The Making of an Immunologist: Heidelberger's Years at the Rockefeller Institute, 1912-1927

Michael Heidelberger rose to scientific prominence during his fifteen years at the Rockefeller Institute of Medical Research (today Rockefeller University), founded in 1901 as the nation's foremost biomedical research facility. As a collaborator in the laboratories of several of the institute's leading investigators, he developed important chemotherapeutic drugs and helped launch modern immunology by introducing biochemical approaches to the field. His seminal discovery with Oswald T. Avery in 1923 that powerful antigens of pneumococcus bacteria are polysaccharides opened up an expansive new area in the study of microorganisms, and for the first time established a connection between the chemical structure of antigens (the microorganisms, particles, or toxins that elicit an immune reaction in animals) and their immunological specificity, or mode of action.

Heidelberger's association with the Rockefeller Institute began before his formal hiring. His former family physician had joined the Institute, and was the first to interview Heidelberger about his interest in becoming a scientist. On the recommendation of the Rockefeller Institute's chemists, Phoebus A. T. Levene, Donald D. Van Slyke, and Walter A. Jacobs, Heidelberger decided in 1911 to spend a year in Richard Willstätter's laboratory at the Federal Polytechnic University in Zurich to complete his training and to increase his chances of obtaining a position at a research university in the United States.

Willstätter, the 1915 Nobel Laureate in chemistry, was doing innovative research on the chemical constitution and function of enzymes and of plant pigments, in particular chlorophyll, which Heidelberger had come to study. Instead, Willstätter set Heidelberger to uncovering the structure of cyclooctatetraene. Heidelberger showed this unstable compound to be the next higher analog of benzene, with eight carbon atoms and four double bonds. Heidelberger worked continuously for nearly 24 hours at low temperatures to obtain a colorless liquid that was both stable and reactive enough for chemical analysis. Heidelberger's results held up in the face of criticism by Hugh S. Taylor and Charles D. Hurd during the 1940s that the substance he and Willstätter had produced was not in fact cyclooctatetraene. As a consequence of their work, cyclooctatetraene became a widely used intermediate in organic chemistry.

After Simon Flexner, the director of the Rockefeller Institute, offered him a position as Fellow, Heidelberger spent more than nine years, from 1912 to 1921, in the laboratory of Walter Jacobs. He described Jacobs as a very skillful experimenter from whom he learned important research techniques, such as how to work on a number of problems at once. They collaborated on the synthesis of several chemotherapeutical drugs, which they hoped would serve as treatments for infectious diseases. At Flexner's direction, they studied a derivative of hexamethylene tetramine, a complex of formaldehyde and ammonia that seemed to prolong the life of monkeys suffering from poliomyelitis, for use in humans. Results appeared promising at first, but later turned out to be due to the loss of virulence of the virus.

During World War I, Jacobs and Heidelberger turned their attention to the aromatic arsenicals for use as chemotherapeutic agents against syphilis and African sleeping sickness. They focused on pentavalent arsenicals, first studied by Paul Ehrlich in the course of research that led to his discovery of Salvarsan, the "magic bullet" against syphilis until the introduction of antibiotics. Ehrlich had examined several compounds in this class, in particular phenylglycine arsenic acid, but had found them too toxic for use as drugs. Jacobs suspected that their toxicity resulted from chemical groupings that were highly reactive in vivo, and reasoned that if these groupings were substituted or chemically masked, the compounds might prove therapeutic. Jacobs and Heidelberger synthesized an amide of phenylglycine arsenic acid in which the carboxyl group was blocked, and the resulting drug, sodium para-phenylglycinamide arsonate, for which Flexner proposed the name tryparsamide, proved effective against trypanosomiasis, i.e. African sleeping sickness. It was successfully tested in humans by Louise Pearce in the Belgian Congo and became the standard chemotherapeutic treatment for the disease. It is still in use today.

During this time Heidelberger also studied the chemotherapy of pneumococcal bacterial infections, the most common cause of lobar pneumonia. Heidelberger and Jacobs believed that to be a useful chemotherapeutic, a substance had to be highly bactericidal. Among the compounds they studied was sulfanilamide, first synthesized by the Viennese chemist Paul Gelmo in 1908. They improved the method for preparing sulfanilamide, but because it was not bactericidal in vitro they did not test it for its therapeutic properties, instead converting it into highly bactericidal but therapeutically ineffective derivatives. Twenty years later, Gerhard Domagk did empirically test Prontosil, an azo dye in which a sulfonamide group was the active ingredient, thereby launching the sulfa drugs and ushering in a new era in the chemotherapy of bacterial infections and other diseases. The discovery of the therapeutic effects of sulfonamide helped save countless lives and earned Domagk the Nobel prize in 1939. In his lectures to first-year medical students at the College of Physicians and Surgeons, Heidelberger always recounted this story as a warning that scientists should not be beholden to one particular theory.

In 1921, after nine-and-a-half years in Jacobs' laboratory, Heidelberger transferred to the laboratory of Donald D. Van Slyke at the Rockefeller Institute Hospital to join Van Slyke and Baird Hastings in their studies on the uptake and release of oxygen by hemoglobin in the blood. He did so at the behest of Flexner, who thought that Heidelberger would have a better chance of gaining an independent research position as a biochemist than as an organic chemist. Heidelberger spent the next two years developing a technique for preparing large quantities of purified, crystalline horse oxyhemoglobin, with its oxygen-carrying capability intact (a capability sometimes lost during purification).

In the course of his work on hemoglobin, Heidelberger designed the first refrigerated centrifuge, with a brine cooling coil wrapped around a standard size 2 centrifuge. His invention was prompted by his concern for his laboratory technician, who contracted recurrent colds by working in the refrigerated room in which a conventional centrifuge had been set up. The refrigerated centrifuge quickly became standard equipment in biochemical laboratories. Heidelberger decided against patenting his invention, with the result that his commercial gain from it amounted to only $50, paid to him by the manufacturer, International Equipment Company, for writing the operating manual.

During these years Heidelberger developed a deeper interest in hemoglobin. Pursuing an idea that was much ahead of its time, Heidelberger in 1920 took some of the first X-ray photos of crystals of the large protein at the General Electrics laboratories in Schenectady, New York, hoping to derive from these photos its three-dimensional structure. He did not achieve useful results--determining the structure of hemoglobin by X-ray crystallography presented many difficulties, and was not achieved until 1953--and his failure prompted him to resume his focus on analytical and biochemistry.

Heidelberger first ventured into the field of immunochemistry during a joint investigation with the Austrian immunologist and future (1930) Nobel Laureate, Karl Landsteiner, who arrived at the Rockefeller Institute in 1922. Their research, as well as Heidelberger's subsequent collaboration with Oswald Avery and, indeed, his work over the next five decades, was designed to elucidate the fact that antibodies, protein molecules that react with a specific antigen, were directed not against an antigen as a whole, but against particular chemical groupings (later called antigenic determinants) on the surface of the antigen to which antibodies bind. Heidelberger thus showed that the specificity of antigens and antibodies--that is, the correlation between a particular kind of antigen and the single kind of antibody whose synthesis it induces (out of more than a million different kinds known today)--was based not on general biological characteristics, as earlier generations of immunologists had concluded, but upon a precisely defined feature of the antigen's chemistry. Although the exact chemical mechanism of attachment of specific antigen to specific antibody was not discovered until the 1950s, Heidelberger's findings explained why certain microorganisms were particularly pathogenic, and why recovery depended on the appearance in patients' serum of antibodies specific for the antigens of the infecting microorganism.

Heidelberger and Landsteiner embarked on studies of the antigenic properties of different types of hemoglobins, which Landsteiner had first isolated and for which he had prepared antisera. Hemoglobins from one species of animal can act as antigens when injected into another, thus triggering an immune response that produces antibodies contained in blood serum, the clear liquid that separates from the blood upon clotting. (Serum is the clear portion of any body fluid. Serum that contains antibodies or antitoxins is called antiserum.)

In two important articles published in 1923, Heidelberger and Landsteiner presented novel immunologic and solubility techniques for distinguishing and comparing hemoglobins of different species. They demonstrated, first, that antigenically distinct hemoglobins could be precipitated by the same antiserum; and second, that horse and donkey hemoglobins cross-reacted with antisera for the other, meaning that there were no immunological differences between them. These experiments taught Heidelberger much about immunological cross reactions, the interaction of an antigen with an antibody formed against a different antigen with which the first antigen shares identical or closely related antigenic determinants. Heidelberger studied cross reactions throughout his career because they give important clues to the chemical structure of antigens and antibodies.

Soon thereafter, Heidelberger was once again drawn into the study of pneumococcus when the bacteriologist Oswald Avery, head of the pneumococcus study group at the Rockefeller Institute, came to see Heidelberger, waving a vial of a dark grey suspension of pneumococcus bacteria and saying: "the whole secret of bacterial specificity is in this little vial. When are you going to work on it?" Until the introduction of the sulfa drugs in the late 1930s and of penicillin in the 1940s, pneumonia caused by pneumococcal infection was a major cause of human illness and death. The only effective treatment was the injection of antipneumococcal antisera produced in rabbits and horses by inoculating them with killed pneumococci. Uncovering the chemical constitution and mode of action--the specificity--of pneumoccocus was of great scientific and clinical importance.

Fred Neufeld had classified pneumococcus isolated from the blood and sputum of infected patients into three stable types (I, II, and III), and had relegated all others (Heidelberger proved over the course of his career that there were many) to group IV. But bacteriologists and clinicians knew little about their chemical composition, or about how antigens of pneumococcus, or indeed of any microorganism or toxin, bind to specific antibodies and cause disease of lesser or greater virulence. Most scientists assumed that antigens were proteins, but since the fine structure of proteins remained mysterious, the specificity of antigens could not be explained in terms of their chemistry.

In order to find out what aspect of its chemistry made pneumococcus such a powerful antigen, and made some of its types even more powerful than others, Avery sought to uncover the chemical composition of precipitates of the "specific soluble substance" he and Alphonse R. Dochez had obtained by incubating virulent pneumococcus bacteria with type-specific antisera. It was this substance, which as Avery and Dochez realized was shed from the capsule surrounding live, virulent pneumococci, that imparted to each pneumoccocal type its immunological specificity. To characterize the capsular material, Avery needed the expertise of a biochemist like Heidelberger.

Applying painstaking techniques required before the invention of paper chromatography, Heidelberger precipitated Avery's specific soluble substance in type II pneumoccocus (a fortunate choice because of the structural simplicity of its capsule) with antiserum specific for this type, and recovered the substance from the precipitate. He then purified it to the point where it no longer contained nitrogen, a component of almost all proteins. Heidelberger and Avery concluded that, rather than protein, the substance consisted of nitrogen-free carbohydrates, namely polysaccharides, sugar compounds that are made up of strings of five or more monosaccharides. The chemical structure of the long, thread-like capsular polysaccharides of each type of pneumococcus was distinct, and served to distinguish between types.

Moreover, Heidelberger and Avery found that virulence in both type II and type III pneumococcus was determined by the repeating sequences of monosaccharides along the length of the molecule. They thereby drew for the first time a definite connection between the chemical constitution of an antigen and its immunological specificity, and gave the first indication of the importance of polysaccharides in immune phenomena. Over the next several decades, Heidelberger determined the carbohydrate structures of several dozen pneumococcal and other polysaccharide antigens, proving that they play a key role in the immunochemistry of many encapsulated microorganisms and several non-encapsulated ones.

In recognition of his and Avery's success, Flexner in 1924 granted Heidelberger a sabbatical--the first ever at the Institute--to tour the leading laboratories of Europe.

After three more years of studying the chemical structure of pneumococcus polysaccharide antigens with Avery and Walther F. Goebel, Heidelberger left the Rockefeller Institute in 1927 in order to become head of his own laboratory, at Mount Sinai Hospital on New York's Upper East Side. There he hoped to continue his research independently, but found a laboratory that was in need of complete reorganization, a task that took up all of his time for the first six months. After that, he was largely preoccupied with the routine testing of hospital specimens, including, among many others, cases of lead-poisoning. He was glad to accept the following year an offer of a research professorship at the College of Physicians and Surgeons at Columbia University. He did, however, meet at Mount Sinai Check M. Soo Hoo, a Chinese chemist whom Heidelberger had been asked to fire, but who instead became his personal laboratory technician for the next thirty-five years and a coauthor of several articles.