Pioneers of Allergy: Personal Reflections Bergmann K-C, Ring J (eds): History of Allergy. Chem Immunol Allergy. Basel, Karger, 2014, vol 100, pp 356–360 DOI: 10.1159/000360100

Kimishige Ishizaka Tokyo, Japan

Place(s) of university education University of Tokyo, School of Medicine (both MD and phD) Who were your most important teachers in allergology? Prof. Keizo Nakamura, University of Tokyo Prof. Dan H. Campbell, California Institute of Technology Please list your 5 most important publications Ishizaka K, Ishizaka T, Hornbrook MM: Physicochemical properties of human reaginic antibody. IV. Presence of a unique immunoglobulin as a carrier of reaginic activity. J Immunol 1966;97:75–85. Ishizaka K, Ishizaka T, Hornbrook MM: Physicochemical properties of reaginic antibody. V. Correlation of reaginic activity with γE globulin antibody. J Immunol 1966;98:840–853. Ishizaka K, Ishizaka T: Identification of γE antibodies as a carrier of reaginic activity. J Immunol 1967;99:1187–1198.

Tomioka H, Ishizaka K: Mechanisms of passive sensitization. II. Presence of receptors for IgE on monkey mast cells. J Immunol 1971;107:971–978. Ishizaka T, Ishizaka K: Biology of immunoglobulin E. Prog Allergy 1975;19:60–121. Have you ever had a function as a President and/ or Secretary General of an allergological society? Collegium International Allergologicum: President (1982–1986) Which position has had the greatest impact on your professional life? Professor of Medicine, Johns Hopkins University, School of Medicine (1970–1989)

Questions about your professional life What brought you to allergy? When I was accepted at the Medical School of the University of Tokyo in 1944, I was expecting to be a physician in the future. However, I became interested in basic science after I spent the summer vacation in 1946 receiving training on bacteriology from Downloaded by: Univ. of California San Diego 198.143.33.33 - 8/19/2015 9:34:13 PM

Year of birth 1925

From whom (academic, teacher) did you learn the most? I got my lifelong research projects as a postdoctoral fellow of Prof. Dan H. Campbell at the California Institute of Technology from 1957 to 1959. His idea was that antigen-antibody complexes may have biological activities, which are lacking in either antigen or antibody alone, and that the formation of such bioactive complexes in vivo may induce anaphylactic reactions. He explained his idea and asked me to test his hypothesis. When I asked him about the experimental systems to be used, he said: ‘That is your business. I gave you a research project. You should decide on appropriate experimental systems to test my hypothesis.’ Fortunately, our studies proved that preformed soluble antigen-antibody complexes induced an increased permeability of guinea pig skin capillaries. Analysis of the antigen-antibody complexes revealed that the bioactive complexes contained two or more antibody molecules, while the complex composed of two antigen molecules and one antibody molecule was inactive. The results suggested to us the possibility that the role of antigen for the formation of bioactive complexes is to bring two antibody molecules into close proximity. I wondered whether the nature of antigen or the nature of antibody molecules is important for the

Personal Reflections

formation of bioactive antigen-antibody complexes. Dr. Campbell agreed with me to proceed with experiments to answer the question; however, he asked me to form a manuscript of the paper before beginning actual experiments, adding: ‘I am not kidding you.’ Carl Landsteiner always wrote his manuscript before he began his experiments, and sent the paper for publication as soon as he got actual data. ‘You can do it!’ It was great advice for me as a professional research scientist. I followed his advice, and wrote a manuscript without any data. The results of the experiments actually showed that the generation of biological activities by the formation of antigen-antibody complexes depends on the nature of antibody molecules rather than the nature of antigen involved. Further studies showed that aggregated normal rabbit γ globulin, which was prepared using a chemical coupling agent, induced skin reactions in the normal guinea pig and fixed complement. Terry (Teruko Ishizaka) and I continued the research after we went back to Japan, and proved that the Fc portion of rabbit antibody molecules contained the structures which are essential for the formation of bioactive antigen-antibody complexes. Indeed, polymerized Fc fragment of rabbit γ globulin (IgG) induced skin reactions in the normal guinea pig and fixed complement, whereas neither the polymerized Fab fragment nor monomeric Fc fragment did. I wondered whether common molecular mechanisms might be involved in both the experimental anaphylaxis in the guinea pig and allergic reactions in humans. At that time, however, no hay fever patient had been reported in Japan. Thus, I visited Prof. Campbell in Pasadena on my way to New York, where the International Congress of Allergology was held in 1961. Following his advice, I accepted an offer from Dr. Sam Bukantz, and moved back to the USA to extend our research to human allergy at the Children’s Asthma Research Institute and Hospital (CARIH) in Denver, Colorado. My plan was to investigate the immunochemical mechanisms of allergic reactions in humans by using ‘reagin’, which had been described by Prausnitz and Küstner in 1921. Retrospectively, I would not have gone into research on the immunochemical mechanisms of human allergy if I had not had the chance to work with Prof. Campbell.

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Prof. Keizo Nakamura at the Institute of Infectious Diseases. He was a bacteriologist but he had a great interest in immunology, and his major research project was the mechanism of anaphylaxis. After the summer vacation, he asked me to translate a book entitled The Chemistry of Antigens and Antibodies written by Prof. J.R. Marrack in 1938. As the content of the book was very impressive for me, I became interested in immunochemistry and made up my mind to focus on immunology after graduation from Medical School. In 1950, the Japanese Society of Allergy was established, and Prof. Nakamura was elected as the President of the Society. As I helped him to organize the first annual scientific meeting of the society, I became acquainted with many allergists in Japan, and had the opportunity to learn problems in allergic diseases.

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ity in the original serum was in the order of 1 μg/ml or less. In the 1960s, isolation and physicochemical characterization of the purified protein was the minimal requirement for claiming the presence of the distinct protein. However, our results indicated that several liters of serum from pollinosis patients would be required for isolation of a sufficient amount of the carrier protein of reaginic activity for physicochemical characterization. At this point, I had to give up the strategy. What was your greatest achievement in allergy? Our greatest achievement in allergy was ‘identification of reagin in vitro, and discovery of a unique immunoglobulin IgE as the carrier protein of reaginic activity’. Since our experiments indicated that the carrier protein of reaginic activity is a minor component of human serum proteins, I switched our strategy to the preparation of rabbit antibodies specific for the carrier protein. If such antibodies were available, it would be possible to identify ‘reagin’ in vitro without purification. Thus, we repeatedly immunized rabbits with a reagin-rich fraction of patients’ serum, and absorbed the antisera with known human immunoglobulins such as IgG, IgA and IgD. After absorption, the antisera did not show a precipitin band with any of the known human immunoglobulins, nor with a normal human serum. However, addition of the antiserum obtained from one of the rabbits to serum samples of hay fever patients resulted in complete removal of reaginic activity in the patients’ sera, indicating that the rabbit antiserum contained the antibodies specific for ‘reagin’. By using the rabbit antiserum and radiolabeled allergen, we identified reagin in vitro in 1965. We tentatively called the carrier protein of reaginic activity γE, because I believed that the antibodies detected by our method were responsible for the induction of erythemawheal reactions. Detection of γE antibodies in vitro in the serum of hay fever patients by using the anti-γE antibodies and radiolabeled allergen facilitated physicochemical characterization of γE. Similar procedures revealed that γE possessed distinct antigenic determinants, which are lacking in the known immunoglobulins, together with the antigenic determinants in

Ishizaka

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What was your greatest disappointment? In early 1962, Heremans and Vaerman from Belgium reported that IgA isolated from the serum of atopic patients had skin-sensitizing activity, as determined by the Prausnitz-Küstner (P-K) reaction. Since the results were confirmed by several other investigators, most immunologists and allergists believed that ‘reagin’ is the IgA antibody against allergen. Thus, I planned to study the mechanisms of reaginic hypersensitivity by using human IgA antibodies. I repeatedly immunized normal type O individuals with ‘blood group A substance’, and isolated IgA, IgG and IgM in their sera. As each purified immunoglobulin contained much anti-A antibody, as determined by radioimmunoassay, I injected each of the purified immunoglobulin, containing 1–2 μg of anti-A antibody, intracutaneously into non-allergic type O individuals, and challenged the skin sites with ‘A substance’ 24 h later. However, no erythemawheal reaction was observed at any of the skin sites. Thus, we repeated the experiment by Heremans and Vaerman. Indeed, IgA isolated from the serum of ragweed-sensitive hay fever patients contained a high titer of reagin, as determined by the P-K reaction. In view of the fact that anti-A IgA antibodies did not have reaginic activity, I added rabbit antibodies specific for human IgA to the purified IgA fraction of the serum of ragweed-sensitive patients, and removed all IgA in the preparation. To my great surprise, essentially all reaginic activity in the purified IgA fraction remained in the supernatant, indicating that the reaginic activity in the so-called ‘pure’ IgA preparations was not associated with IgA, but with the other protein which was not detectable by usual immunological methods. Thus, I had to give up our original plan to use IgA antibodies for elucidation of the mechanisms of reaginic hypersensitivity. The results of the experiments created more difficult problems. After removal of IgA in the purified IgA fraction of the serum of hay fever patients, the concentration of the total human immunoglobulin in the supernatant was 1 μg/ml or less. Yet, the P-K titer of the supernatant was comparable to the titer of the original serum, from which the IgA preparation was obtained. The results suggested that the concentration of the carrier protein of reaginic activ-

What was your most funny experience? I happened to suffer from ‘atopic dermatitis’ as the result of intracutaneous injections of E myeloma protein and anti-IgE antibodies into my forearm. The incident happened after Terry and I had visited Dartmouth University Hospital in Hanover, New Hampshire, in February 1969, to carry out some tests on the E myeloma patient (the second known case). The patient failed to accept passive sensitization with reaginic antibodies for P-K reactions, and reacted only to the intracutaneous injection of a high concentration of anti-IgE. It was also found that his myeloma protein inhibited the passive sensitization of my skin with the serum of a ragweed-sensitive patient for the P-K reaction. As I used myself as a normal control in these experiments, I injected E myeloma protein and anti-IgE into several skin sites of my right forearm. On our way back to Denver I felt itching on my right forearm and recognized that the skin sites that had received anti-IgE had flared up. By the time we arrived at Denver airport red papules had spread all over my right forearm with intense itching. The next morning, I went to CARIH and showed my forearm to Dr. Elliot Middleton, who was the President of the

Personal Reflections

Institute. He said: ‘Oh my God! This is atopic dermatitis! What happened to you?’ The intense itching and papules with erythema lasted for about 1 week, and then gradually subsided. As I had never had an allergic disease before, and I frequently used my forearm for P-K reactions without any trouble, the cause of the dermatitis must have been IgE-anti-IgE complexes formed in my skin. Who would you list among the top 10 allergists of the world? (Please only mention individuals already deceased.) Dr. Bram Rose and Dr. Carl Arbesman. Dr. Rose was Professor of Medicine at McGill University, Montreal, Canada, and Dr. Arbesman was Professor of Medicine at New York State University, Buffalo, USA. When I was at CARIH, Denver, Colorado, both Dr. Rose and Dr. Arbesman had big research and clinical groups for allergy, and physicochemical characterization of reagin was one of the major research projects in both groups. In a sense, we were their competitors, but they helped our studies for several years for the common purpose. Both Dr. Rose and Dr. Arbesman supplied serum samples of ragweed-sensitive patients for our experiments. Dr. Rose was the President of the International Congress of Allergology, which was held in Montreal in October 1967. I greatly appreciated his invitation to the symposium on reaginic antibodies at the congress as one of the main speakers. I believe that ‘γE’ was recognized and accepted internationally at that symposium. As Dr. Arbesman had been interested in detecting ‘reagin’ using monkeys, we collaborated with his group to prove that γE (IgE) antibodies in the serum of ragweed-sensitive patients can be detected by passive cutaneous anaphylaxis in the monkey. He also tried to reproduce our findings on γE. As his group could not prepare anti-γE, I sent him a small amount of our rabbit antiserum specific for human γE upon his request. Thus, he confirmed that our anti-γE could remove reagin in the serum samples of his patients, and he could detect γE antibodies in the sera by using our anti-γE and radiolabeled ragweed antigen. After my presentation before the International

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both κ and λ chains, indicating that γE represents a unique immunoglobulin isotype. Based on the physicochemical properties of γE, we spent 6 months isolating the protein from a large quantity of the serum of ragweed-sensitive patients. On a weight basis, reaginic activity of the purified γE was 1,000 times more active than the original serum, and gave positive P-K reactions at a dilution of 1:80,000. One month later, I received a letter from Dr. Gunnar Johansson and Dr. Hans Bennich in Sweden, and learned that they had obtained an atypical myeloma protein, the physicochemical properties of which were almost identical to those of γE. Subsequent collaboration with them actually showed that our anti-γE reacted with the atypical myeloma protein, while the antibodies specific for the myeloma protein reacted with γE in the serum of ragweedsensitive patients, indicating that the myeloma protein possessed the antigenic determinants characteristic for γE. Thus, γE was officially designated IgE by the World Health Organization in 1968.

What do you see as the greatest problem for allergy in the next 10 years? In the past 40 years so much knowledge has been gained on the immunological, biochemical and molecular mechanism of allergy and allergic diseases. I

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greatly appreciate the progress. However, the greatest problem which remains to be solved is the increase of allergy patients, particularly in developed countries. Environmental exposure to microbial products may play a critical role during the maturation of the immune system of children, and modifications of the pattern of microbial exposure of children may be important factors for the prevalence of atopic disorders. Thus, the so-called ‘hygiene hypothesis’ may partly explain the increase of allergy patients. However, changes in lifestyle may also be an important reason for the increase of patients. Heating or air-conditioning were not available before the end of the Second World War. Changes in lifestyle may have caused epidermal barrier dysfunction. I believe that the greatest problem for allergy in the next 10 years will be finding an effective method for preventing the increase of allergy patients in westernized countries.

Ishizaka

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Congress in Montreal, he made his comments and showed their data to the audience. I believe that his comments convinced the audience of the truth of our conclusion that reagin represents the γE antibodies specific for allergen. Dr. Arbesman was elected President of the International Congress of Allergology held in London. Just before the Congress, however, he called me by telephone and let me know that he was suffering from cancer. He said that he wished to go to London even if he might die on the way. I still remember the long telephone conversation with him at that time.

Kimishige Ishizaka. Tokyo, Japan.

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