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Allergen-Induced mRNA Expression of IL-5, but Not of IL-4 and IFN- , in Peripheral Blood Mononuclear Cells Is a Key Feature of Clinical Manifestation of Seasonal Allergic Rhinitis
Yoshinori Nakai, MD;
Yoshihiro Ohashi, MD;
Yasushi Kakinoki, MD;
Ayaki Tanaka, MD;
Yushi Washio, MD;
Yoshiro Nasako, MD;
Tateo Masamoto, MD;
Hirokazu Sakamoto, MD;
Yasukazu Ohmoto, PhD
Arch Otolaryngol Head Neck Surg. 2000;126:992-996.
ABSTRACT
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Objectives To investigate the allergen-induced messenger RNA (mRNA) expression of interleukin (IL) 4, IL-5 and interferon gamma (IFN- ) in peripheral blood mononuclear cells from individuals sensitized by Japanese cedar (Cryptomeria japonica) pollens, and to elucidate the clinical role of IL-4, IL-5, and IFN- in the allergen sensitization and clinical manifestation of allergic disorders.
Design This study included 30 patients sensitized to the pollen and 14 nonatopic healthy volunteers. Peripheral blood mononuclear cells (1.0 x 106 cells/mL) of each individual were cultured at 37°C for 24 hours in the presence of 10 µg/mL of Cry j 1, a major allergen of the pollens. Total cellular RNA was extracted from the peripheral blood mononuclear cells, and IL-4, IL-5, and IFN- mRNA expression was determined with a reverse transcriptase polymerase chain reaction.
Results From the results of a survey of symptom diary cards and interviews regarding nasal symptoms during the pollen season in 1998, we found that 20 patients (symptomatic group), but not 10 patients (asymptomatic group), had typical symptoms of seasonal allergic rhinitis. Interleukin 4 mRNA was not expressed in the nonatopic subjects but was expressed in 9 asymptomatic patients and in 17 symptomatic patients. Interleukin 5 mRNA was exclusively expressed in the symptomatic patients. Interferon mRNA expression did not differ significantly among the nonatopic subjects, asymptomatic patients, and symptomatic patients.
Conclusions This study has clearly highlighted an interesting and new concept that IL-4 is implicated in allergen sensitization but not in clinical manifestation, and that IL-5 may not be a feature of atopy in itself but seems to be a hallmark of clinical manifestation of ongoing atopic diseases.
INTRODUCTION
CD4-POSITIVE helper T (TH) cells have been categorized into at least 2 distinct subsets (TH1 and TH2) based on their profiles of cytokine secretion.1 An accumulation of TH2-like cells occurs at allergic inflammatory sites and increased levels of TH2 cell–derived cytokines in culture supernatants from peripheral blood lymphocytes.2-3 Cytokines derived by TH2 cells are involved in IgE synthesis and allergic inflammation,4-5 whereas TH1-type cytokines such as interferon gamma (IFN-) inhibit the IgE synthesis and the development of TH2 clones.6-7 The current hypothesis is that imbalances between TH1 and TH2 cells seem to be responsible for allergic conditions.
Particular attention has been focused on the role of interleukin (IL) 4 and IFN- in allergic diseases. Although IL-4 is involved in IgE synthesis, the synthesis of allergen-specific IgE does not inevitably result in the development of clinical allergy. Indeed, the detection of specific IgE does not always coincide with clinical allergic disorders, and merely indicates acquired sensitization to the relevant allergen or a marker of possible future allergic manifestation.8-10 Another important cytokine involved in allergic diseases is IL-5, which is particularly implicated in the allergic inflammatory events because it selectively promotes the differentiation, priming, activation, and survival of eosinophils in vitro.11 A concept has emerged from 3 recent studies12-14 that IL-5 might be a determinant cytokine linked to symptomatic episodes of allergic rhinitis and asthma. Therefore, IL-5 might be a more important cytokine involved in clinical manifestation of allergic diseases than IL-4.
Our hypothesis is that IL-4 is simply involved in allergen sensitization, and IL-5 plays a more important part for ongoing allergic diseases. We thus studied the clini cal role of IL-4, IL-5, and IFN- in the allergen sensitization and clinical manifestation of allergic disorders to test our hypothesis.
SUBJECTS, MATERIALS, AND METHODS
This study design followed the principles outlined in the Declaration of Helsinki.15 It included 44 subjects who gave informed consent for participation. Thirty subjects (sensitized group) were outpatients who visited our hospital during the first week of December 1997, and each of them had detectable levels of specific IgE against Japanese cedar (Cryptomeria japonica) pollens in serum. The remaining 14 subjects were nonatopic healthy volunteers (7 women and 7 men; age range, 18-45 years), and they were chosen on the basis of the following criteria: (1) no history of allergic diseases; (2) no physical findings indicative of allergic diseases; and (3) negative serum IgE antibodies specific to the major allergens in Japan, such as house dust mites, Japanese cedar pollen, ragweed, Japanese cypress pollen, and molds.
The period for pollination of Japanese cedars in 1998 was from the third week of February to the second week of April. Peripheral blood was collected from each subject before the cedar pollen season (during January 1998). No subjects had received specific medications such as antihistamines and steroids for at least 2 months before the collection of peripheral blood. Serum concentrations of the pollen-specific IgE were determined by the use of the Pharmacia CAP system (Pharmacia & Upjohn Co Ltd, Uppsala, Sweden). Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples by Ficoll-density gradient centrifugation. They were then washed twice in phosphate-buffered saline and resuspended in RPMI 1640 (Flow Laboratories, Meckenheim, Germany) containing 10% inactivated fetal calf serum (JRH Biosciences, Lenexa, Kan), 100 µg/mL of streptomycin (Sigma, St Louis, Mo), 100 U/mL of penicillin, and 2-mmol/L glutamine. Peripheral blood mononuclear cells from each subject were cultured and stimulated with 10 µg/mL of Cry j 1, a major allergen of Japanese cedar pollens, for 24 hours at a final concentration of 1.0 x 106 cells/mL in 12-well plates (Iwaki Glass, Tokyo, Japan) at 37°C in a fully humidified, 5% carbon dioxide atmosphere. Cry j 1 protein was isolated from Japanese cedar pollens by affinity chromatography using monoclonal antibody specific for Cry j 1. The justification of the method for the preparation of Cry j 1 and the quality and potency of Cry j 1 used in this study have been described elsewhere.16-17
Total cellular RNA was extracted from the PBMCs with the Isogen (Nippon Gene, Tokyo, Japan) method and dissolved in 20 µL of RNase-free water. One microgram of RNA, 1 µL of 50-pmol/µL random primer (hexadeoxyribonucleotide mixture; Takara Biochemicals, Tokyo, Japan), and water were mixed within an 11-µL total volume. Annealing reactions were performed at 65°C for 10 minutes, and the mixture was placed on ice (approximately 0°C for 5 minutes. Four microliters of 5 x first-strand buffer (Gibco BRL, Rockville, Md), 2 µL of 10-mmol/L deoxynucleotide triphosphates (dNTPs), 2 µL of 0.1-mol/L dithiothreitol (Gibco BRL), 0.5 µL of 200 U/µL Moloney murine leukemia virus reverse transcriptase (RT) (Gibco BRL), and 0.5 µL of RNase inhibitor were added to the tube. Reverse transcriptase was performed at 37°C for 60 minutes, and the solution was kept at 99°C for 10 minutes. Then 30 µL of water was added into the RT products.
Five microliters of each of the RT products was amplified by means of polymerase chain reaction (PCR). In this study, oligonucleotide primers for 3 cytokines and an initial marker were designed to amplify the complementary DNA fragments. For IL-4, the sense primer was 5'-CGGCAACTTTGACCACGGACACAAGTGCGATA-3' (117-148), and the antisense primer was 5'-ACGTACTCTGG TTGGCTTCCTTCACAGGACAG-3' (429-460). For IL-5, the sense primer was 5'-GCTTCTGCATTTGAGTTTGCTAGCT-3' (9-33), and the antisense primer was 5'-TGGCCGTCAATGTATTTCTTTATTAAG-3' (276-302). For IFN-, the sense primer was 5'-ATGAAATATACAAGT TATATCTTGGCTTT-3' (127-155), and the antisense primer was 5'-GATGCTCTTCGACCTCGAAACAGCAT-3' (595-620). For  -actin, the sense primer was 5'-AAGAGAGGCATCCTCACCC T-3' (222-241), and the antisense primer was 5'-TACATGGCTGGGGTGTTGAA-3' (420-439). Complementary DNA was amplified in a 20-µL reaction mixture containing 0.125 µL of 5 U/µL Ex Taq DNA polymerase (Takara Biochemicals), 2.5 µL of 10 x Ex Taq Buffer (Takara Biochemicals), 2 µL of 2.5-mmol/L dNTPs, 1.25 µL of 10-µmol/L of each primer, and 11 µL of water. For IL-4, the PCR profile consisted of 40 cycles of 0.5 minute at 94°C, 2 minutes at 55°C, and 3 minutes at 72°C; for IL-5 and -actin, 33 cycles of 0.5 minute at 94°C, 2 minutes at 60°C, and 3 minutes at 72°C; and for IFN-, 33 cycles of 0.5 minute at 94°C, 2 minutes at 55°C, and 3 minutes at 72°C. Ten microliters each of the PCR products was run on a 1.5% agarose gel and visualized with ethidium bromide staining. Interleukin 4, IL-5, IFN-, and -actin complementary DNA fragments were detected at 344 base pairs (bp), 294 bp, 494 bp, 218 bp, respectively. The results were divided into negative or positive expression of cytokine messenger RNA (mRNA) (Figure 1).
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Figure 1. The results of messenger RNA (mRNA) analysis by reverse transcriptase polymerase chain reaction (RT-PCR) were divided into negative or positive expression of cytokine mRNA. A plus sign indicates a case of positive results; a minus sign, a case of negative results; N, negative control; P, positive control; M, marker 4 (øX174/Hae III); IL, interleukin; and IFN, interferon.
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At the end of the pollen season (the second week of April 1998), nasal symptoms during the pollen season were investigated in each subject in the sensitized group by the use of symptom diary cards and interviews. Each subject was asked to complete the symptom diary cards before and during the pollen season (between February and April 1998). The symptom diary cards included the number of sneezing attacks, the number of nose blowings, and the degree of nasal obstruction. A survey of the diary cards was used to grade the 3 nasal symptoms (sneezing attack, watery rhinorrhea, and nasal obstruction) on a scale of 0 to 3, depending on severity (Table 1).18 The nasal symptoms were classified into 3 categories of severity (mild,  2; moderate, 3-6; and severe,  7) based on the average daily scores of the sum total of the 3 nasal symptom scores (maximum score, 9). The subjects whose severity was moderate or severe were judged as symptomatic. In addition, to confirm the justification of our judgment, the types and severity of nasal symptoms during the pollen season in 1998 were obtained by interview with each subject in the sensitized group. To exclude possible bias or prejudice, the judgment (symptomatic or asymptomatic) and the detection of cytokine mRNA expression were performed by different physicians. In addition, no information about the background of each sample was given to the physicians involved in the detection of cytokine mRNA expression. The Fisher exact probability test and Mann-Whitney U test were used for statistical analysis, and significant difference was accepted in either analysis when P was <.05.
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Grading System Used for Nasal Symptoms
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RESULTS
NASAL SYMPTOMS DURING THE POLLEN SEASON
According to the symptom diary cards, 20 subjects (11 women and 9 men; age range, 18-45 years) in the sensitized group were classified as symptomatic, and the remaining 10 subjects (5 women and 5 men; age range, 20-43 years) were classified as asymptomatic. The severity of each of the 20 subjects in the symptomatic group was judged to be severe. The survey of nasal symptoms during the pollen season 1998 confirmed that the 20 subjects in the symptomatic group had typical symptoms of seasonal allergic rhinitis, and the remaining 10 subjects in the asymptomatic group had no nasal symptoms during the pollen season.
SPECIFIC IgE IN SERUM
The levels (mean + SD) of specific IgE in serum did not differ significantly between the asymptomatic group and the symptomatic group (11.5 + 9.5 U/mL and 19.5 + 16.7 U/mL, respectively; P = .20, Figure 2).
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Figure 2. The specific IgE levels in serum did not differ significantly between the asymptomatic group and the symptomatic group.
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RT-PCR RESULTS
The results of RT-PCR in PBMCs stimulated with Cry j 1 are summarized in Figure 3 and Figure 4. -Actin mRNA expression was detected in all 44 subjects; IL-4 mRNA expression was not detected in any subjects in the nonatopic group. By contrast, IL-4 mRNA was expressed in 9 of the 10 subjects in the asymptomatic group and 17 of the 20 subjects in the symptomatic group. None of the subjects in the nonatopic group or the asymptomatic group expressed IL-5 mRNA, whereas IL-5 mRNA was expressed in 18 of the 20 subjects in the symptomatic group. The number of subjects who expressed IFN- mRNA were 3 in the nonatopic group, 3 in the asymptomatic group, and 7 in the symptomatic group.
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Figure 4. Statistical analysis of Cry j 1–induced messenger RNA (mRNA) expression. Significant differences between the nonatopic group and the asymptomatic group occurred exclusively in the expression of interleukin (IL) 4 mRNA. Significant differences between the nonatopic group and the symptomatic group occurred in the expression of IL-4 mRNA and IL-5 mRNA, but not of interferon gamma (IFN-) mRNA. Significant differences between the asymptomatic group and the symptomatic group occurred exclusively in the expression of IL-5 mRNA.
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Significant differences between the nonatopic group and the asymptomatic group occurred exclusively in the expression of IL-4 mRNA (IL-4 mRNA, P<.001; IL-5 mRNA, P>.999; IFN- mRNA, P = .19). Significant differences occurred between the nonatopic group and the symptomatic group in the expression of IL-4 mRNA and IL-5 mRNA, but not of IFN- mRNA (IL-4 mRNA, P<.001; IL-5 mRNA, P<.001; IFN- mRNA, P = .25). Significant differences between the asymptomatic group and the symptomatic group occurred exclusively in the expression of IL-5 mRNA (IL-4 mRNA, P>.999; IL-5 mRNA, P<.001; IFN- mRNA, P>.999).
COMMENTS
Three of the cytokines derived from T lymphocytes are likely to play particularly important roles in allergic diseases. The first is IL-4, which is critical in IgE synthesis.4-5 The second is IL-5, which is essential for terminal differentiation, activation, and priming of eosinophils.11 The third is IFN-, which inhibits IgE synthesis and the development of TH2 clones.6-7 The importance of each has been well established in the development of allergic diseases, but it remains to be established which one is most linked to the clinical manifestation of allergic diseases in humans. The primary purpose of this study was to compare the allergen-induced IL-4, IL-5, and IFN- mRNA expression in PBMCs between the cedar pollen–sensitive patients with rhinitis and those with no symptoms.
Our present study has demonstrated that IL-4 mRNA was expressed in the allergen-sensitive subjects regardless of symptoms, and that IL-5 mRNA was expressed exclusively in the subjects with symptoms. In addition, the allergen-induced expression of IFN- mRNA did not differ among the nonatopic, asymptomatic, and symptomatic subjects, suggesting that IFN-, a TH1-type cytokine, was not likely to play a key role in the sensitization or clinical manifestation of seasonal allergic rhinitis. Therefore, this study has clearly highlighted an interesting and new concept that IL-4 is implicated in allergen sensitization but not in clinical manifestation, and IL-5 may not be a feature of atopy in itself, but seems to be a hallmark of clinical manifestation of ongoing atopic diseases.
The critical part in allergen-induced IL-5 in symptomatic manifestation has been demonstrated.13-14 Expression of mRNA for IL-5 has occurred in bronchial biopsy specimens from patients with asthma, and the expression of IL-5 mRNA has tended to increase in severe disease.19 It was clearly demonstrated by Mori et al12 that T cells of both atopic and nonatopic patients with asthma produced significantly higher amounts of IL-5 in response to nonspecific stimulation when compared with those of nonatopic individuals. In another study, allergen-induced IL-5 production by PBMCs from sensitized atopic subjects with symptoms (asthma or rhinitis) was elevated compared with that in atopic subjects with no symptoms and nonatopic normal control subjects, suggesting that elevated IL-5 synthesis likely occurred only in the presence of ongoing atopic diseases.13 We also investigated the synthesis of IgE, IL-5, and IFN- by allergen-stimulated PBMCs of nonatopic normal controls and subjects sensitized to Japanese cedar pollens.14 IgE production was significantly elevated in sensitized subjects compared with nonatopic controls, but did not differ between sensitized subjects with symptomatic episodes and those without symptomatic episodes. Synthesis of IFN- did not differ among the 3 groups. Interestingly, an increased synthesis of IL-5 was observed only in sensitized subjects with clinical manifestation of seasonal allergic rhinitis.14
Our results are thus in line with the literature, which indicates that IL-5 synthesis is closely linked to the symptomatic manifestation of allergic disorders, but this is the first document, to our knowledge, to show that allergen-induced IL-5 is more definitely related to allergic manifestation than allergen-induced IL-4 and IFN-. To avoid the possible effects of antiallergic medications, we investigated the allergen-induced cytokine mRNA expression in PBMCs obtained before the pollen season. However, it would be important to further study the allergen-induced cytokine mRNA expression during the pollen season.
CONCLUSIONS
Our study has highlighted an interesting and new concept that IL-4 is implicated in allergen sensitization but not in clinical manifestation, and IL-5 synthesis seems to be a hallmark of clinical manifestation of seasonal allergic rhinitis caused by Japanese cedar pollens. Since IL-5 is fundamentally implicated in the development of hyperactivity through the eosinophilia and allergic inflammatory events in the airway, our findings also indicate the key importance of hyperactivity in the development of airway allergic diseases. In addition, a therapeutic strategy to target allergen-induced IL-5 synthesis would be recommended to control allergic symptoms in sensitized individuals. However, it remains unclear whether the key role of IL-5 is characteristic of exclusively seasonal allergic rhinitis caused by Japanese cedar pollens or whether it can be applied to the other kinds of allergic disorders.
AUTHOR INFORMATION
Accepted for publication February 22, 2000.
This study was supported by a Grant-in-Aid for Scientific Research (11671698) from the Ministry of Education, Science and Culture of Japan, Tokyo.
The authors wish to thank Yoshifumi Taniguchi, PhD, Hayashibara Biochemical Laboratories Inc, Okayama, Japan, for the preparation of Cry j 1.
Reprints: Yoshihiro Ohashi, MD, Department of Otolaryngology, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno, Osaka 545-8585, Japan (e-mail: ohashiyocu{at}med.osaka-cu.ac.jp).
From the Department of Otolaryngology, Osaka City University Medical School, Osaka (Drs Nakai, Ohashi, Kakinoki, Tanaka, Washio, Nasako, Masamoto, Sakamoto), and the Laboratories of Cellular Technology, Otsuka Pharmaceutical Co Ltd, Tokushima (Dr Ohmoto), Japan.
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