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Late phase responses after nasal challenges with allergen and histamine in asthmatic children with perennial nasal allergy
Auris Nasus Larynx 28 (2001) 305– 310 www.elsevier.com/locate/anl
Late phase responses after nasal challenges with allergen and histamine in asthmatic children with perennial nasal allergy Masayoshi Kobayashi *, Kotaro Ukai, Masanori Tatematsu, Toru Matsuura, Yasuo Sakakura Department of Otorhinolaryngology, Mie Uni6ersity School of Medicine, 2 -174, Edobashi, Tsu, Mie 514 -8507, Japan Received 17 July 2000; received in revised form 8 March 2001; accepted 23 March 2001
Abstract Objecti6e: Late phase response (LPR) is difficult to investigate in patients with perennial nasal allergy because of their continuous presentation with nasal symptoms. Contribution of histamine to the LPR is also controversial. In this study, we investigated whether exogenous histamine can induce LPR in asthmatic patients with perennial nasal allergy to house dust. Methods: A total of 40 asthmatic children were divided into clinical, subclinical and non-rhinitis groups based on their daily nasal symptoms. Changes in nasal patency and in inflammatory cells in nasal secretion were quantitatively measured for 6 h by acoustic rhinometry and light microscopy respectively before and after nasal challenge with allergen or histamine. Results: The allergen challenge produced a significant biphasic decrease in nasal patency in the subclinical group and a marginal decrease in the clinical group, with increases in eosinophils 6 h after the challenge. By contrast, histamine challenge induced significant responses in the clinical group and only a slight response in the subclinical group. Eosinophils also accumulated in nasal secretion of the clinical group to significant levels 6 h after histamine challenge. Eosinophil accumulation following histamine challenge was earlier than that after exposure to allergen. Conclusion: We conclude that LPR can be demonstrated in asthmatic children with perennial nasal allergy. Exposure to exogenous histamine also induced LPR, mediated mainly by eosinophil-related mediators. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acoustic rhinometry; Clinical and subclinical groups; Eosinophils; Histamine; House dust; Late phase response
1. Introduction It has been well accepted that recurrent bronchial constriction and erythema occur several hours after an antigen-antibody immediate reaction, the process called late phase response (LPR) involved in type I allergy [1,2]. In nasal allergy, LPR to allergen has also been recognized as a phenomenon where nasal obstruction appears recurrently 6 – 12 h after immediate nasal responses such as sneeze, rhinorrhea and nasal obstruction [3,4]. Nasal LPR has been established as an allergic reaction by the fact that IgE-antibody is produced locally in nasal mucosa during the late phase [5]. Because the continuous presentation of allergic nasal * Corresponding author. Tel.: + 81-59-2315028; fax: +81-592315218. E-mail address: [email protected] (M. Kobayashi).
symptoms may mask the LPR, many studies of LPR have been conducted on patients with pollinosis in a pollen off-season [4,6,7]. There are very few studies, to our knowledge, on examination of perennial nasal allergy. Since administration of steroids or leukotriene antagonists suppresses nasal obstruction, a major symptom of nasal LPR, it is generally accepted that endogenous leukotrienes from eosinophils are the main mediators of nasal LPR [4,8 –10]. Based on the observation that histamine was increased in nasal secretion several hours after allergen challenge, some researchers have suggested that endogenous histamine also contributes to nasal LPR as one of the mediators [6,7,11]. However, this interpretation is in contradiction with other studies reporting that sneezing and rhinorrhea, which are characteristic of histamine challenge, are in fact infrequent features in nasal LPR [3,4,8].
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Recently, it was reported that exogenous leukotriene D4 challenge also induced nasal LPR [12]. To date, however, it remains to be proven whether exogenous histamine induces nasal LPR. Bearing in mind that histamine is chemotactic to eosinophils, histamine can be expected to effect LPR directly or indirectly [13,14]. The present study was first designed to investigate whether nasal LPR can be examined in asthmatic children in the presence of perennial nasal allergy, and secondly to test whether exogenous histamine can induce nasal LPR.
2. Materials and methods
2.1. Subjects A total of 40 hospitalized asthmatic children (32 boys and eight girls) ranging in age from 8 to 15 years with a mean age of 11.2 years were studied in Mie National Hospital. The diagnosis of bronchial asthma was made using the criteria recommended by the National Institute of Health [15]. Patients had not been treated with any medication except ergotherapy for a period of 7 weeks prior to inclusion in this study. The reason why hospitalized asthmatics were chosen as subjects for this study was that most of these patients had a combination of asthma with clinical or subclinical nasal allergy to house dust [16] and that these divided groups were simultaneously examined on the same clean environmental condition. The next of kin of the children provided informed consent for participation in this study and the study was approved by Mie National Hospital. The patients had recorded their grade and frequency of daily nasal allergic symptoms for 7 weeks prior to the nasal challenge described later. A skin test or IgE radioallergosorbent test (RAST) in serum and nasal smear cytology were also conducted. Based on these results, the patients were divided into three groups: clinical, subclinical and non-rhinitis group [16] (Table 1). The clinical group consisted of asthmatics who showed a positive skin test and nasal provocation to house dust in addition to typical daily symptoms of nasal allergy. The subclinical group was made up of asthmatics who had little daily nasal symptoms except under an allergen challenge and who exhibited a posi-
tive skin test. The non-rhinitis group consisted of asthmatic patients without any nasal allergic symptoms and without a positive skin test.
2.2. Nasal challenge tests For allergen challenge, a small round paper disc (3 mm in diameter) was saturated with 250 mg house dust extract (protein nitrogen value: 5 mg; Torii, Tokyo, Japan) and applied for 5 min to bilateral nasal cavities on the inferior turbinates [17]. For histamine challenge, 20 ml of a 103 mg/ml histamine dihydrochloride solution (Nacalai Tesque, Kyoto, Japan) was applied to the bilateral nasal cavities on the same area as that in the allergen challenge using a microinjector. The patients were asked to exhale non-absorbed histamine by blowing their noses after 5 min.
2.3. Measurement of nasal airway patency Nasal airway patency was intermittently measured by acoustic rhinometry for 6 h before and after the allergen or histamine challenge [18]. The data obtained were converted to an area-distance function of the nasal cavity which provides estimates of minimum cross-sectional area (A-min) and volume (VOL) from the free tip of the nosepiece in the nostril to 5 cm into the nasal cavity. The previous study showed that the length of the nasal cavity is 7 cm in the adult and 4.5 cm in the newborn [19], while the distance from the nostril to A-min is 2.2 and 1 cm, respectively. Thus, the length of 5 cm is valid to be adopted as the range of VOL in this study. Measurements were taken three times on each occasion after sufficiently removing secretions in nasal cavities by gentle suction. A-min and VOL of each subject were evaluated as the median of the sum of the bilateral sides for three measurements.
2.4. Inflammatory cells in nasal secretion Estimation of inflammatory cells in nasal secretions was as follows. Nasal secretion on anterior parts of the bilateral inferior turbinates was gently collected by touching with Juhn Tym-Tap (Xomed-Treace, Jacksonville, Fla., USA) just before the measurement of nasal airway patency. The collection was carried out just before and 20 min, 3 and 6 h after the nasal
Table 1 Classification of nasal allergy to house dust and its criterion
Clinical Subclinical Non-nasal allergic
Skin test or specific IgE to allergen
Nasal provocation test to allergen
Daily nasal symptoms
Positive Positive None
Positive Positive None
Severe or moderate None None
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challenge. Though the spread of time intervals was uneven, we regarded this experimental design as appropriate because nasal secretion is always carried to the pharynx within 20 min by a nasal mucociliary clearance mechanism [20] and hence the number of cells counted represents a dynamic measure of cell accumulation at each time point. A total of 20 ml of the secretion was mixed with 800 ml of 0.067 mol/l phosphate buffered saline (pH 7.2) containing 0.01 mol/l dithiothreitol and 200 ml of Hanks’ balanced salt solution. After 3 h at room temperature, the samples were centrifuged at 1200 rpm for 10 min using a cytocentrifuge (Cytospin 2; Shandon Southern, Astmore, Runcorn, Cheshire, UK). Samples smeared onto the slide were air-dried, stained with Wright-Giemsa’s solution and examined by light microscopy [21].
2.5. Statistical analysis All values obtained in the present experiments were expressed as means9S.E. and assessed statistically according to Friedman’s analysis of variance for repeated measures. Data with significant differences were further evaluated using Wilcoxon’s signed rank test. Differences were considered significant when P B 0.05.
3. Results
3.1. A-min and VOL in asthmatic children In 12 asthmatics (four in the clinical group, four in the subclinical group and four in the non-rhinitis group), A-min and VOL had been measured during natural progression for 6 h. Although alternate increases and decreases in nasal patency were found between bilateral nasal cavities, the sum of the patency of the bilateral cavities was constant during 6 h for each individual patient. This result supported the validity of measurement of the sum of bilateral nasal patency in this study.
3.2. Effect of allergen challenge In the clinical group (n =10), the house dust challenge evoked a significant decreases in A-min and VOL 20 min after the challenge (Fig. 1). A-min and VOL recovered to basal levels thereafter. Although there was a trend to a significant decrease at 6 h after the challenge, the change was not statistically significant. In the subclinical group (n =10), the allergen challenge resulted in a significant decreases in A-min and VOL at both 20 min and 6 h after the challenge. In the nonrhinitis group (n=8), no significant changes in A-min or VOL were found after the challenge.
Fig. 1. Changes in nasal patency measured by acoustic rhinometry before (pre) and after (from 20 min to 6 h) allergen challenge. Columns with error bars (mean + S.E.) denote magnitudes of sums of bilateral nasal patency. A: A-min; and B: VOL. Closed columns: clinical group (n =10). Semi-closed columns: subclinical group (n= 10). Open columns: non-rhinitis group (n = 8). *PB 0.05, **P B0.01 compared with pre-challenge.
As illustrated in Fig. 2, a variety of inflammatory cells were increased in nasal secretion fluid 6 h after house dust challenge in both the clinical (n= 10) and the subclinical (n= 10) groups. Most strikingly, the number of eosinophils were significantly increased in secretions from both groups after the challenge. No changes in the number of inflammatory cells were found in non-rhinitis group (n= 8) before or after the challenge.
3.3. Effect of histamine challenge In the clinical group (n= 10), a histamine challenge induced significant decreases in A-min and VOL at 20 min and 6 h, but not at 3 h after administration (Fig. 3). The subclinical group also displayed a similar biphasic response. However, in the latter group the changes were very small and were not statistically significant (n= 10). No changes in A-min or VOL were observed in the non-rhinitis group (n= 8). Of the inflammatory cells which were increased in nasal secretion fluids in the clinical group (n= 10) after histamine challenge, only eosinophils were significantly increased 3 h after challenge (Fig. 4). There was no significant change in the subclinical group (n= 10) until 6 h after the challenge. In the non-rhinitis group (n= 8), no changes in the number of inflammatory cells were found.
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3.4. Correlation between nasal patency and eosinophils Correlation between nasal patency and the number of eosinophils in nasal secretion after allergen or histamine challenge was evaluated. A significant correlation between nasal patency and eosinophil accumulation was observed 6 h after allergen challenge in both the clinical (A-min: r= 0.710, VOL: r= 0.684, n = 10, P B 0.05) and the subclinical (A-min: r =0.682, VOL: r = 0.542, n= 10, P B 0.05) groups. In contrast, there was no correlation between these parameters for both groups following histamine challenge (A-min: r= 0.402, VOL: r = 0.436 in the clinical group, n = 10, NS; A-min: r= 0.322, VOL: r = 0.272 in the subclinical group, n =10, NS). However, there was a moderately significant correlation between nasal patency at 6 h and eosinophil accumulation at 3 h after a histamine challenge in the clinical group (A-min: r= 0.508, VOL: r =0.533, n= 10, P B 0.05), although this was not observed in the subclinical group (A-min: r= 0.308, VOL: r =0.286, n =10, NS).
4. Discussion While LPR has been established in bronchial asthma and atopic dermatitis since about 30 years ago, nasal LPR was controversial [1,2,22]. For the most recent 15 years, however, many studies have gradually revealed the existence and mechanism of nasal LPR [2– 4,12]. The fact
Fig. 2. Time course of number of inflammatory cells in nasal secretion before and after allergen challenge. Closed columns: clinical group (n = 10). Semi-closed columns: subclinical group (n = 10). Open columns: non-rhinitis group (n= 8). *PB 0.05 compared with pre-challenge.
Fig. 3. Changes in nasal patency before and after (20 min to 6 h) histamine challenge measured by acoustic rhinometry. Columns with error bars (mean + S.E.) denote magnitudes of sums of bilateral nasal patency. A: A-min; and B: VOL. Closed columns: clinical group (n =10). Semi-closed columns: subclinical group (n = 10). Open columns: non-rhinitis group (n = 8). *P B 0.05, **PB 0.01 compared with pre-challenge.
that nasal LPR is an allergic response was supported by a report that IgE was increased locally within nasal mucosa during the late phase [5]. In addition, these studies were in agreement that nasal LPR occurs 6 h after allergen challenge at the latest. Based on these findings, the time point at 6 h was selected to examine the LPR. In this study, we were able to demonstrate evidence of nasal LPR in asthmatic patients with perennial nasal allergy due to house dust. Previous studies have only demonstrated these changes in subclinical symptomatic individuals with seasonal pollinosis in the off-season [4,6,7]. In our study, clinical and subclinical patients were different from each other in their nonspecific nasal hypersensitivity. This is in agreement with our previous research which showed that the threshold of nasal allergic symptoms induced by histamine challenge in the subclinical group was higher than those in the clinical patients [16]. Also, in our present study, the concentration of histamine solution which was used produced neither an immediate- nor a late-phase decrease in nasal patency in the subclinical patients, while it induced significant biphasic responses in the clinical patient group. The study of nasal LPR in patients with perennial allergy in the clinical setting has been hampered by the problem that the size of the change in airway resistance in nasal LPR is much smaller than that in LPR of bronchial asthma. Furthermore, patients with perennial allergy always inhale abundant allergen which compels
M. Kobayashi et al. / Auris Nasus Larynx 28 (2001) 305–310
them to present continuous nasal allergic symptoms. In our allergen challenge, the clinical group of patients did not show a significant late-phase decrease in nasal patency, although a decrease in nasal patency on the immediate phase and a tendency to decrease in nasal patency on the late phase was observed. Such clinical patients may scarcely have residual reserves of swelling capacity in nasal mucosa on the late phase due to their constant responses to allergens even if significant allergic response can be induced on the immediate phase. By contrast, subclinical patients with perennial nasal allergy usually present little or no nasal symptoms, similar to patients with nasal pollinosis in the off-season. Therefore, we consider that our investigative protocol is available for studying nasal symptoms such as LPR in patients with perennial allergies and that such a procedure would contribute to the understanding of other responses in nasal allergy. Another interesting finding in our study was that exogenous histamine produced a biphasic nasal response including LPR, which has not been reported previously. Regarding histamine and LPR, several studies reported the increase in concentration of histamine in nasal secretion on the late phase, while it is unknown whether the secondly-released histamine contributes to the induction of nasal LPR [6,7,11]. On the other hand, another study implied that it was of little importance for asthmatic LPR [23]. However, it is possible that exogenous histamine does not induce LPR directly but does so
Fig. 4. Time course of number of inflammatory cells in nasal secretion before and after histamine challenge. Closed columns: clinical group (n = 10). Semi-closed columns: subclinical group (n = 10). Open columns: non-rhinitis group (n= 8). *PB 0.05 compared with pre-challenge.
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indirectly through some mediators which mainly elicit nasal obstruction, bearing in mind that the main symptoms presented in patients with nasal allergies are sneezing and rhinorrhea which are scarcely involved in nasal LPR [3,4,8]. Our results showed that late-phase decreases in nasal patency due to both house dust and histamine challenges are related to the presence of eosinophils in nasal secretions. If histamine indirectly induces LPR, eosinophils and their released cytokines can be expected to be mediators. Some previous studies have demonstrated that histamine has chemotactic properties towards eosinophils [13] and that this provoked marked local accumulation of eosinophils in allergic patients [22], while it is not so chemotactic as other cytokines: plateletactivating factor (PAF) [24], leukotriene B4 (LTB4) [25]. Another possible explanation is that exogenous histamine provoked release of various cytokines from nasal epithelial cells and that these mediators in turn displayed chemoattractant effects on eosinophils. Our laboratory previously reported that the level of histamine H1-receptor mRNA in nasal mucosa of nasal allergic patients was higher than that in non-rhinitis patients [26]. Others reported that nasal epithelial cells released cytokines chemotactic to eosinophils including RANTES (regulated upon activation, normal T expressed and presumably secreted), granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin (IL)-1a, IL-6, IL-8 and tumor necrosis factor-a (TNF-a) [27–29]. Furthermore, eosinophils themselves release inflammatory mediators such as leukotrienes, major basic protein and PAF [10,29]. Leukotrienes in particular enhance vascular permeability and induce extravasation and mucosal edema [30] and are considered to be factors responsible for nasal LPR, since pretreatment with pranlukast, an antagonist to the specific leukotriene receptor, significantly suppresses LPR as well as inducing an immediate decrease in nasal patency [8]. When we compared the nasal eosinophil accumulation following an allergen challenge with that after a histamine challenge, we found that the latter appeared earlier than the former. A similar result was reported for patients with atopic dermatitis [31]. Our present findings may be explained by the following observations. First, since the dose of histamine used in this study (103 mg/ml × 10 ml× 2 sides) was more than that which is usually released from mast cells accumulated in nasal mucosa following natural allergen presentation [32], the eosinophils might accumulate earlier in our histamine challenge than in the allergen challenge. Secondly, it was reported that specific allergen challenge provokes release of various inflammatory mediators and cytokines from basophils, mast cells and lymphocytes in addition to histamine from mast cells [5,10]. Through the sum of these effects, allergen challenge in our study probably produced a significant eosinophil accumulation 6 h
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after the challenge, consequently later than the histamine challenge did. Similarly, we might propose as a tentative explanation that, also in histamine challenge, the late-phase decrease in nasal patency was produced 6 h after the challenge by the sum of the effects of various mediators from not only eosinophils but also other inflammatory cells, while eosinophil accumulation preceded the late-phase decrease in nasal patency.
5. Conclusion We showed that nasal LPR can be examined even in asthmatic children with perennial nasal allergy by selecting and investigating subclinical symptomatic patients. Furthermore, we demonstrated for the first time that nasal exposure to exogenous histamine also induced nasal LPR, which is mediated mainly by eosinophil-related mediators. Clinically, the latter result implies that suppression of histamine release or effect on the immediate phase may affect the LPR.
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[12] Cho JS, Cha CII, Lee DY, Ukai K, Sakakura Y. The effect of leukotriene D4 on patency of nasal passage in the guinea pig. Am J Rhinol 1998;12:421 – 5. [13] Clark RAF, Gallin JI, Kaplan AP. The selective eosinophil chemotactic activity of histamine. J Exp Med 1975;142:1462 –76. [14] Turnbull LW, Kay AB. Eosinophils and mediators of anaphylaxis histamine and imidazole acetic acid as chemotactic agents for human eosinophil leukocytes. Immunology 1976;31:797 –802. [15] National Heart, Lung, and Blood Institute. International asthma management project. Definition, diagnosis, and classification. In: Claude L, editor. International Consensus Report on the Diagnosis and Management of Asthma. Bethesda: National Institute of Health, 1992:1 – 5. [16] Ukai K, Sakakura Y, Miyoshi Y, Uchida Y. Pathophysiological condition of the nose of asthmatic children with subclinical nasal allergy. Rhinology 1986;24:133 – 40. [17] Okuda M. Basic study of nasal provocative test, first report: side, site of the nose, size of site and allergen amount. Arch Otorhinolaryngol 1977;214:241 – 6. [18] Hilberg O, Jackson AC, Swift DL, Pederson OF. Acoustic rhinometry: evaluation of the geometry of the nasal cavity by acoustic reflections. J Appl Physiol 1989;66:295 – 303. [19] Pederson OF, Berkowitz R, Yamagiwa M, Hilberg O. Nasal cavity dimensions in the newborn measured by acoustic reflections. Laryngoscope 1994;104:1023 – 8. [20] Schuhl JF. Nasal mucociliary clearance in perennial rhinitis. J Investig Allergol Clin Immunol 1995;5:333 – 6. [21] Lee HS, Majima Y, Sakakura Y, Kim BW. A technique for quantitative cytology of nasal secretions. Eur Arch Otorhinolaryngol 1991;248:406 – 8. [22] Eidinger D, Wilkinson R, Rose B. A study of cellular responses in immune reactions utilizing the skin window technique. I. Immediate hypersensitivity reactions. J Allergy 1964;35:77 –85. [23] Twentyman OP, Ollier S, Holgate ST. The effect of H1-receptor blockade on the development of early- and late-phase bronchoconstriction and increased bronchial responsiveness in allergen-induced asthma. J Allergy Clin Immunol 1993;91:1169 –78. [24] Wardlaw AJ, Moqbel R, Cromwell O, Kay AB. Platelet-activating factor: a potent chemotactic and chemokinetic factor for human eosinophils. J Clin Invest 1986;78:1701 – 6. [25] Nagy L, Lee TH, Goetzl EJ, Pickett WC, Kay AB. Complement receptor enhancement and chemotaxis of human neutrophils and eosinophils by leukotrienes and other lipoxygenase products. Clin Exp Immunol 1982;47:541 – 7. [26] Iriyoshi N, Takeuchi K, Yuta A, Ukai K, Sakakura Y. Increased expression of histamine H1 receptor mRNA in allergic rhinitis. Clin Exp Allergy 1996;26:379 – 85. [27] Kenney JS, Baker C, Welch MR, Altman LC. Synthesis of interleukin-1a, interleukin-6, and interleukin-8 by cultured human nasal epithelial cells. J Allergy Clin Immunol 1994;93:1060 – 7. [28] Mullol J, Xaubet A, Gaya A, Roca-Ferrer E, Lo´ pez E, Fernandez JC, et al. Cytokine gene expression and release from epithelial cells. A comparison study between healthy nasal mucosa and nasal polyps. Clin Exp Allergy 1995;25:607 – 15. [29] Terada N, Maesako K, Hamano N, Ikeda T, Sai M, Yamashita T, et al. RANTES production in nasal epithelial cells and endothelial cells. J Allergy Clin Immunol 1996;98:S230 –7. [30] Dahlen SE, Hedqvist P, Hammarstrom S, Samuelsson B. Leukotrienes are potent constrictors of human bronchi. Nature 1980;288:484 – 6. [31] Feinberg AR, Feinberg SM, Lee F. Leukocytes and hypersensitivity reactions. I. Eosinophil response in skin window to ragweed extract, histamine, and compound 48/80 in atopic and nonatopic individuals. J Allergy 1967;40:73 – 87. [32] Ohtsuka H, Okuda M. Important factors in the nasal manifestation of allergy. Arch Otorhinolaryngol 1981;233:227 – 35.
Late phase responses after nasal challenges with allergen and histamine in asthmatic children with perennial nasal allergy Masayoshi Kobayashi *, Kotaro Ukai, Masanori Tatematsu, Toru Matsuura, Yasuo Sakakura Department of Otorhinolaryngology, Mie Uni6ersity School of Medicine, 2 -174, Edobashi, Tsu, Mie 514 -8507, Japan Received 17 July 2000; received in revised form 8 March 2001; accepted 23 March 2001
Abstract Objecti6e: Late phase response (LPR) is difficult to investigate in patients with perennial nasal allergy because of their continuous presentation with nasal symptoms. Contribution of histamine to the LPR is also controversial. In this study, we investigated whether exogenous histamine can induce LPR in asthmatic patients with perennial nasal allergy to house dust. Methods: A total of 40 asthmatic children were divided into clinical, subclinical and non-rhinitis groups based on their daily nasal symptoms. Changes in nasal patency and in inflammatory cells in nasal secretion were quantitatively measured for 6 h by acoustic rhinometry and light microscopy respectively before and after nasal challenge with allergen or histamine. Results: The allergen challenge produced a significant biphasic decrease in nasal patency in the subclinical group and a marginal decrease in the clinical group, with increases in eosinophils 6 h after the challenge. By contrast, histamine challenge induced significant responses in the clinical group and only a slight response in the subclinical group. Eosinophils also accumulated in nasal secretion of the clinical group to significant levels 6 h after histamine challenge. Eosinophil accumulation following histamine challenge was earlier than that after exposure to allergen. Conclusion: We conclude that LPR can be demonstrated in asthmatic children with perennial nasal allergy. Exposure to exogenous histamine also induced LPR, mediated mainly by eosinophil-related mediators. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Acoustic rhinometry; Clinical and subclinical groups; Eosinophils; Histamine; House dust; Late phase response
1. Introduction It has been well accepted that recurrent bronchial constriction and erythema occur several hours after an antigen-antibody immediate reaction, the process called late phase response (LPR) involved in type I allergy [1,2]. In nasal allergy, LPR to allergen has also been recognized as a phenomenon where nasal obstruction appears recurrently 6 – 12 h after immediate nasal responses such as sneeze, rhinorrhea and nasal obstruction [3,4]. Nasal LPR has been established as an allergic reaction by the fact that IgE-antibody is produced locally in nasal mucosa during the late phase [5]. Because the continuous presentation of allergic nasal * Corresponding author. Tel.: + 81-59-2315028; fax: +81-592315218. E-mail address: [email protected] (M. Kobayashi).
symptoms may mask the LPR, many studies of LPR have been conducted on patients with pollinosis in a pollen off-season [4,6,7]. There are very few studies, to our knowledge, on examination of perennial nasal allergy. Since administration of steroids or leukotriene antagonists suppresses nasal obstruction, a major symptom of nasal LPR, it is generally accepted that endogenous leukotrienes from eosinophils are the main mediators of nasal LPR [4,8 –10]. Based on the observation that histamine was increased in nasal secretion several hours after allergen challenge, some researchers have suggested that endogenous histamine also contributes to nasal LPR as one of the mediators [6,7,11]. However, this interpretation is in contradiction with other studies reporting that sneezing and rhinorrhea, which are characteristic of histamine challenge, are in fact infrequent features in nasal LPR [3,4,8].
0385-8146/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 8 5 - 8 1 4 6 ( 0 1 ) 0 0 0 8 8 - 8
M. Kobayashi et al. / Auris Nasus Larynx 28 (2001) 305–310
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Recently, it was reported that exogenous leukotriene D4 challenge also induced nasal LPR [12]. To date, however, it remains to be proven whether exogenous histamine induces nasal LPR. Bearing in mind that histamine is chemotactic to eosinophils, histamine can be expected to effect LPR directly or indirectly [13,14]. The present study was first designed to investigate whether nasal LPR can be examined in asthmatic children in the presence of perennial nasal allergy, and secondly to test whether exogenous histamine can induce nasal LPR.
2. Materials and methods
2.1. Subjects A total of 40 hospitalized asthmatic children (32 boys and eight girls) ranging in age from 8 to 15 years with a mean age of 11.2 years were studied in Mie National Hospital. The diagnosis of bronchial asthma was made using the criteria recommended by the National Institute of Health [15]. Patients had not been treated with any medication except ergotherapy for a period of 7 weeks prior to inclusion in this study. The reason why hospitalized asthmatics were chosen as subjects for this study was that most of these patients had a combination of asthma with clinical or subclinical nasal allergy to house dust [16] and that these divided groups were simultaneously examined on the same clean environmental condition. The next of kin of the children provided informed consent for participation in this study and the study was approved by Mie National Hospital. The patients had recorded their grade and frequency of daily nasal allergic symptoms for 7 weeks prior to the nasal challenge described later. A skin test or IgE radioallergosorbent test (RAST) in serum and nasal smear cytology were also conducted. Based on these results, the patients were divided into three groups: clinical, subclinical and non-rhinitis group [16] (Table 1). The clinical group consisted of asthmatics who showed a positive skin test and nasal provocation to house dust in addition to typical daily symptoms of nasal allergy. The subclinical group was made up of asthmatics who had little daily nasal symptoms except under an allergen challenge and who exhibited a posi-
tive skin test. The non-rhinitis group consisted of asthmatic patients without any nasal allergic symptoms and without a positive skin test.
2.2. Nasal challenge tests For allergen challenge, a small round paper disc (3 mm in diameter) was saturated with 250 mg house dust extract (protein nitrogen value: 5 mg; Torii, Tokyo, Japan) and applied for 5 min to bilateral nasal cavities on the inferior turbinates [17]. For histamine challenge, 20 ml of a 103 mg/ml histamine dihydrochloride solution (Nacalai Tesque, Kyoto, Japan) was applied to the bilateral nasal cavities on the same area as that in the allergen challenge using a microinjector. The patients were asked to exhale non-absorbed histamine by blowing their noses after 5 min.
2.3. Measurement of nasal airway patency Nasal airway patency was intermittently measured by acoustic rhinometry for 6 h before and after the allergen or histamine challenge [18]. The data obtained were converted to an area-distance function of the nasal cavity which provides estimates of minimum cross-sectional area (A-min) and volume (VOL) from the free tip of the nosepiece in the nostril to 5 cm into the nasal cavity. The previous study showed that the length of the nasal cavity is 7 cm in the adult and 4.5 cm in the newborn [19], while the distance from the nostril to A-min is 2.2 and 1 cm, respectively. Thus, the length of 5 cm is valid to be adopted as the range of VOL in this study. Measurements were taken three times on each occasion after sufficiently removing secretions in nasal cavities by gentle suction. A-min and VOL of each subject were evaluated as the median of the sum of the bilateral sides for three measurements.
2.4. Inflammatory cells in nasal secretion Estimation of inflammatory cells in nasal secretions was as follows. Nasal secretion on anterior parts of the bilateral inferior turbinates was gently collected by touching with Juhn Tym-Tap (Xomed-Treace, Jacksonville, Fla., USA) just before the measurement of nasal airway patency. The collection was carried out just before and 20 min, 3 and 6 h after the nasal
Table 1 Classification of nasal allergy to house dust and its criterion
Clinical Subclinical Non-nasal allergic
Skin test or specific IgE to allergen
Nasal provocation test to allergen
Daily nasal symptoms
Positive Positive None
Positive Positive None
Severe or moderate None None
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challenge. Though the spread of time intervals was uneven, we regarded this experimental design as appropriate because nasal secretion is always carried to the pharynx within 20 min by a nasal mucociliary clearance mechanism [20] and hence the number of cells counted represents a dynamic measure of cell accumulation at each time point. A total of 20 ml of the secretion was mixed with 800 ml of 0.067 mol/l phosphate buffered saline (pH 7.2) containing 0.01 mol/l dithiothreitol and 200 ml of Hanks’ balanced salt solution. After 3 h at room temperature, the samples were centrifuged at 1200 rpm for 10 min using a cytocentrifuge (Cytospin 2; Shandon Southern, Astmore, Runcorn, Cheshire, UK). Samples smeared onto the slide were air-dried, stained with Wright-Giemsa’s solution and examined by light microscopy [21].
2.5. Statistical analysis All values obtained in the present experiments were expressed as means9S.E. and assessed statistically according to Friedman’s analysis of variance for repeated measures. Data with significant differences were further evaluated using Wilcoxon’s signed rank test. Differences were considered significant when P B 0.05.
3. Results
3.1. A-min and VOL in asthmatic children In 12 asthmatics (four in the clinical group, four in the subclinical group and four in the non-rhinitis group), A-min and VOL had been measured during natural progression for 6 h. Although alternate increases and decreases in nasal patency were found between bilateral nasal cavities, the sum of the patency of the bilateral cavities was constant during 6 h for each individual patient. This result supported the validity of measurement of the sum of bilateral nasal patency in this study.
3.2. Effect of allergen challenge In the clinical group (n =10), the house dust challenge evoked a significant decreases in A-min and VOL 20 min after the challenge (Fig. 1). A-min and VOL recovered to basal levels thereafter. Although there was a trend to a significant decrease at 6 h after the challenge, the change was not statistically significant. In the subclinical group (n =10), the allergen challenge resulted in a significant decreases in A-min and VOL at both 20 min and 6 h after the challenge. In the nonrhinitis group (n=8), no significant changes in A-min or VOL were found after the challenge.
Fig. 1. Changes in nasal patency measured by acoustic rhinometry before (pre) and after (from 20 min to 6 h) allergen challenge. Columns with error bars (mean + S.E.) denote magnitudes of sums of bilateral nasal patency. A: A-min; and B: VOL. Closed columns: clinical group (n =10). Semi-closed columns: subclinical group (n= 10). Open columns: non-rhinitis group (n = 8). *PB 0.05, **P B0.01 compared with pre-challenge.
As illustrated in Fig. 2, a variety of inflammatory cells were increased in nasal secretion fluid 6 h after house dust challenge in both the clinical (n= 10) and the subclinical (n= 10) groups. Most strikingly, the number of eosinophils were significantly increased in secretions from both groups after the challenge. No changes in the number of inflammatory cells were found in non-rhinitis group (n= 8) before or after the challenge.
3.3. Effect of histamine challenge In the clinical group (n= 10), a histamine challenge induced significant decreases in A-min and VOL at 20 min and 6 h, but not at 3 h after administration (Fig. 3). The subclinical group also displayed a similar biphasic response. However, in the latter group the changes were very small and were not statistically significant (n= 10). No changes in A-min or VOL were observed in the non-rhinitis group (n= 8). Of the inflammatory cells which were increased in nasal secretion fluids in the clinical group (n= 10) after histamine challenge, only eosinophils were significantly increased 3 h after challenge (Fig. 4). There was no significant change in the subclinical group (n= 10) until 6 h after the challenge. In the non-rhinitis group (n= 8), no changes in the number of inflammatory cells were found.
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3.4. Correlation between nasal patency and eosinophils Correlation between nasal patency and the number of eosinophils in nasal secretion after allergen or histamine challenge was evaluated. A significant correlation between nasal patency and eosinophil accumulation was observed 6 h after allergen challenge in both the clinical (A-min: r= 0.710, VOL: r= 0.684, n = 10, P B 0.05) and the subclinical (A-min: r =0.682, VOL: r = 0.542, n= 10, P B 0.05) groups. In contrast, there was no correlation between these parameters for both groups following histamine challenge (A-min: r= 0.402, VOL: r = 0.436 in the clinical group, n = 10, NS; A-min: r= 0.322, VOL: r = 0.272 in the subclinical group, n =10, NS). However, there was a moderately significant correlation between nasal patency at 6 h and eosinophil accumulation at 3 h after a histamine challenge in the clinical group (A-min: r= 0.508, VOL: r =0.533, n= 10, P B 0.05), although this was not observed in the subclinical group (A-min: r= 0.308, VOL: r =0.286, n =10, NS).
4. Discussion While LPR has been established in bronchial asthma and atopic dermatitis since about 30 years ago, nasal LPR was controversial [1,2,22]. For the most recent 15 years, however, many studies have gradually revealed the existence and mechanism of nasal LPR [2– 4,12]. The fact
Fig. 2. Time course of number of inflammatory cells in nasal secretion before and after allergen challenge. Closed columns: clinical group (n = 10). Semi-closed columns: subclinical group (n = 10). Open columns: non-rhinitis group (n= 8). *PB 0.05 compared with pre-challenge.
Fig. 3. Changes in nasal patency before and after (20 min to 6 h) histamine challenge measured by acoustic rhinometry. Columns with error bars (mean + S.E.) denote magnitudes of sums of bilateral nasal patency. A: A-min; and B: VOL. Closed columns: clinical group (n =10). Semi-closed columns: subclinical group (n = 10). Open columns: non-rhinitis group (n = 8). *P B 0.05, **PB 0.01 compared with pre-challenge.
that nasal LPR is an allergic response was supported by a report that IgE was increased locally within nasal mucosa during the late phase [5]. In addition, these studies were in agreement that nasal LPR occurs 6 h after allergen challenge at the latest. Based on these findings, the time point at 6 h was selected to examine the LPR. In this study, we were able to demonstrate evidence of nasal LPR in asthmatic patients with perennial nasal allergy due to house dust. Previous studies have only demonstrated these changes in subclinical symptomatic individuals with seasonal pollinosis in the off-season [4,6,7]. In our study, clinical and subclinical patients were different from each other in their nonspecific nasal hypersensitivity. This is in agreement with our previous research which showed that the threshold of nasal allergic symptoms induced by histamine challenge in the subclinical group was higher than those in the clinical patients [16]. Also, in our present study, the concentration of histamine solution which was used produced neither an immediate- nor a late-phase decrease in nasal patency in the subclinical patients, while it induced significant biphasic responses in the clinical patient group. The study of nasal LPR in patients with perennial allergy in the clinical setting has been hampered by the problem that the size of the change in airway resistance in nasal LPR is much smaller than that in LPR of bronchial asthma. Furthermore, patients with perennial allergy always inhale abundant allergen which compels
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them to present continuous nasal allergic symptoms. In our allergen challenge, the clinical group of patients did not show a significant late-phase decrease in nasal patency, although a decrease in nasal patency on the immediate phase and a tendency to decrease in nasal patency on the late phase was observed. Such clinical patients may scarcely have residual reserves of swelling capacity in nasal mucosa on the late phase due to their constant responses to allergens even if significant allergic response can be induced on the immediate phase. By contrast, subclinical patients with perennial nasal allergy usually present little or no nasal symptoms, similar to patients with nasal pollinosis in the off-season. Therefore, we consider that our investigative protocol is available for studying nasal symptoms such as LPR in patients with perennial allergies and that such a procedure would contribute to the understanding of other responses in nasal allergy. Another interesting finding in our study was that exogenous histamine produced a biphasic nasal response including LPR, which has not been reported previously. Regarding histamine and LPR, several studies reported the increase in concentration of histamine in nasal secretion on the late phase, while it is unknown whether the secondly-released histamine contributes to the induction of nasal LPR [6,7,11]. On the other hand, another study implied that it was of little importance for asthmatic LPR [23]. However, it is possible that exogenous histamine does not induce LPR directly but does so
Fig. 4. Time course of number of inflammatory cells in nasal secretion before and after histamine challenge. Closed columns: clinical group (n = 10). Semi-closed columns: subclinical group (n = 10). Open columns: non-rhinitis group (n= 8). *PB 0.05 compared with pre-challenge.
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indirectly through some mediators which mainly elicit nasal obstruction, bearing in mind that the main symptoms presented in patients with nasal allergies are sneezing and rhinorrhea which are scarcely involved in nasal LPR [3,4,8]. Our results showed that late-phase decreases in nasal patency due to both house dust and histamine challenges are related to the presence of eosinophils in nasal secretions. If histamine indirectly induces LPR, eosinophils and their released cytokines can be expected to be mediators. Some previous studies have demonstrated that histamine has chemotactic properties towards eosinophils [13] and that this provoked marked local accumulation of eosinophils in allergic patients [22], while it is not so chemotactic as other cytokines: plateletactivating factor (PAF) [24], leukotriene B4 (LTB4) [25]. Another possible explanation is that exogenous histamine provoked release of various cytokines from nasal epithelial cells and that these mediators in turn displayed chemoattractant effects on eosinophils. Our laboratory previously reported that the level of histamine H1-receptor mRNA in nasal mucosa of nasal allergic patients was higher than that in non-rhinitis patients [26]. Others reported that nasal epithelial cells released cytokines chemotactic to eosinophils including RANTES (regulated upon activation, normal T expressed and presumably secreted), granulocyte-macrophage colonystimulating factor (GM-CSF), interleukin (IL)-1a, IL-6, IL-8 and tumor necrosis factor-a (TNF-a) [27–29]. Furthermore, eosinophils themselves release inflammatory mediators such as leukotrienes, major basic protein and PAF [10,29]. Leukotrienes in particular enhance vascular permeability and induce extravasation and mucosal edema [30] and are considered to be factors responsible for nasal LPR, since pretreatment with pranlukast, an antagonist to the specific leukotriene receptor, significantly suppresses LPR as well as inducing an immediate decrease in nasal patency [8]. When we compared the nasal eosinophil accumulation following an allergen challenge with that after a histamine challenge, we found that the latter appeared earlier than the former. A similar result was reported for patients with atopic dermatitis [31]. Our present findings may be explained by the following observations. First, since the dose of histamine used in this study (103 mg/ml × 10 ml× 2 sides) was more than that which is usually released from mast cells accumulated in nasal mucosa following natural allergen presentation [32], the eosinophils might accumulate earlier in our histamine challenge than in the allergen challenge. Secondly, it was reported that specific allergen challenge provokes release of various inflammatory mediators and cytokines from basophils, mast cells and lymphocytes in addition to histamine from mast cells [5,10]. Through the sum of these effects, allergen challenge in our study probably produced a significant eosinophil accumulation 6 h
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after the challenge, consequently later than the histamine challenge did. Similarly, we might propose as a tentative explanation that, also in histamine challenge, the late-phase decrease in nasal patency was produced 6 h after the challenge by the sum of the effects of various mediators from not only eosinophils but also other inflammatory cells, while eosinophil accumulation preceded the late-phase decrease in nasal patency.
5. Conclusion We showed that nasal LPR can be examined even in asthmatic children with perennial nasal allergy by selecting and investigating subclinical symptomatic patients. Furthermore, we demonstrated for the first time that nasal exposure to exogenous histamine also induced nasal LPR, which is mediated mainly by eosinophil-related mediators. Clinically, the latter result implies that suppression of histamine release or effect on the immediate phase may affect the LPR.
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