(Chamaecyparis Obtusa) Pollen by Immunoblotting with the Sera from Patients with Japanese Cedar Pollinosis

Home , Chamaecyparis, Chamaecyparis obtusa, Cryptomeria

Allergology International (1996) 45: 181-186

Original Article

Analysis of the allergenic components of Hinoki cypress (Chamaecyparis obtusa) pollen by immunoblotting with the sera from patients with Japanese cedar pollinosis

Hirotaka Ito,1 MotohikoSuzuki,1 ShinichiroMamiya,1 IppeiTakagi,1 Shunkichi Baba,1 Keiko Tomita2 and Akira Tanaka2 '1Department of Otorhinolaryngology , Nagoya City University Medical School, Nagoya and 2Pharmacia KK, Tokyo, Japan

ABSTRACT INTRODUCTION The polypeptides in Hinoki cypress (Chamaecyparis obtusa) In Japan, the most important causative allergen of pollinosis in and Japanese cedar (Cryptomeria japonica) extracts were spring is Japanese cedar pollen (Cryptomeria japonica) which analyzed by sodium dodecyl sulfate-polyacrylamide gel pollinates from February to the middle of March. Recently, electrophoresis (SDS-PAGE), and then IgE binding activities Hinoki cypress (Chamaecyparis obtusa) pollen in the air has were examined by immunoblotting using sera from 25 been increasing in quantity from the middle of March to April.' patients with Japanese cedar pollinosis. Fifteen component Some patients with Japanese cedar pollinosis exhibited nasal bands with IgE binding abilities were detected. The most fre- symptoms during the Hinoki cypress pollen season. These nasal quent IgE binding band was observed on the line of 45 kDa symptoms were considered to be caused by Hinoki cypress (92%), followed by 50 (88%) and 74 kDa (52%). The pollen.2 immunoblotting profile of Hinoki cypress was different from A significant correlation between Japanese cedar and Hinoki that of Japanese cedar. In the inhibition immunoblotting test, cypress was shown to exist as a result of skin testing2 and CAP IgE binding activities of 14, 30-35 and 74-85 kDa compo- radioallergosorbent test (CAP RAST).3Yasueda et al.4 and Pan- nents in Japanese cedar extract and 14, 30-35 and 60-74 zani et al.5 demonstrated a cross-allergenicity between these two kDa proteins in Hinoki cypress extract were completely inhib- pollens. However, Yoo et al. reported a lack of cross-reactivity ited with heterologous extracts. However, those of the 41 kDa between the Cupressaceae family and Japanese cedar.b Pre- proteins and 46 kDa in Japanese cedar extract and 45 and viously, we have also reported a cross-allergenicity between 50 kDa components in Hinoki cypress extract were partially these two pollens and species-specific allergens as determined inhibited. These data suggest that 45 and 50 kDa compo- by a CAP RAST inhibition test.3 nents of Hinoki cypress and 41 and 46 kDa components There are many reports regarding the major allergens from of Japanese cedar share some cross-reacting and specific Japanese cedar pollen.7-9 However, few reports on the major epitopes. allergens of Hinoki cypress are available.10 In the study reported here, we investigated the IgE-binding components in Hinoki Key words: allergenic components, CAP radioallergosorbent cypress extracts by immunoblotting. We also performed an test (CAP RAST),Chamaecyparis obtusa, Cryptomeria japon- immunoblot-inhibition test in order to identify the cross-allergic ica, immunoblotting, sodium dodecyl sulfate-polyacrylamide components between these two pollens. gel electrophoresis (SDS-PAGE). METHODS

Patient sera

Serum samples were obtained from 34 nasal allergic patients Correspondence: Dr Hirotaka Ito, Department of Otorhinolaryngology, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, whose sera contained IgE antibodies against both Japanese Mizuho-ku, Nagoya 467, Japan. cedar and Hinoki cypress. A non-atopic serum was used as the Received 14 March 1996. Accepted for publication 3 July 1996. negative control for IgE binding. 182 HITO ET AL.

Measurement of IgE antibody graphic film (Hyperfilm, -MP, Amersham) mounted on card paper in a cassette was set on the NC strips. The cassette was A CAP BAST (Pharmacia, Uppsala, Sweden) was performed allowed to stand at-70℃ for 72h to exposure. The exposed according to the manufacturer's instructions to measure IgE film was developed, fixed and dried. antibodies against the two pollens.

Inhibition immunoblotting Allergen sources Inihibition immunoblotting was performed using sera from three Japanese cedar and Hinoki cypress pollen were collected from patients. Patient serum (0.5mL) was mixed with 1.5mL of 0.05 their flowers during the 1992 pollen season. Extraction was per- mol/L phosphate buffer (pH 7.4) containing an inhibitor (50 mg formed by suspending the pollens in a 0.1mol/L phosphate crude Japanese cedar or Hinoki cypress extract) and incubated buffer (pH 7.4) and stirring for 2h at 4℃. The mixture, at a con- for 2 h before reacting with the NC strips. As a control, 0.5 mL centration of 1/20 (w/v), was centrifuged at 10000g for 30 min patients' sera were mixed with 1.5mL of the same buffer without and the supernatant filtered through filter paper (TOYO 4A; inhibitor concentration. The concentration of the inhibitor for Toyo Roshi Co., Tokyo, Japan). The crude extracts of Japanese the present study was determined by a preliminary experiment cedar and Hinoki cypress were desalted on PD-10 columns using extracts and pooled sera from patients who were sensitive (Pharmacia) and lyophilized. to both Japanese cedar and Hinoki cypress allergens.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The lyophilized crude extract for sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) were recon- Table 1. Specific IgE antibodies in serum samples of patients with stituted at a final concentration of 25 mg dry weight/mL. allergic rhinitis SDS-PAGE was performed according to Naville.11 The extract solutions were mixed with an equal volume of sample buffer (0.08 mol/L Tris-H2SO4[pH 6.1], 4% SDS, 2-mercaptoethanol, 9% glycerol, 0.02% bromophenol blue) and boiled for 5 min.

This mixture (750μg crude extract/lane) was applied on 6% stacking polyacrylamide gels on top of polyacrylamide gradient gels for separation (7.5-20%). The electrophoresis was run at 6mA per gel for 18h with a cooling system. Tris-Borate (0.04 mol/L; pH 8.64) containing 0.1% SDS and 0.42 mol/L Tris-HCI (pH 9.18) was used as upper and lower reservoir buffers, respectively. Protein standards (LMW calibration kit, Pharmacia) were used to estimate the molecular weight of the pollen components.

Immunoblotting

The separated allergen components and the protein standards in the gradient gel were transferred to a nitrocellulose (NC) membrane (Hybond-C 0.45μm, Amersham, Buckinghamshire, UK) in a TransBlot system by electrophoretic force (0.4A for 4h at 4℃). After the transfer, the NC membrane was incubated with a blocking buffer 0.05 mol/L phosphate buffer (pH 7.4) containing O.5%Tween 20 at 37℃ for 30 min to block un一 occupied binding sites on the NC membrane. The NC mem- branewasthen cut into 5mmXllcm strips.The NC stripswere incubated with 2mL of diluted sera (0.5mL serum+1.5mL blocking buffer) at room temperature for 18 h with shaking. After the incubation, the NC strips were washed and re- incubated with 2mL of 1251-anti-human IgE (6-fold diluted Phadebas BASTtracer) at room temperature for 4h with shaking. The NC strips were again washed and dried. A radio- ALLERGENIC COMPONENTS OF HINOKI CYPRESS 183

RESULTS The comparison of IgE immunoblot profiles between the two pollen extracts using six patients' sera is shown in Fig. 3. A dif- IgE antibody titer against Japanese cedar and ferent profile was obtained between Japanese cedar and Hinoki Hinoki cypress cypress extract. IgE antibodies against both Japanese cedar and Hinoki cypress were detected in all sera from 34 patients (Table 1) and the sera Inhibition blotting used in the following studies. The inhibition blotting test was performed to identify cross- reactive IgE binding bands between the two pollens (Fig. 4). IgE immunoblot profile Table 2. IgE-binding components in the extract of Japanese cypress Of the 25 patients who were positive against both allergens in by immunoblotting with 25 patients' sera BAST,23 were positive in the immunoblotting studies of extract from Hinoki cypress pollens (Fig. 1). No reaction was detected in two patients (cases 8, 14). The IgE immunoblots showed a total of 15 IgE binding bands in the Hinoki cypress extract. The most frequent band was obtained with the 45 kDa protein (92%), followed by 50 (88%) and 74 kDa (52%; Table 2). These were considered to be the major allergens of Hinoki cypress pollen. The same 25 patients' sera were tested for IgE immunoblotting with Japanese cedar extract. However, five of these patients (cases 7, 8 12, 13, 25) were negative. Ten IgE binding bands were found in the Japanese cedar extract. The most frequent band was 41 kDa (80%), followed by 29 (36%), 32 (36%) and 46 kDa (36%). The 41 and 46 kDa proteins were considered as Cry) 1 (Fig. 2).

Fig. 1 IgE-binding components in pollen extract from Hinoki cypress detected by sera from patients with Japanese cedar pollinosis (cases 1-25) and a non-atopic serum (negative). 184 HITO ET AL.

Fig. 2 IgE-binding components in pollen extract from Japanese cedar detected by the same sera using Hinoki cypress.

Fig. 3 A comparison of the immunoblotting profile between Hinoki cypress and Japanese cedar. These two pollen extracts were transferred on the some nitrocellulose strip and incubated with the same patients' sera (cases 26-34). ALLERGENIC COMPONENTS OF HINOKI CYPRESS 185

Fig. 4 Inhibition immunoblotting of Hinoki cypress and Japanese cedar extracts by the use of three patients' sera (cases 32-34). -, without inhibitor; Ced, Japanese cedar; Cyp, Hinoki cypress.

Pre-treatment of patient sera with Hinoki cypress extract com- quent band was observed on the 45 kDa protein followed by 50 pletely inhibited the reactivity of 14, 30-35 and 74-88 kDa and 74 kDa in Hinoki cypress extract. These proteins are con- proteins in Japanese cedar extracts. However, those of 41 and sidered to be the major allergens from Hinoki cypress. The 46 kDa proteins were only partially inhibited in all three sera. immunoblotting profile and the major allergen from Hinoki On the other hand, pre-treatment with Japanese cedar cypress were different from those of Japanese cedar. extract completely inhibited the reactivity of 14, 30-35 and The serum samples used for immunoblottinganalysis were pos- 60-74 kDa proteins in Hinoki cypress extract. However, those of itive to both pollens in CAP BAST.However, 5/25 serum samples 45 and 50 kDa proteins were not inhibited completely. were negative by immunoblotting with Japanese cedar extract. Inhibition tests with homologous allergen extract showed This phenomenon may have been caused by the treatment of the complete inhibition by both pollen extracts in all three sera. extract with SDS, 2-mercaptoethanol and boiling. These patients' (cases 7, 8, 12, 13, 15) IgE antibodies might recognize certain conformational allergenic epitopes which may have been dena- DISCUSSION tured by these treatments.8As four of the five serum samples (cases In a previous report, we demonstrated the presence of common 7, 12, 13, 25) were positive in immunoblotting with Hinoki allergens and species-specific allergens between Japanese cypress, the antigenicity of Hinoki cypress may be more resistant cedar and Hinoki cypress.3 Yasueda et al.,4 Panzani et al.5 and against these treatments than that of Japanese cedar. Enomoto and Ohnishi12 also demonstrated the existence of In the inhibition blotting study, IgE binding activities of 14, these two allergen types in the two pollens. 30-35 and 74-88 kDa proteins in Japanese cedar extract were In the present study, we performed IgE-immunoblotting and completely inhibited by pre-treatment of patient sera with Hinoki inhibition blotting to identify the cross-reactive and species- cypress extract. Proteins 14, 30-35 and 60-74 kDa in Hinoki specific components in the two types of pollen. cypress extract were completely inhibited by pre-treatment of To compare IgE binding bands between the two pollens, the patient sera with Japanese cedar extract. The results suggest immunoblotting profile of Hinoki cypress was compared to that that these bands are cross-reactive components between the of Japanese cedar using the same patients' sera. The most fre- two pollens. 186 HITO ET AL.

The IgE binding activities of 41 and 46 kDa components in (cypress) pollen in the cedar-cypress pollination season. Pract. Japanese cedar extract which are presumed to be Cry j 1,8 were Otol. 1991;52: 75-9. 3 Ito H, Nishima J, Suzuki M. Specific IgE to Japanese cypress partially inhibited by pre-treatment of patient sera with Hinoki (Chamaecyparis obtusa) in patients with nasal allergy. Ann. Allergy cypress. The reactivity of the 45 and 50 kDa components in Asthma Immunol. 1995; 74: 299-303. Hinoki cypress were also partially inhibited by pre-treatment 4 Yasueda H, YuiY, Shimizu T, Shida T. Isolation and partial charac- with Japanese cedar extract. The results suggest that these terization of malor allergen from Japanese cedar (Cryptomeria major allergens share certain cross-reacting and species- japonica) pollen. J. Allergy Clin. Immunol. 1983; 71: 77-86. specific epitopes. Takahashi et al.13 reported immunoreactivity 5 Panzani R,Yasueda H, Shimizu T, Shida T. Cross-reactivity between the pollens of Cupressus sempervirens (common cypress) and of of mouse anti-Cry j 1 antibodies to 34 kinds of pollen. Two of Cryptomeria japonica (Japanese cedar). Ann. Allergy 1986; 57: those antibodies reacted with pollen from Pinaceae, Taxodia- 26-30. ceae and Cupressaceae family, the other reacted with the 6 Yoo TY, Spitz E, McGrety JL. Conifer pollen allergy: Studies of immunogenicity and cross-antigenicity of conifer pollens in rabbit pollen of Japanese cedar alone.13 Ide et al. demonstrated that and man. Ann. Allergy 1975; 34: 87-93. 40, 45, 50 and 67 kDa proteins in Hinoki cypress extract were 7 Sakaguchi M, Inoue S, Taniai M, Ando S, Usui M, Matuhashi T. stained by a rabbit anti-Cry j 1 antiserum.10 These results sup- Identification of the second major allergen of Japanese cedar port the present findings. pollen. Allergy 1990; 45: 309-12. During recent years, Hinoki cypress pollen counts have been 8 Kawashima T, Taniai M, Usui M, Ando S, Kurimoto M, Matuhashi T. increasing in spring. Some patients with Japanese cedar polli- Antigenic analyses of Sugi basic protein by monoclonal antibodies. II. Detection of immunoreactive fragments in enzyme-cleaved nosis had nasal symptoms even after Japanese cedar pollen Cry j I. Int. Arch. Allergy Immunol. 1992; 98: 118-26. season and were discovered to have IgE antibodies to Hinoki 9 Taniguchi Y, Ono A, Sawatani M et al. Cry j I, a major allergen of cypress.1 Cross-reactive components between these two pollens Japanese cedar pollen, has pectate lyase enzyme activity. Allergy may contribute to clinical symptoms. 1995; 50: 90-3. In conclusion, the 45 and 50 kDa components of Hinoki 10 Ide T, Yamamoto K, Tabata S, Ashida T. N-terminal amino acid sequence of a major allergen, Cha o 1, of Japanese cypress cypress and 41 and 46 kDa components of Japanese cedar pollen. Jpn. J. Palynol.1995; 41: 69-72 (in Japanese with English share certain cross-reacting and specific epitopes. Further aller- abstract). genic analysis of the 45 and 50 kDa components of Hinoki 11 Naville DM. Molecular weight determination of protein dodecyl cypress pollen is required to complete the comparison with the sulfate complexes by gel electrophoresis in a discontinuous buffer major allergens of Japanese cedar pollen. system. J. Biol. Chem. 1971; 246: 6328-34. 12 Enomoto T, Ohnishi S. Measurement of specific 1gE antibody to False cypress (Chamaecyparis obtusa, Hinoki) pollens using REFERENCES Shionoria specific IgE. Jpn. J. Allergol. 1994; 43: 106-12 (in Japanese with English abstract). 1 Ogasawara H, Yoshimura S, Nakahara T, Fulitani T, Okada H. 13 Takahashi Y, Nagoya T, Watanabe M, Inouye S, Sakaguchi M, Airborne pollen survey of Cryptomeria japonica and Chamaecy- Katagiri S. A new method of counting airborne Japanese cedar paris spp. in Hyogo prefecture. Jpn. J. Palynol. 1991; 37: 145-50 (Cryptomeria japonica) pollen allergens by immunoblotting. (in Japanese with English abstract). Allergy 1993; 48: 94-8. 2 Ito H, Yokota A, Nishimura J. Implication of Chamaecyparis