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’-triiodothyronine, 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

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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.

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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. The test material and the final concentrations in the incubation mixtures used are as follows: KI, His, MIH, DIH and contrast media at a concentration of 10 mM, I-GSA 7.2 at 50 µg/ml, Tyr, MIT, and DIT at 5 mM, and T3 and T4 at a concentration of 0.5 mM. ELISA inhibition data for KI and iodinated protein, I-GSA 7.2, are shown in Fig. 2. As expected from

Fig. 1. ELISA inhibition study on antigen antibody reaction between iodinated guinea pig serum albumin, I-GSA 9.5, and antisera immunized with I-GSA 7.2 by iodinated histidine and tyrosine. Antisera were obtained by immunization of guinea pigs with iodinated guinea-pig serum albumin, I-GSA 7.2, emulsified with complete Freund’s adjuvant. To each 50 mM solution of histidine and tyrosine in borate buffer, pH 8.5 was added with iodine at molar ratios of 0 (control), 1 and 2, and incubated overnight at room temperature. The antisera diluted 125-fold were added with equal volume each 10 mM solution of iodinated histidine or tyrosine and incubated. Antibody Fig. 2. Inhibition of guinea pig anti-iodine antibody binding activities of the mixtures were accessed on the ELISA to solid-phase I-GSA by iodinated guinea pig serum microtiter plates coated with I-GSA 9.5. The final con- albumin (I-GSA, 50 µg/ml) and potassium iodide centration of the antisera was 500-fold dilution. (KI, 10 mM).

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H. SHIONOYA et al. the PCA-eliciting activity of I-GSA 7.2 (See MATERI- T3 and T4 contain a central 3,5-diiodophenyl moiety, ALS AND METHODS), I-GSA 7.2 but not KI inhib- the fact that T3 showed no inhibitory activity reveals ited the antibody activity. The data indicated that antibodies produced by immunization with iodine rec- ognized the iodinated protein. It is known that iodine binds to the imidazole rings of histidine residues and to the phenol rings of protein tyrosine residues (Covelli and Wolff, 1966); the inhibitory activities of iodinated histidines and iodinated tyrosines were therefore studied in subsequent experiments. As shown in Figs 3 and 4, neither of the iodinated histidines, i.e. 4-monoiodo- L-histidine (MIH) and 2,4-diiodo-L-histidine (DIH), inhibited anti-iodine antibody activity. Among the tyrosine residues, only 3,5-diiodo-L-tyrosine (DIT) but not 3-iodo-L-tyrosine (MIT) inhibited the antibody activity. In the next experiment, the effects of thyroid hormones T4 and T3 on the antibody activity were studied. The data, as shown in Fig. 5, clearly show that T4 and DIT but not T3 were the inhibitors. In subsequent experiments, the inhibitory activity of DIT was compared with that of T4 by measuring the percentage inhibition in an ELISA at a constant antibody concentration (at 500-fold serum dilution) incubated with varying concentrations of DIT and T4. As shown in Fig. 6, the concentrations of DIT and T4 resulting in Fig. 4. ELISA inhibitory activities of monoiodotyrosine 50% inhibition were the same (50 µM). Although both (MIT, 5 mM) and diiodotyrosine (DIT, 5 mM) compared with those of tyrosine (Tyr, 5 mM) and PBS.

Fig. 3. ELISA inhibitory activities of monoiodohistidine (MIH, 10 mM) and diiodohistidine (DIH, 10 mM) Fig. 5. ELISA inhibitory activities of 3,5,3’-triiodothyronine compared with those of histidine (His, 10 mM) and (T3, 0.5 mM), thyroxine (T4, 0.5 mM) compared PBS. with those of DIT (5 mM) and PBS.

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Experimental iodine allergy: 1. Specificity of anti-iodine antibody. the possibility of the terminal 3’,5’-diiodo-4-hydrox- yphenoxyl moiety of T4 molecules as an antigenic determinant. The ELISA inhibition activity of various iodinated contrast media was studied at a final concentration of 10 mM. As shown in Figs. 7 and 8, none of the ionic contrast media - iodamide, ioxaglic acid, and amidotrizoic acid - and none of the non-ionic contrast media - iomeprol, iopamidol, and iohexol - showed any inhibitory activity in ELISA.

DISCUSSION

Many iodine-containing preparations, such as iodine tincture, Lugol’s solution, popidone iodine, iodoform, and iothion, are widely used as antiseptics. These antiseptic preparations may induce the iodination of proteins at their histidyl and tyrosyl residues (Covelli and Wolff, 1966). However, only antibodies to iodinated tyrosine residues were detected and no antibodies to iodinated histidine iodohistidine were Fig. 7. Lack of inhibition of guinea pig anti-iodine antibody detected in the iodine allergy model of the present binding to solid phase iodinated guinea pig serum study. As mentioned in MATERIALS AND METH- albumin in ELISA by ionic iodinated contrast media, ODS, guinea pig serum albumin iodinated in vitro,I- iodamide, ioxaglic acid, and amidotrizoic acid at 10 GSA 7.2 produced antibodies to both iodinated mM.

Fig. 6. Comparison of the ELISA inhibition activities of DIT and thyroxine on guinea pig anti-iodine antibody Fig. 8. Lack of inhibition of guinea pig anti-iodine antibody binding to solid-phase I-GSA. The hapten inhibition binding to solid phase iodinated guinea pig serum experiments were performed by adding various con- albumin in ELISA by non-ionic iodinated contrast centrations of DIT or thyroxine to a constant concen- media, iomeprol, iopamodol, and iohexol at 10 mM. tration of pooled serum sample (diluted 1:500).

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H. SHIONOYA et al. tyrosine residues as well as iodinated histidines, and it antibody with a high immunological specificity. The was considered that effective mounts of iodination at result suggests that cross-sensitivity in iodine allergy histidine residues did not occur in vivo to give an anti- does not depend on antigen antibody reaction between genic stimulation in guinea pigs. iodine-containing chemicals and cross-reactive anti- The finding that diiodotyrosine and thyroxine iodine antibodies to the chemicals. Furthermore, the exhibited the same degree of inhibition as shown in findings of the present investigation that antigen recog- Fig. 6 indicates that antibodies recognize only the ter- nition of anti-iodine antibody is specific to iodinated minal residue of diiodinated tyrosine. protein support our hypothesis that iodine allergy is an Specific precipitin reaction inhibition by diiodo- immune response to iodinated self proteins produced tyrosine in a system consisting of iodinated sera and from various iodine-containing chemicals in vivo. their antisera was first reported in 1930 by Wormall Therefore, it seems important to study the existence (Wormall, 1930). Jacobs has confirmed the results and and the specificity of anti-iodine antibodies in humans also reported that potassium iodide did not inhibit the suffering from iodine allergy. precipitin reaction (Jacobs, 1932). However, specificity of anti-iodine antibody to various iodine-containing REFERENCES organic chemicals has not been elucidated. In our studies, the antibody activities of antisera obtained from Covelli, I. and Wolff, J. (1966): Iodohistidine forma- animals immunized with iodine were neither inhibited tion in ribonuclease A. J. Biol. Chem., 241, by the addition of potassium iodide or iodinated his- 4444-4451. tidines nor by any ionic and non-ionic contrast media Hughes, W. L. Jr. and Straessle, R. (1950): Preparation used in ELISA, and clearly discriminated monoiodoty- and properties of serum and plasma proteins. rosine from diiodotyrosine. Thus, the immune recogni- XXIV. Iodination of human serum albumin. J. tion of the antibodies produced by immunizing with Am. Chem. Soc., 72, 452-457. iodine is very specific in the animal model of iodine Ishikawa, M.(1953): Experimental allergic reactions allergy. induced by simple chemical compounds. pp. 17- It has generally been thought that cross-sensitiv- 18, Maruzen, Tokyo. ity is a common phenomenon of iodine allergy. Sub- Jacobs, J.(1932): Serological studies on iodinated sera jects allergic to iodine are usually also allergic to 1. Precipitins and precipitinogens. J. Immunol., iodinated organic compounds. However, we found no 23, 361-374. evidence of a cross-reactive ‘anti-iodine antibody’ in Wormall, A. (1930): Immunological specificity of the animal model used in the present investigation of chemically altered proteins. J. Exp. Med., 51, iodine allergy, and the antibody present in the animals 295-317. immunized with iodine was anti-diiodotyrosyl residue

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