Immunodominance of seven regions of a major allergen, Cry j 2, of Japanese cedar pollen for T-cell immunity

The immunodominant regions of the Japanese cedar pollen allergen Cry j 2 for T-cell immunity were determined with whole peripheral blood lymphocytes (PBL) derived from seven allergic patients and three nonallergic subjects. Cry j 2–stimulated T-cell proliferation was inhibited by anti-HLA-DR, but not by anti-HLA-DQ antibody, indicating that the responding T cells recognized the allergen peptides associated with HLA-DR molecules. It was found that seven regions of Cry j 2. i.e., regions corresponding to amino acid numbers 1–26, 70–84, 151–167, 187–203, 252–279, 283–314, and 345–362, were immunodominant for T-cell proliferation. Thus, Cry j 2 bears a limited number of immunodominant regions despite polymorphic features of HLA-DR in the immune system. This suggests the possibility of molecularly designing Cry j 2 antagonists that could downregulate allergic reactions to Japanese cedar pollen.     

Cry j 2, a major allergen of Japanese cedar pollen, shows polymethylgalacturonase activity

We examined Cry j 2, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, for polygalacturonase enzyme activity, since a nucleotide sequence of cDNA of Cry j 2 showed a significant homology with that of tomato polygalacturonase. Polygalacturonase is well known to depolymerize preferentially polygalacturonic acid (PGA) by hydrolysis. However, Cry j 2 did not act on PGA, but was found to depolymerize pectin and methylesterified PGA in a dose-dependent manner. The substrate specificity of Cry j 2 was different from that of polygalacturonase derived from Aspergillus niger. The depolymerizing activity of Cry j 2 reached a maximum at 50%-60% of methylesterification of PGA. In contrast, polygalacturonase showed its maximum activity to PGA, and the activity decreased as the degree of methylesterification increased. Interestingly, the pectin-depolymerizing activity of Cry j 2 was due to a hydrolysis, but not a lyase, activity which splits the glycosidic bonds by β-elimination, since no unsaturated uronides were found by measurement of absorbance at 235 nm in the reaction mixture. The enzyme activity was markedly inhibited by anti-Cry j 2 antibodies. These results indicate that Cry j 2 probably has polymethylgalacturonase enzyme activity, as postulated by von Neukom in 1963, although existence of this activity has not yet been proven.     

Cry j I, a major allergen of Japanese cedar pollen, has pectate lyase enzyme activity

In the course of analyzing the partial amino acid sequences of Cry j I, a major allergen of Japanese cedar (Cryptomeria japonica) pollen, we found a peptide fragment which has a significant homology to some pectate lyase isozymes secreted by plant pathogenic bacteria. Therefore, we investigated whether Cry j I has pectate lyase activity. Cry j I reacted with polygalacturonic acid, resulting in the release of unsaturated uronide products. The optimum temperature and pH for the reaction were 60–70°C and pH 10. The enzymatic reaction had an absolute Ca²⁺ ion requirement. These characteristics were very compatible with the character of the pectate lyase isozymes reported previously. These results clearly show that Cry j I has pectate lyase activity.     

Th1/Th2 response profiles to the major allergens Cry j 1 and Cry j 2 of Japanese cedar pollen

Cry j 1 and Cry j 2 are known to be the major allergens of Japanese cedar pollen. A comparative study was carried out on the immune responses to stimulation with Cry j 1 and Cry j 2 in 24 symptomatic patients and six nonallergic subjects. In T-cell proliferation assays, mean stimulation indexes (SI) were 10.6 for Cry j 1 and 11.7 for Cry j 2 stimulation, respectively, in the allergic patients. Two of the nonallergic subjects showed strong T-cell proliferation to both allergens, while the remainder did not. All the allergic subjects (17/17) showed high titers of anti-Cry j 1 IgE antibody at a mean value of 165 U/ml, whereas only 64% responded to Cry j 2 with low titers at a mean value of 26 U/ml. Nonallergic subjects did not respond with IgE production. Allergic subjects were further examined for their cytokine production profiles. All allergic subjects tested (16/16) produced high levels of interferon-gamma (IFN-γ) in response to Cry j 1 with a mean value of 918 pg/ml, while only five subjects showed significant elevation of IFN-γ production in response to Cry j 2 with a mean value of 679 pg/ml. The remainder produced small amounts of IFN-γ. Cry j 1 induced higher levels of interleukin (1L)-10 gene expression than did Cry j 2 stimulation, while both allergens induced IL-4 expression at a similar level. The IL-12 p35 gene was constitutively expressed, whereas the IL-12 p40 gene expression in Cry j 1-stimulated cells was elevated eightfold over that of nonstimulated cells. Increased expression of the IL-12 p40 gene was negligible in Cry j 2-stimulated cells. Thus, Cry j 1 stimulated mixed features of Th1 and Th2-like responses, while Cry j 2 played a minor role in inducing IgE production and cytokine (IFN-γ, IL-10, and IL-12) production, except for IL-2 production and strong T-cell proliferative activity. Therefore, it was concluded that Cry j 1 is the more important allergen, and that T-cell proliferation assays do not necessarily reflect the level of allergenicity.     

Existence of exine-free airborne allergen particles of Japanese cedar (Cryptomeria japonica) pollen

We investigated whether exine-free pollen allergen particles exist together with the intact pollen grains of Japanese cedar (Cryptomeria japonica) in the air during the pollen season in Yamagata City. First, we separated the allergen particles in an Andersen multi-stage air-sampler according to their aerodynamic diameters. The amount of major allergen (Cry j I) on each stage of the sampler was determined by a sensitive fluorometric sandwich ELISA, and the pollen count of the same samples was done by light microscopy after Carberla staining. Cry j I was found in stages 1 to 6, whereas most of intact and ruptured pollen grains were microscopically observed only in stages 1 and 2. Second, we suctioned the air through a tandem membrane filter system (the first filter, Nuclepore filter with 5 microns-pores; and the second, Millipore filter with 0.3 micron-pores). None of the pollen grains was detectable on the 0.3 micron-pore filter with light microscopy. However, Cry j I was detectable in the aqueous extract from the second filter. From these results, we concluded that pollen-free Cry j I existed in the air of Yamagata City during the pollen season.     

Identification of the second major allergen of Japanese cedar pollen

We isolated and characterized the second major allergen (Cry j II) from Japanese cedar pollen. We found that most patients with this pollinosis had IgE antibody to this protein in addition to IgE antibody to Cry j I; however, some sera reacted only with Cry j I or Cry j II. IgE-ELISA inhibition studies revealed that Cry j I and Cry j II had no cross-allergenicity. Cry j II did not react with anti-Cry j I monoclonal antibodies. In SDS-PAGE under a non-reducing condition, Cry j II showed a band at the 37 kDa position, compared with the 45-50 kDa bands of Cry j I. N-terminal amino acid sequence of Cry j II was completely different from that of Cry j I.     

Comparison of concentrations of Cry j 1 and Cry j 2 in diploid and triploid Japanese cedar (Cryptomeria japonica) pollen extracts

Japanese cedar ( Cryptomeria japonica ) pollinosis has been a serious allergic disease in Japan. There are two kinds of Japanese cedar trees; the popular one is diploid, the less popular is triploid. These trees are not very different morphologically. However, the relative allergenicity of their pollens is unknown, although both major allergens, Cry j 1 and Cry j 2, have been purified and cloned from the diploid line. Triploid trees are sterile and the allergenicity of their pollen may differ. Using Japanese-cedar-allergic patient sera, we compared the concentration of these two major allergens in both kinds of pollen. Pollen collected from different years and regions was also studied. The results indicate that triploid tree pollen extract has lower concentrations of both major allergens; therefore, the pollen may be less allergenic.     

Identification and characterization of a group 2 conifer pollen allergen from Chamaecyparis obtusa, a homologue of Cry j 2 from Cryptomeria japonica

BACKGROUND: Not only Cryptomeria japonica (Japanese cedar) pollen but also that of Chamaecyparis obtusa (Japanese cypress) induces the allergic symptoms of Japanese cedar pollinosis. However, allergens from C. obtusa pollen have not been as well characterized as those from C. japonica pollen. OBJECTIVE: We sought to identify and characterize a homologue of the second major allergen of C. japonica pollen, Cry j 2, from the pollen of C. obtusa. METHODS: An allergen homologous to Cry j 2 was identified in C. obtusa pollen extract by immunoblot analysis, probed with anti-Cry j 2 monoclonal antibodies and purified by a series of column chromatographic steps. RESULTS: The allergen isolated from the extract showed a slightly diffuse band of 45 kDa and closely spaced double-bands of 42 and 45 kDa on SDS-PAGE, under reducing and non-reducing conditions, respectively; the bands were approximately 5-7 kDa larger than those of Cry j 2. In 24 of 30 residues, the N-terminal amino acid sequence of the allergen was identical with corresponding sequence in Cry j 2. Most patients with pollinosis who were IgE antibody-positive to Cry j 2 were shown to be IgE antibody-positive to this allergen, and the IgE antibody levels to both allergens were highly correlated. CONCLUSION: The results indicate that the allergen isolated from C. obtusa pollen in this study is a homologue of Cry j 2. The allergen was designated as Cha o 2 according to the WHO/IUIS Allergen Nomenclature Subcommittee recommendation.     

Molecular cloning and characterization of a new Japanese cedar pollen allergen homologous to plant isoflavone reductase family

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis, and more than 10% of Japanese people suffer from this allergic disorder. However, only two major pollen allergens, Cry j 1 and Cry j 2, have been identified and exclusively characterized. OBJECTIVE: The aim of this study was to explore and identify important Japanese cedar pollen allergens other than Cry j 1 or Cry j 2. METHODS: C. japonica cDNA library was immunoscreened by rabbit antiserum raised against a partially purified cedar pollen allergen fraction. An isolated cDNA clone was inserted into a glutathione S-transferase (GST)-tagged Escherichia coli expression vector to obtain recombinant GST fusion protein. Non-fusion recombinant protein was purified by glutathione Sepharose affinity chromatography in conjunction with factor Xa cleavage of the GST moiety. IgE-binding ability of the recombinant protein was then evaluated by western blot analysis and enzyme-linked immunosorbent assay (ELISA). RESULTS: The cDNA encodes 306 amino acids with significant sequence similarity to those of plant isoflavone reductase-like proteins, which include a recently identified birch pollen allergen Bet v 5. Western blot analysis demonstrated that recombinant protein was recognized by cedar pollinosis patient IgE. In contrast to Bet v 5 being reported as a minor allergen, the recombinant protein exhibited 76% IgE binding frequency (19/25) against pollinosis patients. CONCLUSION: Here we identified the third member of Japanese cedar pollen allergen homologous to isoflavone reductase. Its high IgE-binding frequency implicates that the isoflavone reductase homologue might be an additional major pollen allergen in C. japonica.     

Molecular cloning of a class IV chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen and competitive inhibition of its immunoglobulin E-binding capacity by latex C-serum

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergic diseases in Japan. Only three C. japonica allergens, Cry j 1, Cry j 2, and CJP-6, have been characterized. The full IgE-binding spectrum of C. japonica pollen allergens demonstrates that many allergens remain to be identified. OBJECTIVE: The aim of this study was to characterize a novel allergen with a high frequency of IgE binding. METHODS: The cDNA coding for a high-frequency IgE-binding protein, designated CJP-4, was cloned from the total mRNA of C. japonica pollen. The corresponding native allergen was purified by affinity precipitation with colloidal chitin and gel chromatography. The IgE-binding ability of purified native CJP-4 was characterized by ELISA and ELISA inhibition. RESULTS: The CJP-4 cDNA encoded 281 amino acids with significant sequence homology to class IV chitinases. Purified native CJP-4, migrated as a homogeneous 34-kDa protein on SDS-PAGE, revealed endochitinase activity on native PAGE. The purified protein displayed the ability to bind IgE from all patients tested (31/31) in ELISA, whereas Cry j 1 bound to IgE at a 71% frequency (22/31). Pre-incubation with latex C-serum completely inhibited the reaction of pooled sera IgE from patients with C. japonica pollinosis and/or latex allergy to purified CJP-4. CONCLUSION: We identified CJP-4 as a novel and fourth C. japonica chitinase allergen with high IgE-binding frequency. The competitive IgE-binding profile between C. japonica chitinase and latex C-serum indicated that C. japonica chitinase should be an important pan-allergen in C. japonica pollen.     

IgE reactivity and cross-reactivity of Japanese monkeys (Macaca fuscata) to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens

BACKGROUND: The natural occurrence of Japanese cedar (Cryptomeria japonica, CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata). However, the reactivity to Japanese cypress (Chamaecyparis obtusa, CO) pollen allergens in these monkeys has not yet been reported. OBJECTIVES: The present study was designed to investigate the reactivity to CO pollen allergens in monkeys sensitized to CJ pollen allergens. METHODS: Serum samples from 40 monkeys naturally sensitized to CJ pollen allergens were collected from four troops. We measured the specific IgE to CO pollen allergens and examined the reactivity to the allergens by intradermal test. Cross-reactivity between CJ and CO pollen allergens was examined by ELISA inhibition method. Furthermore, we examined the sensitivity to the allergens by histamine release assay from leucocytes. RESULTS: All 40 monkeys had specific IgE to crude and purified major allergens (Cha o 1) of CO pollen. The monkeys showed a positive reaction to CO pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in CJ pollen) was also observed. Specific histamine release to both the major allergens was noted in two monkeys with CJ pollinosis. CONCLUSION: Japanese monkeys sensitized to Japanese cedar pollen allergens also demonstrate reactivity to Japanese cypress pollen allergens.     

Isolation and characterization of native Cry j 3 from Japanese cedar (Cryptomeria japonica) pollen

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollinosis is the most prevalent allergy in Japan. Recently, the Japanese cedar pollen allergen Cry j 3 was cloned as a homologue of Jun a 3, which is a major allergen from mountain cedar (Juniperus ashei) pollen. However, native Cry j 3 has not been isolated and there are no reports on its allergenic activity. The aims of this study were to isolate native Cry j 3 and assess its immunoglobulin E (IgE)-binding capacity in patients with Japanese cedar pollinosis. METHODS: Native Cry j 3 was purified from Japanese cedar pollen by multidimensional chromatography. We assessed the IgE-binding capacity using sera from patients allergic to Japanese cedar pollen by immunoblot analysis and ELISA. Moreover, we assayed the capacity of Cry j 3 to induce histamine release from the patients' leukocytes. We cloned cDNA corresponding to purified Cry j 3 from a cDNA library of Japanese cedar pollen. RESULTS: We isolated native Cry j 3 as a 27-kDa protein. The IgE-binding frequency of Cry j 3 from the sera of patients allergic to Japanese cedar pollen was estimated as 27% (27/100) by ELISA. Cry j 3 induced the release of histamine from leukocytes. We cloned the cDNA and named it Cry j 3.8. Cry j 3.8 cDNA encoded 225 amino acids and had significant homology with thaumatin-like proteins. CONCLUSIONS: Cry j 3 is a causative allergen in Japanese cedar pollinosis and may play crucial roles in the cross-reactivity with oral allergy syndrome.     

Pilot study of Japanese cedar pollen exposure using a novel artificial exposure chamber (OHIO Chamber)

For basic and clinical studies of Japanese cedar (JC) pollinosis and its treatment, experimental facilities for exposure to pollen under stable environmental conditions are becoming increasingly desirable. We developed an artificial exposure chamber (OHIO Chamber) that allows the dispersal of fixed concentrations of JC pollen in stable environments with a unique pollen supply system, air flow system for fixing the concentration of JC pollen, system for monitoring the number of pollen grains, and automated pure water washing and drying system. In the chamber, temperature and relative humidity (RH) could be successfully maintained at 22±1.1 °C and 45±5%, respectively. The spatial distribution of pollen concentrations in the chamber was within 10% of target, including when subjects were present. Only a few or no pollen grains were detected in the chamber after automatic washing and drying. We conducted a pilot tolerability and safety study in 15 JC pollinosis patients who were exposed to 15 000 pollen grains/m³ for 2 h. Symptoms manifested on average 33 min after start of exposure. The subjects experienced no serious side-effects, and pollen exposure at 15 000 grains/m³ was confirmed safe. After exposure, the number of intranasal and intraocular pollen grains was 469 and 602, respectively. The lower number of pollen grains in the nose than in the eyes was considered due to sneezing and nasal discharge. Further studies are needed to clarify the number of pollen grains required for the occurrence of symptoms in the OHIO Chamber.     

Identification of a sequential B-cell epitope on major allergen (Cry j 1) of Japanese cedar (Cryptomeria japonica) pollen in mice

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides. The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. METHODS: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. RESULTS AND CONCLUSION: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 ((141)GVEPVHPQDGDALTLRTATN(160)), though those antibodies did not react with other peptides. IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be (145)VHPQDGDA(152).     

Changes in quantity of Cry j1, a major cedar pollinosis allergen,in Cryptomeria japonica pollen during spring in Japan

The changes in quantity of Cry j1 in Cryptomeria japonica pollen were assessed during the course of spring in Japan. C. japonica pollen was collected from the same trees in Hiroshima and Yamanashi Prefectures, and Cry j1 was quantified by enzyme-linked immunosorbent assay. The quantities of Cry j1 were found to gradually decrease, and this trend was similar in both Hiroshima and Yamanashi Prefectures. In Yamanashi Prefecture, no significant differences in the quantities of Cry j1 between the upper and lower parts of the tree were observed in samples collected from March 1 to 14. Furthermore, the quantities of Cry j1 on March 1, 5, 10 and 14 were significantly lower than those on February 1. These results suggest that expression of C. japonica pollen levels in terms of particle numbers is not sufficient and that quantities of Cry j1 vary throughout the course of spring.     

Sensitivity to two major allergens (Cry j I and Cry j II) in patients with Japanese cedar (Cryptomeria japonica) pollinosis

Background: Japanese cedar (Cryptmeria japonica: CJ) pollinosis is one of the most important allergic diseases in Japan. Recently, the second major allergen (Cry j II) was isolated from CJ pollen. There have been no prevalence studies of sensitivity to Cry j I and Cry j II among a large number of patients with pollinosis. Objective: This study was conducted to evaluate the prevalence of sensitivity to Cry j I and Cry j II. We measured specific IgE antibodies to these allergens in the sera of 145 patients. Furthermore, comparison of the sensitivity to Cry j I and Cry j II was examined by the hisiamine release assay. Methods: Specific IgE antibodies to Cry j I and Cry j II were assayed by a fluorometric ELISA. Allergen-specific histamine release was measured by a radioimmunoassay kit, Results: More than 90% of 145 patients had specific IgE antibodies to both allergens. the remainder had specific IgE to either one or the other. There were seasonal changes in the level of specific IgE. The changes in the levels of anti-Cry j II IgE antibodies were parallel to those of anti-Cry j I IgE. The histamine release assay with leucocytes from the patients demonstrated that the allergenic potency of the two allergens is almost the same. Conclusion: Cry j II is an as important a major allergen as Cry j I.     

The IgE-binding epitopes of rPar j 2, a major allergen of Parietaria judaica pollen, are heterogeneously recognized among allergic subjects

Pollen allergens are multivalent proteins that cross-link IgE antibodies on mast or basophil cells, inducing secretion of biologic mediators, and resulting in various allergic symptoms. The IgE-binding regions of the Parietaria judaica (Pj) pollen major allergen rPar j 2 were investigated. Twenty-nine single sera from Pj-allergic subjects were tested by Western blot against five recombinant peptides. At least four putative IgE-binding epitopes were identified. The analysis of their diffusion suggested a heterogeneous IgE-binding response. In fact, 75% of the sera reacted with peptide 1-54, 48% with peptide 48-101, 24% with peptide 1-30, 7% with peptide 29-54, and none with peptide 48-76. These five peptides were analyzed with the histamine-release assay. Only peptide 48-101 was capable of inducing degranulation and release of histamine. These results suggest that the recombinant rPar j 2 allergen contains IgE epitopes that are heterogeneously recognized by sensitive patients, and that therefore the therapeutic approach based on the use of haptenic peptides needs a careful evaluation.     

Epitope specificity of IgE antibodies to a major allergen (Cry j 1) of Japanese cedar pollen in sera of humans and monkeys with pollinosis.

Japanese cedar (Cryptomeria japonica) pollinosis has been reported to occur naturally in Japanese monkeys (Macaca fuscata) as well as humans. Using monoclonal antibodies (mAb) specific to Cry j 1, a major allergen in Japanese cedar pollen, we identified five independent epitopes (EP-1 to EP-5) on the molecule. The epitopes recognized by IgE antibodies in the sera of humans and monkeys with the pollinosis were analysed by an IgE enzyme-linked immunosorbent assay inhibition method with these mAb. In human patients, the mAb to EP-1 strongly blocked the binding of IgE antibodies in all patients' sera to Cry j 1. The reaction patterns of IgE antibodies in monkeys, however, varied among the troops of monkeys. In some troops, the mAb to EP-1 showed a blocking pattern similar to that for human patients. In other troops, mAb to EP-4 and EP-5 blocked binding of IgE. These results indicate that some, but not all, monkeys have antibody responses to the major allergen similar to those of humans.     

Identification of human T cell epitopes in Japanese cypress pollen allergen, Cha o 1, elucidates the intrinsic mechanism of cross-allergenicity between Cha o 1 and Cry j 1, the major allergen of Japanese cedar pollen, at the T cell level

BACKGROUND: Pollens from species of Cupressaceae family are one of the most important causes of respiratory allergies worldwide. In Japan, many patients with pollinosis have specific IgE to both pollens of Japanese cypress (Chamaecyparis obtusa) and Japanese cedar (Cryptomeria japonica). The sequences between Cha o 1 and Cry j 1, the major allergens of Japanese cypress and Japanese cedar pollens, respectively, are 80% identical. OBJECTIVE: This study was undertaken to identify T cell epitopes in Cha o 1, and to elucidate the mechanism of cross-allergenicity between Cha o 1 and Cry j 1, at the T cell level. METHODS: T cell epitopes in Cha o 1 were identified by the reactivity of T cell lines, generated from 19 patients, to stimulation with overlapping peptides. The subsets of T cell clones specific to rCha o 1 were determined according to their ability to produce IL-4 and IFN-gamma. Peptide specificities of two T cell clones were determined by stimulation with the peptides from Cha o 1 and Cry j 1. RESULTS: Four dominant and six subdominant T cell epitopes were identified in Cha o 1. While four T cell epitopes, p11-30, p211-230, p251-270 and p331-350, were common to Cha o 1 and Cry j 1, 4 T cell epitopes, p61-80, p71-90, p311-330 and p321-340, were considered to be unique to Cha o 1. The subsets of T cell clones were predominantly of T helper2-type. One T cell clone recognized p16-30, which is common to Cha o 1 and Cry j 1, but another recognized p321-330, which is unique to Cha o 1. CONCLUSION: Presence of both T cells reactive to T cell epitopes common to Cha o 1 and Cry j 1 and T cells specific to T cell epitopes unique to Cha o 1 in patients with pollinosis contributes to prolonged symptoms after the cedar pollen season in March and the following cypress pollen season in April.