Studies on Experimental Iodine Allergy: 1. Antigen Recognition of Guinea Pig Anti-Iodine Antibody

The Journal of Toxicological Sciences, 131 Vol.29, No.2, 131-136, 2004 STUDIES ON EXPERIMENTAL IODINE ALLERGY: 1. ANTIGEN RECOGNITION OF GUINEA PIG ANTI-IODINE ANTIBODY Hiroshi SHIONOYA1 Yoshiki SUGIHARA2, Kazuo OKANO3, Fumio SAGAMI1, Takashi MIKAMI1 and Kouichi KATAYAMA3 1Department of Drug Safety Research, Eisai Kawashima Research Laboratories, Eisai Co., Ltd., 1 Takehaya-cho, Kawashima-cho, Hashima-gun, Gifu 501-0061, Japan 2Department of Drug Safety Research, Eisai Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tohkodai, Tsukuba-shi, Ibaraki 300-2635, Japan 3Exploratory Division, Eisai Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tohkodai, Tsukuba-shi, Ibaraki 300-2635, Japan (Received September 3, 2003; Accepted February 18, 2004) ABSTRACT — It has generally been thought that iodine allergy is cross-sensitive to various iodine-containing chemicals. However, this concept seems to deviate from the immunological principle that immune recognition is specific. To solve this contradiction, we hypothesize that iodine allergy is an immunological reaction to iodinated autologous proteins produced in vivo by iodination reaction from various iodine-containing chemicals. Antisera to iodine were obtained from guinea pigs immunized subcutaneously with iodine-potassium iodide solution emulsified in complete Freund’s adjuvant (CFA). The specificity of guinea pig anti-iodine antiserum was determined by enzyme-linked immunosorbent assay (ELISA) inhibition experiments using microplates coated with iodinated guinea pig serum albumin (I-GSA). Antibody activities were inhibited by I-GSA, diiodo-L-tyrosine, and thyroxine, but not by potassium iodide, monoiodo-L-tyrosine, 3,5,3’-tri- iodothyronine, monoiodo-L-histidine, or diiodo-L-histidine, or by ionic or non-ionic iodinated contrast media. The results that antigen recognition of anti-iodine antibody is specific to iodinated protein support our hypothesis. While protein iodination usually takes place both at histidine residues as well as at tyrosine residues, only iodinated tyrosine acted as an antigenic determinant and no antibody activities to iodinated histidine were detected in our experimental iodine allergy model. KEY WORDS: Iodine allergy, Anti-iodine antibody, Iodotyrosine, Guinea pig, Iodocontrast media INTRODUCTION In order to solve this contradiction, we hypothesize that iodine allergy is an immunological reaction to The term iodine allergy has been defined gener- iodinated autologous proteins produced in vivo by iodi- ally as “an allergic reaction induced by the use of nation reaction from various iodine-containing chemi- iodine-containing drugs” such as inorganic iodine cals. Iodination of protein by iodine occurs readily, preparations used as a skin disinfectant or various even at physiological pH (Hughes and Straessle, 1950). organic iodine-containing drugs. Accordingly it has Iodine molecules are substituted for hydrogen atoms on generally been thought that iodine allergy is cross-sen- the phenol rings of tyrosyl residues and on the imida- sitive to various iodine-containing chemicals. However, zole rings of histidyl residues (Covelli and Wolff, this concept seems to deviate from the immunological 1966). Experimental studies on immunogenicity of principle that immune recognition is specific. iodine (Ishikawa, 1953) and iodinated homologous Correspondence: Yoshiki SUGIHARA Vo l . 2 9 N o . 2 132 H. SHIONOYA et al. serum proteins (Jacobs, 1932) have been reported. Spe- was injected subcutaneously at a total volume of 0.5 ml cific precipitin reaction inhibition by diiodotyrosine in (0.125 ml per site at four separate positions or 0.25 ml a system consisting of iodinated sera and their antisera per site at two separate positions) into the back of five was first reported in 1930 by Wormall (Wormall, guinea pigs at intervals of 2 weeks. The dose of iodine 1930). Jacobs has confirmed these results and also was therefore 1 mg per animal for each immunization. reported that potassium iodide did not inhibit the pre- After five-time immunizations, animals were bled and cipitin reaction (Jacobs, 1932). However, there has the sera separated were pooled and preserved at −20°C. been no report on the specificity of antigen recognition An antibody titer of pooled sera by passive cutaneous of anti-iodine antibody, especially in relation to cross- anaphylaxis (PCA) test was 4000 using an iodinated sensitization of iodine allergy. Accordingly, it is impor- guinea pig serum albumin (I-GSA 7.2 as mentioned in tant to reveal the specificity of anti-iodine antibody for the next paragraph) as an eliciting antigen. better understanding of the mechanism of iodine allergy. Therefore, we performed this study to observe Preparation of iodinated proteins the antigen recognition of anti-iodine antibody using Iodinated guinea pig serum albumin for ELISA an experimental iodine allergy model. inhibition experiment and PCA test was prepared at pH 7.2, (as for in vivo iodination of protein) as follows. MATERIALS AND METHODS Thirty µl of 0.5 M iodine was added to 1 ml of a 10 mg/ ml solution of GSA in phosphate buffer saline (PBS, Animals and reagents pH 7.2) and incubated at 37°Covernight;pHwas Female Hartley strain guinea pigs, at the age of 4 maintained at 7.2 by adding 1 M sodium carbonate. weeks, were purchased from Nippon SLC The residual iodine in the reactants was reduced by (Hamamatsu, Japan). They were acclimated for 2 adding 0.1 M sodium thiosulfate and the reactants were weeks before use. Iodine (I2) and potassium iodide then dialyzed against PBS. The preparation was (KI) were obtained from Katayama Chemical Indus- referred to as I-GSA 7.2. Because the maximum iodi- tries (Osaka, Japan). L-Tyrosine disodium salt (Tyr), 3- nation of protein takes place at an alkaline pH (Hughes iodo-L-tyrosine (MIT), L-histidine (His), 3,5,3’-tri- and Straessle, 1950), iodinated GSA used in ELISAs iodothyronine (T3), thyroxine (T4), guinea pig serum was prepared as follows. An aliquot of 50 µl of 0.5 M albumin (GSA), were purchased from Sigma (St Louis, iodine was added to 1 ml of 5 mg/ml solution of GSA MO, USA). 3,5-Diiodo-L-tyrosine (DIT) was pur- in 0.1 M borate buffer, pH 9.5, and incubated at 37°C chased from Nacalai Chemicals (Kyoto, Japan). 4- overnight. The residual iodine in the reactant was Monoiodo-L-histidine (MIH) and 2,4-diiodo-L-histi- reduced and dialyzed against 0.1 M borate buffer at pH dine (DIH) were synthesized in our laboratories. The 9.5. The preparation was referred to as I-GSA 9.5. λ 1% ionic radiocontrast media and non-ionic contrast media max andE 1CM of I-GSA 9.5 were 311 nm and 16.9, used were amidotrizoic acid (Urografin; Schering respectively. The shift of λ max from 280 of GSA to Japan, Osaka, Japan), iodamide (Conraxin; Bracco, 311 nm of I-GSA 9.5 compares favorably with that of Milan, Italy), ioxaglic acid (Hexablix; Tanabe Pharm., tyrosine from 293 nm to 311 nm by di-iodination Osaka, Japan), iopamidol (Iopamiron; Schering (Hughes and Straessle, 1950). Under the iodination Japan), iohexol (Omnipaque; Daiichi Pharm., Tokyo, conditions employed, all the tyrosine residues and Japan) and iomeprol (Iomeron; Eisai, Tokyo, Japan). almost all the histidine residues were considered to be Protein assays were performed using BCA protein changed to di-iodinated ones when human serum albu- assay reagent (Pierce, Rockford, IL, USA). Bovine min (containing 18 tyrosine and 16 histidine residues serum albumin (BSA) standard solution attached to the per mol) was used (Hughes and Straessle, 1950). For assay reagent was used as a standard. the purpose of confirming potency of I-GSA 9.5 to detect antibodies which were produced by immuniza- Immunization of guinea pigs tion of guinea pigs with iodinated protein, I-GSA 7.2, Animal handling for all procedures such as injec- ELISA inhibition experiments were performed using tions and bleedings was performed under pentobarbital iodinated histidine and tyrosine as an inhibiting hap- sodium anesthesia. Iodine solution (4 mg/ml; I2/KI = ten. The results are shown in Fig. 1, indicating that I- 1.269: 2.50, W/W) was emulsified with an equal vol- GSA 9.5 detected antibodies to either iodinated histi- ume of CFA (Difco, Detroit, MI, USA). The emulsion dine or tyrosine. Vo l . 2 9 N o . 2 133 Experimental iodine allergy: 1. Specificity of anti-iodine antibody. ELISA inhibition experiments for anti-iodine anti- The absorbance at 490 nm was measured with a model body 3550 microplate reader (BioRad, Richmond, CA, I-GSA 9.5 was coated onto microtiter plates USA). Each sample was assayed in triplicate. (Coster, Cambridge, MA, USA). After blocking unoc- cupied plastic sites with bovine serum albumin (BSA, RESULTS Sigma), anti-iodine antisera previously incubated at 4°C overnight with test compounds for ELISA inhibi- The effects of inorganic iodine (KI) and iodinated tion were added to each well, followed by incubation at organic derivatives on antigen-antibody reactions were 37°C for 1 hr. After washing the plates, they were incu- studied in ELISA inhibition experiments using antisera bated with peroxidase-conjugated goat anti-guinea pig obtained from guinea pigs immunized with iodine and IgG (heavy and light chain-specific, Cooper Biochem- microplates coated with I-GSA 9.5. ical, Malvern, PA, USA) and washed. Peroxidase sub- A fourfold serial dilution of the antisera was per- strate solution containing o-phenylenediamine (Sigma) formed with PBS containing 1% BSA, 0.05% Tween and hydrogen peroxide was then added to each well. 20, and 3% sodium chloride as a diluent. To 0.1 ml of The plates were kept for 30 min at room temperature, the diluted sera, 0.1 ml of test material solution for and 3N hydrochloric acid was then added to each well. ELISA inhibitory activity was added and incubated at 4°C overnight. Antibody activities were then assayed on the microplates coated with I-GSA 9.5.

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