionline.org. For RNA, measurements of piston volume displacement and cylinder pressure were used to calculate the impedance of the respiratory system, as described by Pillow et al.16 All data were analyzed with FlexiVent software (Scireq, Montreal, Quebec, Canada). For measurements in the early-phase reaction after OVA challenge, RNA was continuously monitored from 2 to 10 minutes after allergen challenge. RNA in the late-phase response (persistent nasal obstruction) was measured 24 hours after the last OVA challenge.
RESULTS Nasal resistance increases after repetitive intranasal allergen challenge in sensitized mice Sensitization to OVA followed by 3 or 6 intranasal challenges significantly increased serum levels of antiOVA IgE and IgG1 and total IgE in WT mice compared with that seen in control mice receiving no treatment or OVA intranasal challenges alone (Table I and Table E1 in the Online Repository in the online version of this article at www.jacionline.org). Nasal tissue inflammation and, in particular, eosinophil accumulation were assessed by using immunohistochemistry with anti–major basic protein antibody. Sensitization followed by 6 daily challenges elicited a significant increase in eosinophil numbers in the nasal mucosa in WT mice compared with that seen in the control groups (Table I). Fig 1 shows RNA values after intranasal OVA challenge in WT mice. RNA was determined 24 hours after the sixth OVA challenge. Sensitization followed by 6 daily OVA challenges induced significant and persistent (24 hours after the last challenge) increases in RNA compared with
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Group
IgE (EU/mL)
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FIG 1. RNA 24 hours after 6 nasal challenges. RNA was assessed 24 hours after the last nasal challenge in untreated mice (PBS/PBS), nonsensitized but challenged mice (PBS/OVA6), and sensitized and challenged mice (OVA/OVA6). Nasal resistance was measured as described in the Methods section (n 5 8 in each group). Means 6 SEM are shown. *P < .01 compared with PBS/PBS or PBS/OVA6 groups.
values seen in nonsensitized, nonchallenged and nonsensitized, challenged mice (Fig 1).
Sensitization followed by nasal challenge does not affect lower airway responses To confirm that the approach to sensitization and challenge was confined to the upper airways, we assessed lower airway responses. Fig E2, A (available in the Online Repository in the online version of this article at www. jacionline.org), shows the baseline values of lower airway resistance and changes in lower airway resistance in response to inhaled methacholine (MCh) in all 3 groups of mice. The values for lower airway resistance in sensitized mice and mice challenged 6 times daily (0.52 6 0.02 cm H2O/mL/s) were not different when compared with those of untreated mice (0.54 6 0.02 cm H2O/mL/s) or mice that were only challenged (0.52 6 0.02 cm H2O/mL/s). Lower airway responsiveness to inhaled MCh in sensitized mice and mice challenged 6 times daily similarly did not show any difference compared with that seen in PBS/OVA and PBS/PBS mice. Bronchoalveolar lavage fluid from sensitized and challenged mice showed no lower airway eosinophilic inflammation, similar to that seen in control mice (Fig E2, B, in the Online Repository in the online version of this article at www.jacionline.org). These data suggest that this approach to sensitization and intranasal challenge in the absence of anesthesia did not increase lower airway resistance or inflammation. RF decreases after repetitive intranasal allergen challenge in allergen-sensitized mice Fig 2, A, illustrates RF after 3 or 6 intranasal OVA challenges. We measured RF in the same mice with WBP before or 24 hours after the last of 3 or 6 OVA challenges. RF values in OVA-sensitized mice were significantly decreased 24 hours after the sixth OVA challenge (Fig 2, A). In contrast, nonsensitized mice did not show any changes in RF values after 3 or 6 OVA or PBS challenges. Both expiratory (Fig 2, B) and inspiratory (Fig 2, C) time were increased after 6 OVA challenges, and both contributed to the decrease in RF.
FIG 2. Changes in respiratory parameters after 3 or 6 OVA challenges. RF (A), expiratory time (B), and inspiratory time (C) were monitored 24 hours after 3 or 6 nasal challenges, as described in the Methods section. Respiratory parameters were assessed in untreated mice (PBS/PBS), nonsensitized but challenged mice (PBS/OVA), and OVA-sensitized and OVA-challenged mice (OVA/OVA; n 5 8 in each group). Means 6 SEM are shown. *P < .01 compared with PBS/PBS or PBS/OVA groups.
Decreases in RF correlate with increases in RNA To determine whether the decreases in RF values correlated with the increases in RNA, we monitored RNA immediately after measurements (within 5 minutes) of RF in the same animals with WBP. We compared RF and RNA in PBS/PBS, OVA/OVA4, OVA/OVA5, and OVA/OVA6 mice. Increases in RNA paralleled the decreases in RF values monitored with WBP (Fig E3 in
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FIG 3. Comparison of RNA and RF. After 4 to 6 challenges, individual RNA and RF values were compared. RNA and RF were assessed in untreated mice (PBS/PBS) and in sensitized mice after 4, 5, or 6 challenges. The correlation analysis was carried out in untreated mice (n 5 4) and sensitized mice and mice challenged 4, 5, or 6 times (n 5 4-8). RNA negatively correlated with RF.
FIG 4. RF 24 hours after repeated allergen challenges. Twenty-four hours after the last of 4 to 12 challenges, RF was monitored in previously sensitized mice (n 5 8 in each group). Means 6 SEM are shown. *P < .05 compared with baseline values.
the Online Repository in the online version of this article at www.jacionline.org). Comparison of RNA and RF in the same mice indicated the significant inverse correlation between these 2 measurements (Fig 3).
Changes in RF and RNA after repetitive allergen challenge We next investigated whether decreases in RF were also maintained after repetitive challenges of sensitized mice. Using WBP, we were able to follow the consequences of increasing numbers of intranasal OVA challenges on RF in OVA-sensitized mice (Fig 4). We recorded daily changes in RF values in the same mouse for up to 12 days of challenges. RF values decreased progressively after up to 7 challenges and then remained low throughout the 12-day monitoring period. A similar pattern was observed when we monitored RNA directly in the invasive system. Increases in RNA remained significant after 12 challenges (1.7 6 0.17 cm H2O/mL/s) compared with those in PBS/ PBS mice (0.9 6 0.03 cm H2O/mL/s, P < .05).
FIG 5. A, RNA was monitored over 10 minutes after a fourth OVA challenge in sensitized (OVA/OVA3) or nonsensitized (PBS/OVA3) mice. B, RF was monitored over 60 minutes after a fourth allergen challenge in WT or FceRI2/2 mice (n 5 8 in each group). Means 6 SEM are shown for RNA and RF as a percentage of the value at 2 minutes and baseline values before challenge. *P < .05, **P < .01, and ***P < .001 compared with PBS/OVA3 or PBS/ PBS3 groups. #P < .05, ##P < .01, and ###P < .001 compared with OVA/OVA3 FceRI2/2 mice.
Kinetics of early-phase nasal hyperresponsiveness to allergen We next determined whether an early-phase nasal response after nasal allergen challenge could be detected in sensitized mice. We prepared mice by means of sensitization and then exposed them to 3 OVA challenges. Twenty-four hours after the third challenge, a fourth challenge was administered, and the kinetics of the immediate response were monitored. RNA values after the fourth OVA challenge increased rapidly in sensitized and challenged mice compared with in mice that were only challenged (Fig 5, A). The experiments were stopped after 10 minutes because of increased nasal secretions. We evaluated changes in RF in this early phase as well. Sensitized and challenged mice showed an early decrease in RF after the fourth intranasal OVA challenge, with a nadir at 4 to 7 minutes (Fig 5, B). This early decrease in RF was associated with prolongation of both inspiratory and expiratory times (Fig E4 in the Online Repository in the online version of this article at www.jacionline.org).
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OVA/OVA WT mice showed significant increases in RNA and decreases in RF compared with PBA/OVA WT mice after 6 challenges (Fig 7). Sensitized and challenged FceRI2/2 mice had significant increases in RNA and decreases in RF compared with those in nonsensitized but challenged mice, but the extent of these changes in the FceRI2/2 mice were significantly lower than those in sensitized and challenged WT mice.
Mechanisms of asthma and allergic inflammation
FIG 6. Tissue infiltration with major basic protein–positive eosinophils 24 hours after 6 nasal challenges in WT and FceRI2/2 mice. Immunohistochemical (major basic protein) localization of nasal septal tissue eosinophils was determined 24 hours after 6 nasal challenges in sensitized or nonsensitized mice, as described in the Methods section. Eosinophil infiltration in nonsensitized but challenged WT mice (A), sensitized and challenged WT mice (B), nonsensitized but challenged FceRI2/2 mice (C), and sensitized and challenged FceRI2/2 mice (D) is shown. Bar 5 100 mm.
In contrast, PBS/PBS3 and PBS/OVA3 mice did not show any decreases in RF within 60 minutes after OVA challenge (Fig 5, B). In the OVA/OVA3 mice RF levels returned to normal by 1 hour.
Sensitization and nasal challenge with OVA increases total IgE and OVA-specific IgE and IgG1 levels in FceRI2/2 mice To assess the effect of sensitization and 6 challenges on Ig production in FceRI2/2 mice, we measured serum levels of total IgE and OVA-specific IgE and IgG1. OVA-sensitized and challenged FceRI2/2 mice showed increased serum levels of total IgE and OVA-specific IgE and IgG1 (Table I), which were similar to those in OVA-sensitized and challenged WT mice and significantly higher than those in nonsensitized but challenged FceRI2/2 mice. Nasal eosinophil accumulation in FceRI2/2 mice After 6 days of OVA challenges, sensitized WT mice demonstrated significant increases in nasal eosinophil numbers compared with those seen in the nonsensitized but challenged mice (Table I). Similar results were observed in FceRI2/2 mice: after 6 nasal challenges, sensitized FceRI2/2 mice showed significant increases in numbers of nasal eosinophils compared with those seen in the nonsensitized but challenged FceRI2/2 mice (Fig 6 and Table I). The number of eosinophils in sensitized and challenged FceRI2/2 mice were not different from those in sensitized and challenged WT mice. RNA and RF responses are significantly modified in FceRI2/2 mice Fig 7 shows the changes in RNA and RF 24 hours after the sixth OVA challenge in both WT and FceRI2/2 mice.
FceRI2/2 mice do not have early-phase nasal hyperresponsiveness To assess whether FceRI plays a role in the development of early-phase nasal hyperresponsiveness, we sensitized and challenged FceRI2/2 mice 3 times. OVA- sensitized and OVA-challenged FceRI2/2 mice, unlike FceRI1/1 mice, demonstrated no significant decrease in RF after the fourth OVA challenge, and the responses were similar to those of the nonsensitized but challenged WT mice (Fig 5, B). IL-13 levels in nasal tissue homogenates are significantly lower in FceRI2/2 mice compared with those in WT mice after sensitization and challenge IL-13 has been shown to be an important mediator in the development of allergic responses.17,18 To assess whether IL-13 is upregulated after sensitization and nasal challenges, we measured levels of IL-13 in nasal tissue homogenates adjusted to weight of samples. WT mice that were sensitized and challenged (6 times) exhibited increased levels of IL-13 in tissue homogenates 24 hours after the sixth nasal challenge compared with levels in nonsensitized but challenged WT mice (Fig 8). Sensitized and challenged FceRI2/2 mice showed some increase in the levels of IL-13 compared with those in nonsensitized but challenged FceRI2/2 mice, but the levels were significantly lower than those in sensitized and challenged WT mice (P < .01). IL-4 and IL-5 levels were increased in sensitized and challenged WT and FceRI2/2 mice to a similar degree, whereas IFN-g levels in both WT and FceRI2/2 mice were similar to those in mice that were only challenged. DISCUSSION Indirect methods for assessing the nasal responses in mice have relied on counting the number of sneezing and nasal rubbing episodes or the threshold concentration of histamine to evoke sneezing.14,19,20 Measurements of RNA have been reported in other animal models, such as guinea pigs and dogs,21,22 but direct measurement of nasal resistance in mice was technically difficult because of the anatomically smaller nasal cavities. In this study we characterized 2 methods to measure nasal airway responses in vivo, using both invasive and noninvasive approaches. We used custom-designed ventilation equipment, which can measure the resistance of an open canal by means of
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FIG 7. Nasal resistance and RF 24 hours after repeated allergen challenge in FceRI2/2 mice. RNA (A) and RF (B) were measured 24 hours after the sixth allergen challenge in nonsensitized (PBS/OVA6) and sensitized (OVA/ OVA6) WT and FceRI2/2 mice (n 5 8 in each group). Means 6 SEM are shown. #P < .05 and ##P < .01 compared with OVA/OVA FceRI2/2 mice. *P < .05 and **P < .01 compared with PBS/OVA group.
piston volume displacement and cylinder pressure.16 This method enabled us to directly measure RNA in the small nasal cavities of mice. We also predicted that allergeninduced nasal blockage should influence the respiratory pattern of mice because they are obligate nasal breathers. In parallel to RNA, we monitored RF in unrestrained conscious mice using WBP. This box signal measures differences in pressure between a main chamber containing the animal and a reference chamber and provides data on a number of different respiratory parameters.23 Using this noninvasive method, we demonstrated that increases in RNA were significantly correlated with decreases in RF, suggesting that RF is a valid, albeit indirect, indicator of nasal resistance or blockage in mice. To some extent, the responses differed on the basis of the number of challenges. After up to 4 challenges in sensitized mice, changes in RF returned to baseline by 24 hours. After additional challenges, congestion or sustained nasal blockage persisted, resulting in higher baseline values the following day (eg, after 6 challenges). The advantage of monitoring RNA is that it is not influenced by circadian rhythm or movement of the animals but is a direct measure of intranasal pressure. On the other hand, the advantage of the indirect and noninvasive measurement of nasal resistance with WBP is that the animals do not need to be killed once the measurements are finished, and several measurements on the same animals can be performed, even over days, allowing longitudinal studies and investigation of treatment protocols. One problem with measuring nasal obstruction with WBP is the uncertainty of the site of obstruction.24 Previously, we documented the noninvasive measurement of lower airway function in a mouse model of allergen sensitization followed by allergen inhalation challenge. In that study we demonstrated that the increase in lower airway resistance to inhaled MCh was associated with a decrease in RF.25 In the present study we carried out intranasal challenges, instilling an OVA solution into the nostril but without anesthesia. In studies of lower airway
FIG 8. Levels of IL-13 in nasal tissue after sensitization and challenge. Levels of IL-13 in nasal tissue homogenates were determined 24 hours after the sixth nasal challenge, as described in the Methods section (n 5 8 in each group). Means 6 SEM are shown. *P < .01 compared with OVA/OVA FceRI2/2 mice. #P < .01 compared with PBS/OVA6 and PBS/PBS6 groups.
function, intranasal challenges are performed in anesthetized mice, which aspirate the solution. In contrast, nonanesthetized mice swallow the solution. Saito et al19 have shown that no trypan blue staining was observed below the tracheal region after nasal instillation in nonanesthetized mice and that the mice sensitized and nasally challenged without anesthesia do not show lower airway hyperresponsiveness to MCh. Similarly, we confirmed that intranasal allergen challenge of mice using the same method did not evoke lower airway inflammation or airway hyperresponsiveness to inhaled MCh. Sensitized mice subjected to intranasal allergen challenge without anesthesia showed decreases in RF and increases in RNA without changes in lower airway resistance. These data establish that the decreases in RF in our protocol were induced by nasal blockage but not lower airway obstruction. Early nasal blockage is likely caused by increases in nasal secretions and swelling of nasal membranes. After aspiration of the nasal cavity after initial measurements, RNA decreased immediately in both the OVA/OVA and
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PBS/OVA groups. In the PBS/OVA group RNA returned to baseline values, but in the OVA/OVA group RNA remained higher. In contrast, the late nasal response might be caused by persistent swelling of nasal membranes because nasal suction did not influence RNA values 24 hours after the last challenge. Many lines of evidence have led to the concept that the interaction between IgE and mast cells and basophils is the primary effector pathway in allergic responses, at least early-phase responses and possibly late-phase responses as well. Mast cell activation occurs after cross-linking of cell surface–bound IgE with multivalent allergens, resulting in the release of mediators, such as histamine, as well as cytokines, such as IL-13, which directly induce allergic symptoms.26 Immediate and late-phase allergic reactions induced by IgE-dependent mast cell activation trigger a cascade that leads to chronic allergic inflammation.27 IgE-producing plasma cells have been identified in the submucosal tissue of the nose,28 and allergen-specific IgE has been detected in nasal mucosa and nasal washes from human subjects with AR.29,30 The role of FceRI in AR has been implied but has not been well studied. FceRI2/2 mice were prepared by disruption of the FceRIa gene.31 These mice were essentially normal in all aspects of their development and maturation, including the presence of functional mast cells.31,32 The FceRI2/2 mice were able to fully mount an immune response to allergen exposure, as shown by increased levels of total IgE and allergen-specific IgE and IgG1. It has been reported that FceRI2/2 mice are resistant to passive cutaneous anaphylaxis and systemic anaphylaxis,31 whereas they showed normal development of lower airway hyperresponsiveness after sensitization (together with alum) and challenge comparable with that seen in WT mice.33 In the present study sensitized and challenged FceRI2/2 mice did not show any nasal response in the early-phase response to allergen challenge and had only small increases in RNA during the late-phase response (24 hours after 6 challenges). These data suggest that nasal reactivity in AR might be more dependent on the IgE–FceRI–mast cell module than certain models of allergen-induced airway hyperresponsiveness and inflammation, which can develop in the absence of IgE,10 FceRI,33 or mast cells.9 Sensitized and challenged FceRI2/2 mice did show increases in IL-13 levels in the nasal mucosa 24 hours after the last of 6 allergen challenges, but the levels were significantly lower than in sensitized and challenged WT mice. IL-13 has been shown to be an important mediator in the development of allergic responses.17,18 Our results indicate that the presence of FceRI is required, at least to some degree, for the production of IL-13 in vivo. Mast cells might be one source of IL-13, but this might not be the only or critical source.34 More important potentially are other mast cell–derived mediators (eg, leukotriene B4), which might recruit other IL-13–producing cell types to the target organ.35,36 After sensitization and challenge, FceRI2/2 mice showed different results in terms of eosinophil infiltration, dependent on the mode of allergen exposure.33,37 On the other hand, in a mouse model of AR,
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nasally sensitized, IgE-deficient mice showed no difference in the intensity of eosinophil infiltration in the nasal membranes compared with that seen in WT mice, suggesting that rhinitis could arise in the absence of IgE.15 Here we showed that FceRI2/2 mice also demonstrated a similar level of eosinophil infiltration in the nasal membrane as WT mice. Nonetheless, the mice did not have earlyphase nasal reactivity and exhibited reduced late-phase nasal blockage. Because the numbers of eosinophils were not different, the role eosinophils play in these nasal responses remains to be defined. In summary, this report describes novel invasive and noninvasive methods to evaluate nasal responses to allergen challenge. Development of decreased RF, assessed by means of WBP, was highly correlated with directly measured increases in RNA. No lower airway hyperresponsiveness developed when mice were sensitized and challenged by means of nasal instillation in conscious animals. Two phases of the response were detected, an early phase within minutes that resolved by 60 minutes and a late or chronic sustained nasal blockage, which persisted for at least 24 hours after 6 or more challenges. On the basis of the responses in FceRI2/2 mice, we demonstrated that the integrity of FceRI was essential to the development of an early-phase nasal response and was a major contributor to the late-phase response and IL-13 production. The measurement of both RNA and RF provides a promising approach for defining critical components in the early- and late-phase responses in AR and the means for identifying new approaches to the prevention and treatment of AR. We thank Dr J. J. Lee (Mayo Clinic, Scottsdale, Ariz) for providing the anti–major basic protein antibody and D. Nabighian (National Jewish Medical and Research Center) for her assistance.
REFERENCES 1. Naclerio RM. Allergic rhinitis. N Engl J Med 1991;325:860-9. 2. Skoner DP. Allergic rhinitis: definition, epidemiology, pathophysiology, detection, and diagnosis. J Allergy Clin Immunol 2001;108(suppl):S2-8. 3. Corrado OJ, Ollier S, Phillips MJ, Thomas JM, Davies RJ. Histamine and allergen induced changes in nasal airways resistance measured by anterior rhinomanometry: reproducibility of the technique and the effect of topically administered antihistaminic and anti-allergic drugs. Br J Clin Pharmacol 1987;24:283-92. 4. Mygind N, Anggard A. Anatomy and physiology of the nose—pathophysiologic alterations in allergic rhinitis. Clin Rev Allergy 1984;2: 173-88. 5. Sherwood JE, Hutt DA, Kreutner W, Morton JB, Chapman RW. A magnetic resonance imaging evaluation of histamine-mediated allergic response in the guinea pig nasopharynx. J Allergy Clin Immunol 1993;92:435-41. 6. Juliusson S, Bende M. Allergic reaction of the human nasal mucosa studied with laser Doppler flowmetry. Clin Allergy 1987;17:301-5. 7. Mygind N, Brofeldt S, Ostberg B, Cerkez V, Tos M, Marriott C. Upper respiratory tract secretions: pathophysiology. Eur J Respir Dis Suppl 1987;153:26-33. 8. Gawin AZ, Emery BE, Baraniuk JN, Kaliner MA. Nasal glandular secretory response to cholinergic stimulation in humans and guinea pigs. J Appl Physiol 1991;71:2460-8. 9. Takeda K, Hamelmann E, Joetham A, Shultz LD, Larsen GL, Irvin CG, et al. Development of eosinophilic airway inflammation and airway hyperresponsiveness in mast cell-deficient mice. J Exp Med 1997;186:449-54.
10. Hamelmann E, Takeda K, Schwarze J, Vella AT, Irvin CG, Gelfand EW. Development of eosinophilic airway inflammation and airway hyperresponsiveness requires interleukin-5 but not immunoglobulin E or B lymphocytes. Am J Respir Cell Mol Biol 1999;21:480-9. 11. Taube C, Duez C, Cui ZH, Takeda K, Rha YH, Park JW, et al. The role of IL-13 in established allergic airway disease. J Immunol 2002;169: 6482-9. 12. Miyahara N, Swanson BJ, Takeda K, Taube C, Miyahara S, Kodama T, et al. Effector CD81 T cells mediate inflammation and airway hyperresponsiveness. Nat Med 2004;10:865-9. 13. Lee JJ, Dimina D, Macias MP, Ochkur SI, McGarry MP, O’Neill KR, et al. Defining a link with asthma in mice congenitally deficient in eosinophils. Science 2004;305:1773-6. 14. Iwasaki M, Saito K, Takemura M, Sekikawa K, Fujii H, Yamada Y, et al. TNF-alpha contributes to the development of allergic rhinitis in mice. J Allergy Clin Immunol 2003;112:134-40. 15. van de Rijn M, Mehlhop PD, Judkins A, Rothenberg ME, Luster AD, Oettgen HC. A murine model of allergic rhinitis: studies on the role of IgE in pathogenesis and analysis of the eosinophil influx elicited by allergen and eotaxin. J Allergy Clin Immunol 1998;102:65-74. 16. Pillow JJ, Korfhagen TR, Ikegami M, Sly PD. Overexpression of TGF-alpha increases lung tissue hysteresivity in transgenic mice. J Appl Physiol 2001;91:2730-4. 17. Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM, et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998;282:2261-3. 18. Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282:2258-61. 19. Saito H, Howie K, Wattie J, Denburg A, Ellis R, Inman MD, et al. Allergen-induced murine upper airway inflammation: local and systemic changes in murine experimental allergic rhinitis. Immunology 2001;104: 226-34. 20. Asakura K, Saito H, Watanabe M, Ogasawara H, Matsui T, Kataura A. Effects of anti-IL-5 monoclonal antibody on the murine model of nasal allergy. Intl Arch Allergy Immunol 1998;116:49-52. 21. Mizuno H, Kawamura Y, Iwase N, Ohno H. Effects of flutropium on experimental models of drug- and allergy-induced rhinitis in guinea pigs. Jpn J Pharmacol 1991;55:321-8. 22. Tiniakov RL, Tiniakova OP, McLeod RL, Hey JA, Yeates DB. Canine model of nasal congestion and allergic rhinitis. J Appl Physiol 2003; 94:1821-8. 23. Epstein MA, Epstein RA. A theoretical analysis of the barometric method for measurement of tidal volume. Respir Physiol 1978;32: 105-20.
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24. Bellofiore S, Di Maria GU, Martin JG. Changes in upper and lower airway resistance after inhalation of antigen in sensitized rats. Am Rev Respir Dis 1987;136:363-8. 25. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, et al. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 1997;156:766-75. 26. Galli SJ. New concepts about the mast cell. N Engl J Med 1993;328: 257-65. 27. Iliopoulos O, Proud D, Adkinson NF Jr, Norman PS, Kagey-Sobotka A, Lichtenstein LM, et al. Relationship between the early, late, and rechallenge reaction to nasal challenge with antigen: observations on the role of inflammatory mediators and cells. J Allergy Clin Immunol 1990;86: 851-61. 28. Tada T, Ishizaka K. Distribution of gamma E-forming cells in lymphoid tissues of the human and monkey. J Immunol 1970;104:377-87. 29. KleinJan A, Godthelp T, van Toornenenbergen AW, Fokkens WJ. Allergen binding to specific IgE in the nasal mucosa of allergic patients. J Allergy Clin Immunol 1997;99:515-21. 30. Diaz-Sanchez D, Dotson AR, Takenaka H, Saxon A. Diesel exhaust particles induce local IgE production in vivo and alter the pattern of IgE messenger RNA isoforms. J Clin Invest 1994;94:1417-25. 31. Dombrowicz D, Flamand V, Brigman KK, Koller BH, Kinet JP. Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor alpha chain gene. Cell 1993;75:969-76. 32. Dombrowicz D, Flamand V, Miyajima I, Ravetch JV, Galli SJ, Kinet JP. Absence of Fc epsilon RI alpha chain results in upregulation of Fc gammaRIII-dependent mast cell degranulation and anaphylaxis. Evidence of competition between Fc epsilonRI and Fc gammaRIII for limiting amounts of FcR beta and gamma chains. J Clin Invest 1997;99:915-25. 33. Mayr SI, Zuberi RI, Zhang M, de Sousa-Hitzler J, Ngo K, Kuwabara Y, et al. IgE-dependent mast cell activation potentiates airway responses in murine asthma models. J Immunol 2002;169:2061-8. 34. Burd PR, Thompson WC, Max EE, Mills FC. Activated mast cells produce interleukin 13. J Exp Med 1995;181:1373-80. 35. Tager AM, Bromley SK, Medoff BD, Islam SA, Bercury SD, Friedrich EB, et al. Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment. Nat Immunol 2003;4:982-90. 36. Ott VL, Cambier JC, Kappler J, Marrack P, Swanson BJ. Mast cell-dependent migration of effector CD81 T cells through production of leukotriene B4. Nat Immunol 2003;4:974-81. 37. Taube C, Wei X, Swasey CH, Joetham A, Zarini S, Lively T, et al. Mast cells, Fc epsilon RI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant. J Immunol 2004;172:6398-406.
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Background: There have been few reports using animal models to study the development of allergic rhinitis. Characterization of such a model in mice would be advantageous given the availability of reagents and gene-manipulated strains. Objective: We sought to develop a murine model of allergic rhinitis in the absence of lower airway changes. Methods: After sensitization and challenge, both wild-type and FceRI-deficient mice were studied for their ability to develop early- and late-phase nasal responses. In the invasive approach, direct measurements of nasal airway resistance (RNA) were obtained; in the noninvasive approach using whole-body plethysmography, respiratory frequency and expiratory and inspiratory times were monitored. In both approaches, nasal responses were determined either acutely after challenge (early phase) or 24 hours after challenge (late phase). Results: After challenge of sensitized mice, RNA significantly increased. In parallel, respiratory frequency significantly decreased and was highly correlated with the increases in RNA. Sensitized wild-type mice had an early-phase nasal response and persistent nasal blockage (late-phase response) after allergen challenge. In contrast, sensitized and challenged FceRI a-chain–deficient mice did not have an early-phase nasal reaction and exhibited reduced nasal blockage and lower IL-13 levels in nasal tissue homogenates. Conclusions: These data indicate that FceRI is essential to development of an early-phase nasal response and contributes to the development of the late-phase nasal response. These invasive and noninvasive approaches provide new opportunities to evaluate the mechanisms underlying the development of nasal responses to allergen and to assess various therapeutic interventions. (J Allergy Clin Immunol 2005;116:1020-7.) Key words: Rodent, rhinitis, hyperresponsiveness, allergy
Allergic rhinitis (AR), the most common form of atopic disease, has an estimated prevalence ranging from 5% to 22%,1 with enormous associated costs for treatment.2 The typical symptoms of AR in human subjects are well known, mainly sneezing and nasal blockage.3,4 The 3
From the Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center. Supported by National Institutes of Health grants HL-36577, HL-61005, and AI-42246 and US Environmental Protection Agency grant R835702. Received for publication January 18, 2005; revised August 8, 2005; accepted for publication August 10, 2005. Reprint requests: Erwin W. Gelfand, MD, National Jewish Medical and Research Center, 1400 Jackson St, Denver, CO 80206. E-mail: gelfande@ njc.org. 0091-6749/$30.00 Ó 2005 American Academy of Allergy, Asthma and Immunology doi:10.1016/j.jaci.2005.08.020
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Abbreviations used AR: Allergic rhinitis MCh: Methacholine OVA: Ovalbumin RF: Respiratory frequency RNA: Nasal airway resistance WBP: Whole-body plethysmography WT: Wild-type
major causes of the nasal blockage are thought to be dilitation of capacitance vessels in the nasal septum and turbinates, edematous swelling of nasal membranes, and the direct result of secretions.5-8 The mechanisms underlying the development of bronchial asthma have been analyzed, measuring lower airway function. By using murine strains with targeted mutations of an array of relevant genes, the role of a number of factors contributing to the pathogenesis of lower airway hyperresponsiveness in asthma has been reported.9-13 In contrast, there have been relatively few reports on the development of nasal hyperresponsiveness in AR using an animal model.14,15 The physiologic assessment of AR in mice, especially monitoring early-phase responses and nasal blockage, have not been well established. Nasal reactivity in AR has been shown to occur in 2 phases: early-phase and late-phase responses. Earlyphase responses occur within minutes of exposure to the allergen and tend to produce sneezing, itching, and clear rhinorrhea; the late-phase response reaction occurs 6 to 24 hours after local allergen challenge of subjects with atopic rhinitis and is characterized by congestion, fatigue, malaise, and irritability.2 Persistent tissue edema and eosinophils, mast cells, TH2-type lymphocytes, and macrophages are thought to be involved.2 However, the detailed mechanisms underlying the development of these nasal responses have not been well defined. Given the complexity of the potential contributors to the development of AR and the difficulty in dissecting the mechanisms in human subjects, an animal model of AR is essential, especially one that can address physiologic changes in this model. Furthermore, a model that was limited to changes in the upper airways without lower airway changes was believed to be important. To this end, we used both invasive and noninvasive methods to monitor early and late nasal responses accompanied by
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TABLE I. Antibody responses and nasal eosinophils after 6 nasal challenges OVA-specific: Sensitization
Challenge
None None OVA None OVA
None OVA OVA OVA OVA
<10 27 6 13 674 6 50 à <10 664 6 93§
PBS/PBS WT PBS/OVA6 WT OVA/OVA6 WT PBS/OVA6 FceRI2/2 OVA/OVA6 FceRI2/2
IgG1 (EU/mL)
Total IgE (ng/mL)
<10 646 6 204* 2552 6 72 à 273 6 174 2255 6 31§
862 23 6 5* 221 6 13 à <10 308 6 28§
Eosinophils in nasal septum (/mm2)
16 325 1493 186 1102
6 6 6 6 6
13 136 212 à 56 255§
OVA-specific antibody and total Ig levels in the serum and tissue infiltration with major basic protein–positive eosinophils in nose tissue after 6 nasal challenges are shown. Mice were sensitized and challenged as described in the Methods section. Serum Ig and antibody levels were assessed 24 hours after the sixth nasal challenge in untreated mice (PBS/PBS), mice that were challenged only (PBS/OVA6), and sensitized and challenged mice (OVA/OVA6). The number of eosinophils is shown per square millimeter of nasal septal membrane, excluding cartilage (n 5 8 in each group). Means 6 SEM are shown. *P < .05 and P < .01 compared with untreated group (PBS/PBS). àP < .01 compared with the group that was only challenged (PBS/OVA6). §P < .01 compared with the group that was only challenged (PBS/OVA6) in FceRI2/2 mice.
increased nasal airway resistance (RNA) in mice and in the absence of lower airway changes. We demonstrate the development of allergen-induced increases in RNA and show that decreases in respiratory frequency (RF) were highly correlated with increases in RNA. We monitored early- and late-phase nasal responses in wild-type (WT) and FceRI-deficient (FceRI2/2) mice and showed that sensitized and challenged FceRI2/2 mice did not have an early-phase nasal response to allergen, as well as having a significantly lower late-phase response.
Measurement of respiratory parameters with whole-body plethysmography Twenty-four hours after the last antigen challenge, RF, expiratory time, and inspiratory time were measured in unrestrained conscious animals by using single-chamber whole-body plethysmography (WBP; Buxco, Troy, NY). For measurement of respiratory parameters during the early-phase reaction, mice received OVA (20 mL of 25 mg/mL) through the nostril after measurement of baseline values and were then placed back into the box. RF, inspiratory time, and expiratory time were measured at 4, 10, 15, 20, 25, 30, and 60 minutes.
METHODS
Statistical analysis
See the Methods section in the Online Repository in the online version of this article at www.jacionline.org for additional material.
All results were expressed as the mean 6 SEM. ANOVA was used to determine the levels of difference between all groups. Pairs of groups were compared by using the unpaired 2-tailed Student t test. The time course change in the same groups was compared by using the paired t test. The P value for significance was set at .05.
OVA sensitization and nasal challenge Mice were assigned to either the control or treatment groups on the basis of the following: (1) the ovalbumin (OVA)/OVA group received OVA sensitization followed by OVA intranasal challenge every 24 hours; (2) the PBS/OVA group received OVA intranasally daily without intraperitoneal sensitization; and (3) the PBS/PBS group received intraperitoneal PBS followed by PBS intranasal instillation. OVA/OVA3 indicates that these mice were sensitized to OVA and received 3 nasal challenges. Mice were sensitized by means of intraperitoneal injection of 20 mg of OVA (Grade V; Sigma Chemical, St Louis, Mo) emulsified in 2.25 mg of alum (AlumImuject; Pierce, Rockford, Ill) in a total volume of 100 mL on days 0 and 14. This was followed by daily challenge by instillation of OVA diluted in PBS (20 mL of 25 mg/mL) in the nostril without anesthesia from day 28 onward. The solutions were shown to contain less than 1 EU of LPS per milligram of protein.
Measurement of RNA For more information, see Fig E1 in the Online Repository in the online version of this article at www.jacionline.org. For RNA, measurements of piston volume displacement and cylinder pressure were used to calculate the impedance of the respiratory system, as described by Pillow et al.16 All data were analyzed with FlexiVent software (Scireq, Montreal, Quebec, Canada). For measurements in the early-phase reaction after OVA challenge, RNA was continuously monitored from 2 to 10 minutes after allergen challenge. RNA in the late-phase response (persistent nasal obstruction) was measured 24 hours after the last OVA challenge.
RESULTS Nasal resistance increases after repetitive intranasal allergen challenge in sensitized mice Sensitization to OVA followed by 3 or 6 intranasal challenges significantly increased serum levels of antiOVA IgE and IgG1 and total IgE in WT mice compared with that seen in control mice receiving no treatment or OVA intranasal challenges alone (Table I and Table E1 in the Online Repository in the online version of this article at www.jacionline.org). Nasal tissue inflammation and, in particular, eosinophil accumulation were assessed by using immunohistochemistry with anti–major basic protein antibody. Sensitization followed by 6 daily challenges elicited a significant increase in eosinophil numbers in the nasal mucosa in WT mice compared with that seen in the control groups (Table I). Fig 1 shows RNA values after intranasal OVA challenge in WT mice. RNA was determined 24 hours after the sixth OVA challenge. Sensitization followed by 6 daily OVA challenges induced significant and persistent (24 hours after the last challenge) increases in RNA compared with
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Group
IgE (EU/mL)
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FIG 1. RNA 24 hours after 6 nasal challenges. RNA was assessed 24 hours after the last nasal challenge in untreated mice (PBS/PBS), nonsensitized but challenged mice (PBS/OVA6), and sensitized and challenged mice (OVA/OVA6). Nasal resistance was measured as described in the Methods section (n 5 8 in each group). Means 6 SEM are shown. *P < .01 compared with PBS/PBS or PBS/OVA6 groups.
values seen in nonsensitized, nonchallenged and nonsensitized, challenged mice (Fig 1).
Sensitization followed by nasal challenge does not affect lower airway responses To confirm that the approach to sensitization and challenge was confined to the upper airways, we assessed lower airway responses. Fig E2, A (available in the Online Repository in the online version of this article at www. jacionline.org), shows the baseline values of lower airway resistance and changes in lower airway resistance in response to inhaled methacholine (MCh) in all 3 groups of mice. The values for lower airway resistance in sensitized mice and mice challenged 6 times daily (0.52 6 0.02 cm H2O/mL/s) were not different when compared with those of untreated mice (0.54 6 0.02 cm H2O/mL/s) or mice that were only challenged (0.52 6 0.02 cm H2O/mL/s). Lower airway responsiveness to inhaled MCh in sensitized mice and mice challenged 6 times daily similarly did not show any difference compared with that seen in PBS/OVA and PBS/PBS mice. Bronchoalveolar lavage fluid from sensitized and challenged mice showed no lower airway eosinophilic inflammation, similar to that seen in control mice (Fig E2, B, in the Online Repository in the online version of this article at www.jacionline.org). These data suggest that this approach to sensitization and intranasal challenge in the absence of anesthesia did not increase lower airway resistance or inflammation. RF decreases after repetitive intranasal allergen challenge in allergen-sensitized mice Fig 2, A, illustrates RF after 3 or 6 intranasal OVA challenges. We measured RF in the same mice with WBP before or 24 hours after the last of 3 or 6 OVA challenges. RF values in OVA-sensitized mice were significantly decreased 24 hours after the sixth OVA challenge (Fig 2, A). In contrast, nonsensitized mice did not show any changes in RF values after 3 or 6 OVA or PBS challenges. Both expiratory (Fig 2, B) and inspiratory (Fig 2, C) time were increased after 6 OVA challenges, and both contributed to the decrease in RF.
FIG 2. Changes in respiratory parameters after 3 or 6 OVA challenges. RF (A), expiratory time (B), and inspiratory time (C) were monitored 24 hours after 3 or 6 nasal challenges, as described in the Methods section. Respiratory parameters were assessed in untreated mice (PBS/PBS), nonsensitized but challenged mice (PBS/OVA), and OVA-sensitized and OVA-challenged mice (OVA/OVA; n 5 8 in each group). Means 6 SEM are shown. *P < .01 compared with PBS/PBS or PBS/OVA groups.
Decreases in RF correlate with increases in RNA To determine whether the decreases in RF values correlated with the increases in RNA, we monitored RNA immediately after measurements (within 5 minutes) of RF in the same animals with WBP. We compared RF and RNA in PBS/PBS, OVA/OVA4, OVA/OVA5, and OVA/OVA6 mice. Increases in RNA paralleled the decreases in RF values monitored with WBP (Fig E3 in
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FIG 3. Comparison of RNA and RF. After 4 to 6 challenges, individual RNA and RF values were compared. RNA and RF were assessed in untreated mice (PBS/PBS) and in sensitized mice after 4, 5, or 6 challenges. The correlation analysis was carried out in untreated mice (n 5 4) and sensitized mice and mice challenged 4, 5, or 6 times (n 5 4-8). RNA negatively correlated with RF.
FIG 4. RF 24 hours after repeated allergen challenges. Twenty-four hours after the last of 4 to 12 challenges, RF was monitored in previously sensitized mice (n 5 8 in each group). Means 6 SEM are shown. *P < .05 compared with baseline values.
the Online Repository in the online version of this article at www.jacionline.org). Comparison of RNA and RF in the same mice indicated the significant inverse correlation between these 2 measurements (Fig 3).
Changes in RF and RNA after repetitive allergen challenge We next investigated whether decreases in RF were also maintained after repetitive challenges of sensitized mice. Using WBP, we were able to follow the consequences of increasing numbers of intranasal OVA challenges on RF in OVA-sensitized mice (Fig 4). We recorded daily changes in RF values in the same mouse for up to 12 days of challenges. RF values decreased progressively after up to 7 challenges and then remained low throughout the 12-day monitoring period. A similar pattern was observed when we monitored RNA directly in the invasive system. Increases in RNA remained significant after 12 challenges (1.7 6 0.17 cm H2O/mL/s) compared with those in PBS/ PBS mice (0.9 6 0.03 cm H2O/mL/s, P < .05).
FIG 5. A, RNA was monitored over 10 minutes after a fourth OVA challenge in sensitized (OVA/OVA3) or nonsensitized (PBS/OVA3) mice. B, RF was monitored over 60 minutes after a fourth allergen challenge in WT or FceRI2/2 mice (n 5 8 in each group). Means 6 SEM are shown for RNA and RF as a percentage of the value at 2 minutes and baseline values before challenge. *P < .05, **P < .01, and ***P < .001 compared with PBS/OVA3 or PBS/ PBS3 groups. #P < .05, ##P < .01, and ###P < .001 compared with OVA/OVA3 FceRI2/2 mice.
Kinetics of early-phase nasal hyperresponsiveness to allergen We next determined whether an early-phase nasal response after nasal allergen challenge could be detected in sensitized mice. We prepared mice by means of sensitization and then exposed them to 3 OVA challenges. Twenty-four hours after the third challenge, a fourth challenge was administered, and the kinetics of the immediate response were monitored. RNA values after the fourth OVA challenge increased rapidly in sensitized and challenged mice compared with in mice that were only challenged (Fig 5, A). The experiments were stopped after 10 minutes because of increased nasal secretions. We evaluated changes in RF in this early phase as well. Sensitized and challenged mice showed an early decrease in RF after the fourth intranasal OVA challenge, with a nadir at 4 to 7 minutes (Fig 5, B). This early decrease in RF was associated with prolongation of both inspiratory and expiratory times (Fig E4 in the Online Repository in the online version of this article at www.jacionline.org).
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OVA/OVA WT mice showed significant increases in RNA and decreases in RF compared with PBA/OVA WT mice after 6 challenges (Fig 7). Sensitized and challenged FceRI2/2 mice had significant increases in RNA and decreases in RF compared with those in nonsensitized but challenged mice, but the extent of these changes in the FceRI2/2 mice were significantly lower than those in sensitized and challenged WT mice.
Mechanisms of asthma and allergic inflammation
FIG 6. Tissue infiltration with major basic protein–positive eosinophils 24 hours after 6 nasal challenges in WT and FceRI2/2 mice. Immunohistochemical (major basic protein) localization of nasal septal tissue eosinophils was determined 24 hours after 6 nasal challenges in sensitized or nonsensitized mice, as described in the Methods section. Eosinophil infiltration in nonsensitized but challenged WT mice (A), sensitized and challenged WT mice (B), nonsensitized but challenged FceRI2/2 mice (C), and sensitized and challenged FceRI2/2 mice (D) is shown. Bar 5 100 mm.
In contrast, PBS/PBS3 and PBS/OVA3 mice did not show any decreases in RF within 60 minutes after OVA challenge (Fig 5, B). In the OVA/OVA3 mice RF levels returned to normal by 1 hour.
Sensitization and nasal challenge with OVA increases total IgE and OVA-specific IgE and IgG1 levels in FceRI2/2 mice To assess the effect of sensitization and 6 challenges on Ig production in FceRI2/2 mice, we measured serum levels of total IgE and OVA-specific IgE and IgG1. OVA-sensitized and challenged FceRI2/2 mice showed increased serum levels of total IgE and OVA-specific IgE and IgG1 (Table I), which were similar to those in OVA-sensitized and challenged WT mice and significantly higher than those in nonsensitized but challenged FceRI2/2 mice. Nasal eosinophil accumulation in FceRI2/2 mice After 6 days of OVA challenges, sensitized WT mice demonstrated significant increases in nasal eosinophil numbers compared with those seen in the nonsensitized but challenged mice (Table I). Similar results were observed in FceRI2/2 mice: after 6 nasal challenges, sensitized FceRI2/2 mice showed significant increases in numbers of nasal eosinophils compared with those seen in the nonsensitized but challenged FceRI2/2 mice (Fig 6 and Table I). The number of eosinophils in sensitized and challenged FceRI2/2 mice were not different from those in sensitized and challenged WT mice. RNA and RF responses are significantly modified in FceRI2/2 mice Fig 7 shows the changes in RNA and RF 24 hours after the sixth OVA challenge in both WT and FceRI2/2 mice.
FceRI2/2 mice do not have early-phase nasal hyperresponsiveness To assess whether FceRI plays a role in the development of early-phase nasal hyperresponsiveness, we sensitized and challenged FceRI2/2 mice 3 times. OVA- sensitized and OVA-challenged FceRI2/2 mice, unlike FceRI1/1 mice, demonstrated no significant decrease in RF after the fourth OVA challenge, and the responses were similar to those of the nonsensitized but challenged WT mice (Fig 5, B). IL-13 levels in nasal tissue homogenates are significantly lower in FceRI2/2 mice compared with those in WT mice after sensitization and challenge IL-13 has been shown to be an important mediator in the development of allergic responses.17,18 To assess whether IL-13 is upregulated after sensitization and nasal challenges, we measured levels of IL-13 in nasal tissue homogenates adjusted to weight of samples. WT mice that were sensitized and challenged (6 times) exhibited increased levels of IL-13 in tissue homogenates 24 hours after the sixth nasal challenge compared with levels in nonsensitized but challenged WT mice (Fig 8). Sensitized and challenged FceRI2/2 mice showed some increase in the levels of IL-13 compared with those in nonsensitized but challenged FceRI2/2 mice, but the levels were significantly lower than those in sensitized and challenged WT mice (P < .01). IL-4 and IL-5 levels were increased in sensitized and challenged WT and FceRI2/2 mice to a similar degree, whereas IFN-g levels in both WT and FceRI2/2 mice were similar to those in mice that were only challenged. DISCUSSION Indirect methods for assessing the nasal responses in mice have relied on counting the number of sneezing and nasal rubbing episodes or the threshold concentration of histamine to evoke sneezing.14,19,20 Measurements of RNA have been reported in other animal models, such as guinea pigs and dogs,21,22 but direct measurement of nasal resistance in mice was technically difficult because of the anatomically smaller nasal cavities. In this study we characterized 2 methods to measure nasal airway responses in vivo, using both invasive and noninvasive approaches. We used custom-designed ventilation equipment, which can measure the resistance of an open canal by means of
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FIG 7. Nasal resistance and RF 24 hours after repeated allergen challenge in FceRI2/2 mice. RNA (A) and RF (B) were measured 24 hours after the sixth allergen challenge in nonsensitized (PBS/OVA6) and sensitized (OVA/ OVA6) WT and FceRI2/2 mice (n 5 8 in each group). Means 6 SEM are shown. #P < .05 and ##P < .01 compared with OVA/OVA FceRI2/2 mice. *P < .05 and **P < .01 compared with PBS/OVA group.
piston volume displacement and cylinder pressure.16 This method enabled us to directly measure RNA in the small nasal cavities of mice. We also predicted that allergeninduced nasal blockage should influence the respiratory pattern of mice because they are obligate nasal breathers. In parallel to RNA, we monitored RF in unrestrained conscious mice using WBP. This box signal measures differences in pressure between a main chamber containing the animal and a reference chamber and provides data on a number of different respiratory parameters.23 Using this noninvasive method, we demonstrated that increases in RNA were significantly correlated with decreases in RF, suggesting that RF is a valid, albeit indirect, indicator of nasal resistance or blockage in mice. To some extent, the responses differed on the basis of the number of challenges. After up to 4 challenges in sensitized mice, changes in RF returned to baseline by 24 hours. After additional challenges, congestion or sustained nasal blockage persisted, resulting in higher baseline values the following day (eg, after 6 challenges). The advantage of monitoring RNA is that it is not influenced by circadian rhythm or movement of the animals but is a direct measure of intranasal pressure. On the other hand, the advantage of the indirect and noninvasive measurement of nasal resistance with WBP is that the animals do not need to be killed once the measurements are finished, and several measurements on the same animals can be performed, even over days, allowing longitudinal studies and investigation of treatment protocols. One problem with measuring nasal obstruction with WBP is the uncertainty of the site of obstruction.24 Previously, we documented the noninvasive measurement of lower airway function in a mouse model of allergen sensitization followed by allergen inhalation challenge. In that study we demonstrated that the increase in lower airway resistance to inhaled MCh was associated with a decrease in RF.25 In the present study we carried out intranasal challenges, instilling an OVA solution into the nostril but without anesthesia. In studies of lower airway
FIG 8. Levels of IL-13 in nasal tissue after sensitization and challenge. Levels of IL-13 in nasal tissue homogenates were determined 24 hours after the sixth nasal challenge, as described in the Methods section (n 5 8 in each group). Means 6 SEM are shown. *P < .01 compared with OVA/OVA FceRI2/2 mice. #P < .01 compared with PBS/OVA6 and PBS/PBS6 groups.
function, intranasal challenges are performed in anesthetized mice, which aspirate the solution. In contrast, nonanesthetized mice swallow the solution. Saito et al19 have shown that no trypan blue staining was observed below the tracheal region after nasal instillation in nonanesthetized mice and that the mice sensitized and nasally challenged without anesthesia do not show lower airway hyperresponsiveness to MCh. Similarly, we confirmed that intranasal allergen challenge of mice using the same method did not evoke lower airway inflammation or airway hyperresponsiveness to inhaled MCh. Sensitized mice subjected to intranasal allergen challenge without anesthesia showed decreases in RF and increases in RNA without changes in lower airway resistance. These data establish that the decreases in RF in our protocol were induced by nasal blockage but not lower airway obstruction. Early nasal blockage is likely caused by increases in nasal secretions and swelling of nasal membranes. After aspiration of the nasal cavity after initial measurements, RNA decreased immediately in both the OVA/OVA and
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PBS/OVA groups. In the PBS/OVA group RNA returned to baseline values, but in the OVA/OVA group RNA remained higher. In contrast, the late nasal response might be caused by persistent swelling of nasal membranes because nasal suction did not influence RNA values 24 hours after the last challenge. Many lines of evidence have led to the concept that the interaction between IgE and mast cells and basophils is the primary effector pathway in allergic responses, at least early-phase responses and possibly late-phase responses as well. Mast cell activation occurs after cross-linking of cell surface–bound IgE with multivalent allergens, resulting in the release of mediators, such as histamine, as well as cytokines, such as IL-13, which directly induce allergic symptoms.26 Immediate and late-phase allergic reactions induced by IgE-dependent mast cell activation trigger a cascade that leads to chronic allergic inflammation.27 IgE-producing plasma cells have been identified in the submucosal tissue of the nose,28 and allergen-specific IgE has been detected in nasal mucosa and nasal washes from human subjects with AR.29,30 The role of FceRI in AR has been implied but has not been well studied. FceRI2/2 mice were prepared by disruption of the FceRIa gene.31 These mice were essentially normal in all aspects of their development and maturation, including the presence of functional mast cells.31,32 The FceRI2/2 mice were able to fully mount an immune response to allergen exposure, as shown by increased levels of total IgE and allergen-specific IgE and IgG1. It has been reported that FceRI2/2 mice are resistant to passive cutaneous anaphylaxis and systemic anaphylaxis,31 whereas they showed normal development of lower airway hyperresponsiveness after sensitization (together with alum) and challenge comparable with that seen in WT mice.33 In the present study sensitized and challenged FceRI2/2 mice did not show any nasal response in the early-phase response to allergen challenge and had only small increases in RNA during the late-phase response (24 hours after 6 challenges). These data suggest that nasal reactivity in AR might be more dependent on the IgE–FceRI–mast cell module than certain models of allergen-induced airway hyperresponsiveness and inflammation, which can develop in the absence of IgE,10 FceRI,33 or mast cells.9 Sensitized and challenged FceRI2/2 mice did show increases in IL-13 levels in the nasal mucosa 24 hours after the last of 6 allergen challenges, but the levels were significantly lower than in sensitized and challenged WT mice. IL-13 has been shown to be an important mediator in the development of allergic responses.17,18 Our results indicate that the presence of FceRI is required, at least to some degree, for the production of IL-13 in vivo. Mast cells might be one source of IL-13, but this might not be the only or critical source.34 More important potentially are other mast cell–derived mediators (eg, leukotriene B4), which might recruit other IL-13–producing cell types to the target organ.35,36 After sensitization and challenge, FceRI2/2 mice showed different results in terms of eosinophil infiltration, dependent on the mode of allergen exposure.33,37 On the other hand, in a mouse model of AR,
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nasally sensitized, IgE-deficient mice showed no difference in the intensity of eosinophil infiltration in the nasal membranes compared with that seen in WT mice, suggesting that rhinitis could arise in the absence of IgE.15 Here we showed that FceRI2/2 mice also demonstrated a similar level of eosinophil infiltration in the nasal membrane as WT mice. Nonetheless, the mice did not have earlyphase nasal reactivity and exhibited reduced late-phase nasal blockage. Because the numbers of eosinophils were not different, the role eosinophils play in these nasal responses remains to be defined. In summary, this report describes novel invasive and noninvasive methods to evaluate nasal responses to allergen challenge. Development of decreased RF, assessed by means of WBP, was highly correlated with directly measured increases in RNA. No lower airway hyperresponsiveness developed when mice were sensitized and challenged by means of nasal instillation in conscious animals. Two phases of the response were detected, an early phase within minutes that resolved by 60 minutes and a late or chronic sustained nasal blockage, which persisted for at least 24 hours after 6 or more challenges. On the basis of the responses in FceRI2/2 mice, we demonstrated that the integrity of FceRI was essential to the development of an early-phase nasal response and was a major contributor to the late-phase response and IL-13 production. The measurement of both RNA and RF provides a promising approach for defining critical components in the early- and late-phase responses in AR and the means for identifying new approaches to the prevention and treatment of AR. We thank Dr J. J. Lee (Mayo Clinic, Scottsdale, Ariz) for providing the anti–major basic protein antibody and D. Nabighian (National Jewish Medical and Research Center) for her assistance.
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