History of Immunology

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Chapter 2 The History of Immunology

EARLY THEORIES OF ACQUIRED IMMUNITY

It is clear that humankind must have known the ravages of epidemic disease from the very start of its social organization. Thus, the Babylonian Epic of Gilgamesh from about 2000 B.C. records visitations of disease and pestilence, and severe epidemics are recorded in the annals of the early dynasties of Egypt. In most early societies, however, and even today among more primitive peoples, both humans and nature were thought to function under the magical influences of spirits and demons, or under the mystical influences of the gods. It was only natural, then, that disease came to be considered a punishment for some infraction of a tribal taboo or some sin against the gods. Both the Babylonians and the ancient Egyptians included in their pantheons a god of disease, and throughout the Old Testament, God is continually smiting those who trespass against Him, often employing pestilential disease as a punishment. Thus, God brought down a pestilence not only on His own chosen people as punishment of David抯 sin of numbering the people (II Samuel 24), but also on their various enemies, including the Egyptians (Exodus 9:9), the Philistines (I Samuel 5:6), and King Sennacherib抯 Assyrians (Isaiah 37:36). Even in ancient Greece the sun god, Phoebus Apollo, was supposed to have caused the plague of Thebes because it had been tainted by the misdeeds of Oedipus Rex. Apollo was supposed also to have rained plague arrows on the Greek army before Troy because their leader Agamemnon had abducted the daughter of Apollo抯 priest.

Although the nature of these various epidemics and their relationship to one another were unknown, the keen observer could not fail to notice that those who had once survived a disease might often be spared further involvement on its return. This phenomenon was described well by the historian Thucydides in his account of the plague of Athens of 430 B.C., when he wrote, 揧et it was with those who had recovered from the disease that the sick and the dying found most compassion. These knew what it was from experience, and had now no fear for themselves; for the same man was never attacked twice梟ever at least fatally.?Although this 損lague?was most probably not due to Pasteurella pestis, the plague of Justinian some thousand years later was more likely to have been bubonic plague, and of this the historian Procopius said, 揂t a later time it [the plague] came back; then those who dwelt round about this land, whom formerly it had afflicted most sorely, it did not touch at all.?In time, this resistance to reinfection came to be known by the term immunity, from the Latin immunitas, which in ancient Rome originally described the exemption of an individual from service or duty to the state.

The view of disease as originating from a vengeful deity contains within it an implicit theory of immunity. If disease be considered a punishment for sin, then being spared during a raging epidemic (i.e., natural immunity) would automatically be viewed as the inevitable result of having led a pious life. But a significant change occurred in early Christian times. Now not only did God punish the sins of humans with disease, but He might also employ disease to cleanse humans of their sins. If, then, disease was viewed as an expiation and purgative, then the recovery from a deadly plague would imply not only that the sins had been minor, but also that once cleansed of these sins the individual would not deserve further punishment when the plague returned (acquired immunity). Given the fervent religiosity of the times, such a concept of immunity may have been so ingrained as not to require explicit statement.

It was only during the last millennium that explicit theories of acquired immunity were advanced, most of them highly imaginative and each of them eminently consistent with the then-prevailing notion of disease pathogenesis. Because smallpox was one of the earliest diseases to be identified clinically, and because the lifelong immunity that it conferred could hardly escape notice, it is not surprising that most early theories of immunity would be couched in terms of this disease.

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Chapter 2 The History of Immunology

EARLY THEORIES OF ACQUIRED IMMUNITY

Expulsion Theories

The first clear clinical description of smallpox was given by the tenth century Islamic physician Rhazes. Not only did Rhazes differentiate smallpox from measles and other exanthematous diseases for the first time, but he also stated clearly that recovery from smallpox infection provides lasting immunity. To explain this phenomenon, he advanced the first explicit theory of acquired immunity that we have been able to find in the literature. Like all of his contemporaries, Rhazes followed the Hippocratic tradition, which held that disease was due to quantitative imbalances among the four humors (blood, phlegm, yellow bile, and black bile), to changes in their temperature and consistency, or even to their fermentation. Smallpox was believed to affect the blood, and Rhazes claimed that the disease was due to a fermentation of this humor, which helped to dispel the 揺xcess moisture?that he thought was present in the blood of the young. He proposed that the pustules that formed on the skin during this disease and broke to release fluid were the mechanism whereby the body expelled the excess moisture contained in the blood. He compared the maturation of an individual to the fermentation of wine from grape juice (must) and even suggested that smallpox disease itself might assist in this normal process! Thus he wrote:

I say then that every man, from the time of his birth till he arrives at old age, is continually tending to dryness; and for this reason the blood of children and infants is much moister than the blood of young men, and still more so than that of old men. . . . Now the smallpox arises when the blood putrefies and ferments, so that superfluous vapors are thrown out of it and it is changed from the blood of infants, which is like must, into the blood of young men, which is like wine perfectly ripened; as to the blood of old men, it may be compared to wine which has now lost its strength and is beginning to grow vapid and sour; and the smallpox itself may be compared to the fermentation and the hissing noise which takes place in must at that time. And this is the reason why children, especially males, rarely escape being seized with this disease, because it is impossible to prevent the blood抯 changing from this state into its second state, just as it is impossible to prevent must . . . from changing.

This curious theory appeared to explain well all that was known about smallpox: (a) Substantially everyone is affected, especially during youth (since then the blood is most moist); (b) the disease is seldom seen in adults and almost never in old age (because by then the normal aging process would have sufficiently dried the blood, so that it no longer could support the infection); and (c) a single infection would lead to lasting immunity, and recurrence of the disease would be impossible (since the initial attack would have expelled all of the 揺xcess moisture?that the theory required as a prerequisite for the disease process). It is interesting that Rhazes presented the smallpox of the tenth century as an almost benign childhood disease, and even as a salutory phenomenon that appeared to aid the normal development from infancy to adulthood.

During the eleventh century, Avicenna hinted at another interesting theory of acquired immunity, which was expanded on some 500 years later by the Italian physician Girolamo Fracastoro in his 1546 book On Contagion. Fracastoro claimed that all disease was caused by small seeds or germs (seminaria) which might spread from person to person, each of which possesses a specific affinity for a given plant or animal, and for a given organ or humor. He claimed that the germ of smallpox has an affinity for and caused the fermentation only of that trace of menstrual blood contaminant that he supposed tainted all mammalian young in utero. When a (young) person was infected, the menstrual contaminant would ferment, rise to the surface beneath the skin in the form of pustules, and be expelled when the pustules broke. He wrote,

This ebulition is a kind of purification of the blood . . . infection contracted by the child from the menstrual blood of the mother抯 womb is localized by means of this sort of ebulition and its putrefaction, and the blood is thus purified by a sort of crisis provided by nature. That is why almost all of us suffer from this malady, . . . and this fever is of itself seldom fatal [sic!], but is rather a purgation . . . the malady usually does not recur because the infection has already been secreted in the previous attack.

Thus Fracastoro抯 theory appeared also to explain satisfactorily all that was then known about smallpox. In this instance, acquired immunity would result from the expulsion during the first illness of the menstrual blood contaminant that he thought we all are born with, and without which the disease could not recur. But Fracastoro felt that his theory also implied a simultaneously acquired immunity to other exanthematous diseases, such as measles, and this was criticized by Heironymus Mercurialis, who made perhaps the earliest statement about immunologic specificity. Mercurialis pointed out that Fracastoro抯 theory could not be correct because, among other objections, measles and smallpox could not both be based on a menstrual blood contaminant, since infection with one should have cleansed the blood of the contaminant and conferred immunity to the other disease also; such 揷ross-immunity?was, Mercurialis noted, contrary to fact.

Several other variants of the menstrual blood expulsion theory should be noted, each proposing an analogous pathogenesis of smallpox and a comparable basis for acquired immunity. Thus, in place of menstrual blood contaminant, it has variously been proposed that amniotic fluid or umbilical blood is the culprit. In each instance, the disease was felt to involve the putrefaction of the contaminating substance, its expulsion via the pustules, and lifelong immunity due to the absence of further substrate upon which a new infection might act.

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Chapter 2 The History of Immunology

EARLY THEORIES OF ACQUIRED IMMUNITY

Depletion Theories

The introduction of variolation as a prophylactic measure (the inoculation of live virus from a diseased smallpox victim) led to renewed interest in the nature and in the mechanism of acquired immunity early in the eighteenth century. The inoculation of crusts derived from the pustules of 揻avorable?cases of smallpox had apparently been practiced widely in the folk medicine of many cultures in Asia and Africa and even in the rural areas of Western Europe. The practice may have originated among the Chinese, who advised the blowing of the infected matter into the nose of the recipient through a silver tube, the left nostril being used for males and the right one for females. Elsewhere, it was more customary to make a small incision in the skin and therein to insert the infected crust, or to run into the skin a thread that had been dipped in infected pustular fluid.

Although the practice of inoculation was inveighed against on both religious and medical grounds, it did attain a degree of acceptance, especially in England, thanks to the example set by the Prince and Princess of Wales in 1722 in permitting their children to be inoculated. Inoculation proved especially popular during periods of epidemic smallpox, when the mortality rate often reached 15% to 20% of those infected and the rate of disfigurement was even higher. In contrast, inoculation protected well against reinfection, most frequently involved no facial scarring, and was accompanied by at most a 2% to 3% death rate. The famous Voltaire observed the practice during his travels in England and waxed enthusiastic about its efficacy in his Lettres Philosophiques, crediting (probably erroneously) Lady Mary Wortley Montagu with its introduction into England. Voltaire speculated that inoculation might have originated with the Circassians to protect the beauty of their daughters, whom they could not sell pock-marked into the harems of the Ottoman Empire.

Thus it was that when the practice of inoculation was given currency in the pages of the Philosophical Transactions of the Royal Society, and especially after the prestigious demonstrations that attended the inoculation of the royal family, many were tempted to try the new procedure, and a few were led to speculate on its meaning. As early as 1721, the New England divine Cotton Mather convinced his friend Dr. Zabdiel Boylston to undertake the practice during an epidemic in Boston. Mather subsequently advanced a theory of acquired immunity in the following florid prose:

Whereas, the Miasms of the Small-Pox being admitted in the Way of Inoculation, their Approaches are made only by the Outerworks of the Citadel, and at a Considerable Distance from the Center of it. The Enemy [smallpox], tis true, getts in so far as to make Some Spoil, yea, so much as to satisfy him, and leaves no Prey in the Body of the Patient, for him ever afterwards to seize upon . . . tho?not without a Surrender of those Humours in the Blood, which the Invader makes a Seizure on, they oblige him to march out the same way he came in, and are sure of never being troubled with him any more.

Mather thus suggests that some unidentified substrate is depleted during either natural infection or following inoculation, its absence thenceforth inhibiting the development of the disease a second time.

It was in this context of three-quarters of a century of smallpox inoculation that Edward Jenner published in 1798 his epoch-making report on a safer and even more efficacious vaccine against smallpox, derived from cowpox pustules. The rapidity with which Jennerian vaccination (Latin vaccus, cow) swept the world is truly impressive; only a few years later, when Jenner interceded with the French enemy on behalf of an English prisoner, Napoleon remarked that he could refuse nothing to this great benefactor of humankind. Unfortunately, Jenner appears never to have speculated on why his vaccine caused immunity, perhaps influenced by the earlier advice of his eminent teacher John Hunter: 揥hy think? Why not try the experiment??

One of the more fanciful concepts of disease pathogenesis, and thus of disease immunity, that arose during the seventeenth and eighteenth centuries was that of the 搃nnate seed.?Humans (and animals) were held to be born with the seeds (ovula) for every different disease to which they were subject, each of which could be fertilized specifically by the appropriate contagious agent to produce the given disease. As Thomas Fuller so elegantly summarized the theory:

Because these Ovula are of distinct Kinds, . . . therefore the Pestilence can never breed the Small Pox, nor the Small Pox the Measles. . . . The Ovula always lie quiet and unprolific, till impregnated, and therefore these Distempers seldom come without Infection, which is as it were the Male, and the active Cause. The Ovula of each particular Fever, are all, and every individual one of them, usually impregnated at once. . . . And when these have been impregnated, and delivered of their morbid Foetus, there is an End of them; . . . Upon this Account no Man can possibly . . . be infected with any of the respective Distempers any more than once.

Thus Fuller not only recognized specific etiology, but also offered a plausible explanation of acquired immunity that is at once specific and also long-lasting.

This view that immunity was based on the depletion of some substance necessary to support the disease process was repeated often during the eighteenth century. Thus, one finds statements in the literature such as the following by M. Maty in 1755: 揑 lately tried this experiment [inoculation] upon myself, . . . and it had no effect upon my blood, as it had been sufficiently defecated 15 years before.?Again, a contemporary commentator, Angelo Gatti, compared susceptibility to smallpox with a body which a single spark might set afire, but which thenceforth has become 搃ncombustible?although surrounded by flames, and thus immune to further infection.

The 1870s saw increasing acceptance of the germ theory of disease and, thanks to the efforts of Louis Pasteur, Robert Koch, and others, the identification of the specific agents of many diseases and the elucidation of their modes of action. The newer concepts of disease pathogenesis, and especially Pasteur抯 demonstration in 1880 that acquired immunity could be induced against fowl cholera with an attenuated strain of organisms, overthrew all earlier concepts of the mechanism of immunity. Stimulated by his observations on immunity, and knowing something of the kinetics of bacterial growth in culture, the ever-imaginative Pasteur advanced his own explanation for acquired immunity, which held sway briefly. Pasteur recognized that bacterial growth in vitro rapidly terminates following its initial log phase multiplication and attributed this to the depletion of critical trace substances for which each bacterial species was thought to have specific requirements. At a time when the only known vaccines consisted of live attenuated organisms, Pasteur suggested that infection of the host either naturally or by immunization would rapidly deplete the body of those unique nutrients required for the continued growth of the infectious agent. Absent these necessary substances in the previously depleted host, it would be impossible to establish a second infection with the same agent, and acquired immunity would persist in the host so long as the substances were not renewed. The subsequent demonstrations by Theobald Smith that dead organisms would suffice for the vaccine, and by Emil von Behring and Shibasaburo Kitasato that even the supernatants from the culture growth of diphtheria or tetanus organisms would confer immunity, quickly demonstrated the inadequacy of Pasteur抯 depletion theory.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Preventive Immunization

The science of immunology was born in the laboratory of Louis Pasteur in the context of Pasteur抯 dedicated commitment to the germ theory of disease. Pasteur抯 earlier work on the agents responsible for certain diseases in the French silkworm and wine industries had convinced him that each disease is the reproducible result of an infection by a specific microorganism. Moreover, he held that not only does spontaneous generation not exist, but that these pathogenic agents are constant and specific in their ability to cause a given disease and cannot undergo transformation to yield some other disease picture. Then, in collaboration with Emile Roux, Pasteur discovered variations in the pathogenicity of different strains of a given organism, such that one strain might produce a much less severe disease than another. Pasteur and Roux devised techniques for the attenuation of cultures of virulent bacteria, working most notably with the organism responsible for the disease chicken cholera. By one of those happy instances of serendipity in science, it was discovered that chickens that had recovered from a mild attack of chicken cholera induced by an attenuated strain were thenceforth protected from challenge with more lethal strains.

This report in 1880 was the first generalization on Edward Jenner抯 use of cowpox vaccine to protect against smallpox, and it opened up an entirely new research program of prophylactic immunization. Pasteur was quick to seize upon these possibilities, as his subsequent work on anthrax, rabies, and other diseases amply testifies. Over the next quarter-century, as the specific pathogens of different diseases were reported with increasing frequency in the journals, scientists throughout the world endeavored to develop appropriate preventive vaccines, using Pasteurian approaches.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Cellular Immunity

The second significant step in the expansion of the immunological research program of the nineteenth century came in 1884 with Ilya Metchnikoff抯 cellular theory of immunity. Based upon purely Darwinian evolutionary principles, Metchnikoff suggested that the primitive intracellular digestive functions of lower animal forms had persisted in the capacity of the mobile phagocytes of metazoa and higher forms to ingest and digest foreign substances. Metchnikoff proposed that the phagocytic cell is the primary element in natural immunity (the first line of defense against infection) and is critical also for acquired immunity (the heightened protection conferred by preventive immunization or prior infection).

Metchnikoff抯 theory had several far-reaching consequences for biology and medicine. First, it introduced the notion that interspecific conflict might contribute as importantly to evolution as the classical Darwinian notion of intraspecific competition. Here, the struggle for survival was between the infected host and the offending pathogen, with the phagocyte entering the lists as champion of the former.

Another notable contribution of the phagocytic theory was to the field of general pathology. At the time, most believed that inflammation was a damaging component of the disease process itself; Metchnikoff, on the other hand, suggested that the inflammatory response was in fact an evolutionary mechanism designed to protect the organism. Whereas Metchnikoff抯 idea of the protective role of inflammation eventually triumphed, his cellular theory of immunity stimulated much opposition from those who claimed that humoral (blood-borne) factors were by far the more important. The debate between these two camps over the next two to three decades was fierce, as will be outlined below.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Serotherapy

A notable advance was achieved in 1888, when Emile Roux and Alexandre Yersin demonstrated that a soluble toxin could be isolated from the supernatants of cultures of the diphtheria organism. This toxin alone produced all of the symptoms of typical diphtheria in experimental animals, and it became evident that at least in some situations it was not the organism per se, but an exotoxin elaborated by it that caused the disease. It did not take long for von Behring and his colleague Kitasato to exploit this observation, with their reports in 1890 that animals immunized with diphtheria and tetanus toxins produced something in their blood that could neutralize or destroy the toxin, thus preventing disease. Antitoxic sera from experimental animals were quickly tested in infected children and were shown to produce remarkable and rapid cures, especially when administered during the early stages of the disease. The substance that acted against the toxin was called an antitoxin, and soon the more general and noncommittal term antibody was used to describe this new class of substances. The material responsible for generating these antibodies came to be known as the antigen.

von Behring抯 demonstration of antitoxic therapy took the medical world by storm, and the generality of the approach appeared to receive support from Paul Ehrlich抯 finding that neutralizing antitoxins could be formed by the immunization of animals with the plant toxins ricin and abrin. Here was a remarkable new addition to the medical armamentarium, and it offered great therapeutic promise in combating a variety of infectious diseases. The new so-called serotherapy stimulated an explosion of laboratory and clinical experimentation and of high expectation, in recognition of which von Behring received the first Nobel Prize in 1901.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Cytotoxic Antibodies and Autoimmunity

The fourth significant area that occupied early immunologists stemmed from the demonstration by Jules Bordet in 1899 that antibodies specific for erythrocytes could cause their destruction (hemolysis) in conjunction with the nonspecifically acting serum factor complement. Here was a clear explanation of one of the important mechanisms of protective immunity梩he direct destruction of bacterial pathogens through the cooperation of these two immunologic factors. But other far-reaching implications were seen in Bordet抯 observation. For the first time, the cells and tissues of the immunized host itself were seen possibly to be at risk by an 揳berrant?immune response against self components. With little delay, scientists in almost every active laboratory began to immunize experimental animals with suspensions or extracts of almost every tissue or organ in the body, in an attempt to find cytotoxic antibodies that might be responsible for one or another local disease. Soon the journals were filled with reports of such experiments, and indeed much of the 1900 issue of Annales de l扞nstitut Pasteur was devoted to this question. While it was quickly discovered that xenoantibodies and isoantibodies were often formed, and might be cytotoxic against the target tissue or organ, autoantibodies were, with few exceptions, rarely produced. This led Paul Ehrlich to formulate his famous dictum of horror autotoxicus, which held that, for reasons unknown, an individual is unable to mount a destructive immune response against self-constituents. Nevertheless, for years thereafter, the possibility was seriously entertained that such cytotoxic antibodies might play an important role in the pathogenesis of a number of diseases, both as pure autoimmune phenomena and as secondary contributors to the lesions seen in such diseases as syphilis and sympathetic ophthalmia. Indeed, Donath and Landsteiner reported in 1904 on the first observation of a true autoimmune disease梡aroxysmal cold hemoglobinuria.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Serology and Immunodiagnosis

The discovery in 1896 of the phenomenon of bacterial agglutination was quickly recognized to provide a powerful tool for the bacteriologist. Not only could bacteria be identified and differentiated with the use of appropriate antisera, but the serum of patients could be tested for their ability to agglutinate a given organism, thus testing for prior exposure to that organism and for the degree of immunity against infection that the individual might possess. The discovery of the precipitin reaction extended this approach even further, now to include the assay of antigens and antibodies in systems involving bacterial products, or even nonbacterial agents. This technique was nowhere more elegantly applied than by G. H. F. Nuttall, who showed by the reactions and cross-reactions of antisera prepared against animal and plant proteins that immunology might be usefully applied to the study of taxonomic relationships, and even for forensic purposes.

When Bordet showed that immune hemolysis might be mediated by antierythrocyte antibodies and complement, and that the components of the reaction could be titrated precisely, he opened the door to a new approach to the diagnosis of disease. Now the blood of patients could be examined for the presence of even those antibodies that do not agglutinate or precipitate their respective antigens, but do fix complement. Thus, not only could prior exposure to a pathogen be assessed, but the course of a disease might even be followed serologically. This approach was brilliantly exploited by August von Wassermann and his colleagues in the development of a serodiagnostic complement fixation test for syphilis, and soon thereafter many other adaptations of complement fixation were proposed for the qualitative and quantitative analysis of both antibodies and antigens.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Allergy and Immunopathology

A seminal discovery in the history of immunology was made in 1902 by physiologists Paul Portier and Charles Richet. Until that time, the immune response had been viewed as a purely benign set of mechanisms whose only function was to protect the organism against exogenous pathogens; the work of those searching for cytotoxic antibodies had done little to alter this view. It had been found only a few years earlier that an immune response could be stimulated by other than bacterial antigens and toxins. Now came Portier and Richet to demonstrate that even bland substances could, when injected into presensitized individuals, cause severe systemic shocklike symptoms and even death. They termed this phenomenon anaphylaxis, in an attempt to distinguish it from the usual prophylactic results expected of the immune system. Shortly thereafter, Maurice Arthus demonstrated that bland antigens could cause local necrotizing lesions when they react with specific antibody in the skin of test animals, the so-called Arthus phenomenon. Then, in 1906 Clemens von Pirquet and Bela Schick demonstrated that the pathogenesis of so-called serum sickness depends upon an antibody response by the host to the injection of large quantities of foreign protein antigens, such as accompanied the administration of horse antidiphtheria toxin according to von Behring抯 serotherapeutic doctrine. It could not be argued that these were only artificial laboratory phenomena; soon thereafter it was demonstrated that two of the significant curses of humankind梙ayfever and asthma梐lso belong to this same group of specific antibody-mediated diseases.

It is interesting to consider the ambivalence with which early immunology viewed the many manifestations of allergy or hypersensitivity. Here was a system, presumably evolved for defensive functions, that somehow 搘ent astray?to produce a variety of pathological conditions. This teleologic view of immunity was so deeply ingrained that for over half a century the mechanisms of allergy were treated as quite separate from those of immunity. Only with an increased understanding of the immunologic contributions to the pathogenesis of such diseases as tuberculosis and leprosy, and the development of experimental models of such diseases as Masugi nephritis, experimental allergic encephalomyelitis, and lymphocytic choriomeningitis, was immunopathology incorporated into the broader context of immunologic phenomena.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Allergy and Immunopathology

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Immunohematology

The initial demonstration in the late 1890s that antibodies directed against erythrocytes might mediate their agglutination and hemolysis early focused attention on these cells as antigens, and many studies were undertaken to immunize animals with erythrocytes, both within and across species lines. It was soon found that many animal sera contained 搉atural?isoantibodies capable of agglutinating the erythrocytes of certain other members of the same species. In a series of studies starting in 1901, and for which he gained the Nobel Prize, Karl Landsteiner showed that humans could be divided into several groups, depending on the presence in their sera of agglutinins specific for the erythrocytes of other humans. These groupings were quite distinct and served as the basis for the ABO system of blood types, later shown to be genetically determined by three allelic genes. The theoretical importance of this observation on genetically determined antigenic polymorphism was soon overshadowed by its clinical implications, since it permitted the beginning of a rational approach to blood typing and modern blood transfusion techniques. Landsteiner followed up this observation in the 1920s by discovering, with Philip Levine, the M, N, and P blood groups, and in 1940 he and Alexander Wiener discovered the rhesus factor, important in both blood transfusions and as the principal contributor to the transplacental disease of the newborn, erythroblastosis fetalis. Since that time, many other minor erythrocyte antigens have been identified, and immunohematology has contributed significantly to theoretical immunology, to forensic medicine, and to anthropologic studies of racial relationships and mass migrations.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Ehrlich抯 Side-Chain Theory

The term antibody (antik鰎per) was originally a noncommittal one, employed to designate whatever it was in immune serum that had the capacity to neutralize toxins and pathogenic bacteria. With the demonstration of the ability to transfer immunity passively by means of serum, it quickly became apparent that antibody must be a discrete substance somehow formed within the immune host, and thus the mechanism of its formation became a valid topic for speculation and study. At the outset, the plausible explanation was advanced that antigen itself carried the information for antibody specificity by somehow being incorporated into the antibody molecule in such a manner that it would thenceforth react specifically with other similar molecules of antigen. Such a theory could not long survive the early quantitative studies that showed that much more antibody was formed than could be accounted for by the quantity of antigen injected, and that antibody formation would continue, once started, without further administration of antigen. It remained for Paul Ehrlich to propose a comprehensive theory of antibody formation in 1897, which he first appended to his epoch-making study on the measurement of diphtheria toxin and antitoxin, and which was later elaborated in great detail by Ehrlich and his students.

Ehrlich believed that antibodies are macromolecules whose specificity for antigen and even complement depends upon the presence of certain sterochemical configurations, whose complementarity with analogous structures on the antigen permits specific interaction. He suggested that these antibodies are naturally occurring products of the body that function as specific receptors on the surface membrane of the cell, there to fulfill normal physiologic functions similar to those served by hypothetical food receptors in digestion (or by drug receptors, in his later theories of chemotherapy). Ehrlich postulated that these antibody receptors would be selected for specifically by an appropriate antigen, leading to their loss from the surface and thereby stimulating a compensatory overproduction of receptors that would appear in the blood as circulating antibody. Ehrlich抯 imaginative theory held great sway for many years and, especially in Germany, influenced conceptual thought in many different fields of medicine. Few were troubled at the time by any hint that the potential size of the immunologic repertoire of antigens and antibodies presented any problem, since the only antibodies known in the mid-1890s were thought to be antitoxins directed against a rather limited number of human and animal pathogens. But two changes occurred in immunology over the succeeding decades to cast doubt on Ehrlich抯 theory. The first of these was the flood of studies that showed that antibodies could be produced against a wide variety of even benign, naturally occurring animal and plant substances, including many to which the host would normally never be exposed. Then, in the second decade of the twentieth century came the observation of F. Obermeyer and E. P. Pick, greatly extended by Karl Landsteiner, that showed that antibodies could be formed against almost any artificial chemical capable of being coupled as a hapten onto a protein carrier. Thus, it began to appear unreasonable that an individual could make specific antibodies spontaneously against so great a number of foreign and even artificial structures.

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

The Cellular Versus Humoral Controversy

Historians of science recognize that very often the eras that mark the most significant advances in a field are those characterized by disputes between two opposing schools, in which each is stimulated to devise experiments designed to uphold its own position and to challenge the opposing view. Such conflicts arose during the early days of immunology, involving the nature of the antigen朼ntibody interaction and the mode of action of complement, and these provided notable stimuli for the rapid advance of immunologic knowledge. But perhaps no dispute lasted so long and had such important consequences for the future development of immunology as did that between the proponents of a cellular theory of immunity and those who argued that all immunity was based on the action of humoral elements. This immunologic dispute was, however, not an isolated event; it was rather part of a larger conceptual revolution in medicine in the nineteenth century that concerned the very basis of normal and abnormal physiologic processes. For more than 2,000 years, medicine had been under the domination of the Greek humoralist view that disease was based on quantitative or qualitative imbalances in the essential bodily humors. Only in the nineteenth century was the importance of the cells that comprise the various tissues appreciated, and Virchow抯 cellular pathology (which held that disease is based on abnormal cellular function) was scarcely 30 years old when immunologists chose sides in their own modification of this larger conflict.

It was the zoologist Ilya Metchnikoff who first suggested clearly in 1884 that leukocytes might play an important role in the body抯 defense against infectious diseases, by virtue of their phagocytic capabilities. Metchnikoff based his thesis on observations that even marine invertebrates possess macrophages capable of ingesting and destroying foreign substances or invading bacteria, or at least of walling them off by the formation of giant cells and granulomatous reactions. Metchnikoff suggested that the vertebrate phagocytic cells perform a similar protective function and in fact are the most important contributors to both natural and acquired immunity. This work impressed Pasteur, who invited Metchnikoff to join him at the newly constructed Pasteur Institute in Paris, where Metchnikoff and a succession of distinguished students spent the next decades working productively and imaginatively to verify and extend the cellular (phagocytic) theory of immunity.

Metchnikoff抯 cellular theory quickly excited opposition. In the first place, it was advanced at a time when most pathologists considered the inflammatory reaction, and the microphages and macrophages that accompanied it, to be deleterious rather than protective responses. It was even thought at the time that although phagocytic cells might indeed ingest infectious organisms, the result was not the destruction of these agents but rather their transport throughout the body to cause dissemination of the disease. Then, in 1888 Nuttall observed that the serum of normal animals contains substances that are naturally toxic for certain microorganisms and that these antibacterial properties are much enhanced in the immunized host. Then Koch抯 student Richard Pfeiffer described the phenomenon that bears his name, in which circulating antibody (even that passively transferred to a normal recipient) would cause the specific lysis of cholera vibrios injected into the peritoneal cavity of immune guinea pigs. Two different humoral substances were found to cooperate in bacterial lysis: (a) heat-stable serum antibody and (b) the thermolabile factor called complement, or alexin (Greek aleksein, to defend).

But perhaps the most telling blow to the cellular theory of immunity came with the description by von Behring and Kitasato in 1890 of immunity to diphtheria and tetanus, clearly mediated by circulating antibody rather than by phagocytic cells. As time went on, circulating antibodies were found for most of the new pathogenic organisms that were rapidly being discovered, and Paul Ehrlich not only showed in the diphtheria antitoxin system how these antibodies might be measured, but also published pictures of them that made readers feel that they understood what an antibody was and how it acted. Finally, when Metchnikoff抯 own student Bordet described the lysis of erythrocytes by humoral antibody and complement, most investigators were tempted to agree with Koch that the humoralists had carried the day.

Metchnikoff and his students were by no means silent in the face of these strong attacks against the phagocytic theory. In paper after paper, these workers showed that there is often no relationship between the bactericidal powers of the blood and host resistance to infection against a given organism. Rather, species resistance can often be directly correlated with the ability of its phagocytes to ingest the pathogen, as in the case of anthrax, and ingenious experiments were devised to show that microorganisms enclosed within little sacks of filter paper that protected them from phagocytes would remain virulent, although bathed in antibody-containing tissue fluids. Metchnikoff also showed that the creation of a macrophage-rich peritoneal exudate, with the attendant activation of these macrophages, would protect the host against intraperitoneal injection of otherwise lethal doses of different bacterial pathogens, an early forerunner of the modern practice of nonspecific immunotherapy. But the tide had obviously turned against the phagocytic theory during the 1890s, and Metchnikoff抯 last-ditch attempt to reestablish the importance of phagocytosis with the publication in 1901 of his famous book, Immunity in the Infectious Diseases, came too late. The book was widely admired for its scholarship, but made few converts among the unbelievers.

If we analyze the immunologic literature of the first decade of the twentieth century, it becomes apparent that by their choice of subjects, most investigators had voted for the humoral theory of immunity and against the cellular theory. Except for Metchnikoff and his immediate adherents, most chose to study antibodies, which were more easily measured and worked with, and few devoted themselves to the more difficultly manipulable cells. Even so, two attempts were made during this period to mediate the cellular杊umoral dispute. In 1908, the Swedish Academy conferred the Nobel Prize in Medicine jointly to Metchnikoff, the champion of cellularism, and to Ehrlich, the then-leading exponent of humoralist doctrines. In England somewhat earlier, Sir Almroth Wright and S. R. Douglas attempted to rationalize the differences between these two schools by their extensive work on the process of opsonization (Greek opsonein, to render palatable). These investigators claimed that both humoral and cellular factors were equally important and interdependent, in that humoral antibody appears to interact specifically with its target microorganism to render it more susceptible to phagocytosis by macrophages.

Wright抯 espousal of this doctrine became so popular in England that his friend Bernard Shaw used it as the subject of his play The Doctor抯 Dilemma. In his Preface on Doctors, Shaw summarized Wright抯 approach in an otherwise scathing castigation of the medical profession:

Sir Almroth Wright, following up one of Metchnikoff抯 most suggestive biological romances, discovered that the white corpuscles or phagocytes, which attack and devour disease germs for us, do their work only when we butter the disease germs appetizingly for them with a natural sauce which Sir Almroth named opsonin.

But partly because his techniques were so difficult to perform and their results difficult to reproduce, Wright抯 opsonic indices and therapeutic approaches soon fell out of favor, and his efforts to revivify the cellular theory of immunity had little long-lasting effect.

The fact that the humoral theory of immunity carried the day over the cellular theory around the turn of the century had long-term implications for future developments in the young discipline of immunology. It is generally the case in science that the most imaginative and productive investigators tend to choose their problems based on what they (or their teachers) feel is most significant in their field, and during the early decades of the twentieth century it was clear to most workers that antibody held the key to an understanding of immunity. In the humoralist context of the times, many approachable problems in cellular immunology were thus neglected as being 搖ninteresting.?

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Chapter 2 The History of Immunology

THE ORIGINS AND RESEARCH PROGRAM OF EARLY IMMUNOLOGY

Transplantation and Immunogenetics

Transplantation research occupies a curious place in the history of immunology. Pursued since the turn of the century, it produced throughout its course valuable information of great potential significance to immunology. However, those who worked in the area were not immunologists but surgeons, oncologists, biologists, and geneticists with little connection to contemporary immunology, and thus the significance of their results generally went unrecognized by mainstream immunology for over 50 years.

Surgeons have dreamed of replacing missing or defective tissues and organs since the Middle Ages, and the miracle of the transplantation of a leg by Saints Cosmas and Damian was often celebrated by Renaissance painters. But through the centuries, all attempts failed, save for the occasional success with corneal grafts. Then, at the end of the nineteenth century, it was shown that tumors could be passaged in experimental animals. But these grafts usually failed梩he recipients were held to be 搃mmune敆and tumor biologists saw in this phenomenon an approach to the solution of the problem of human cancer. If they could unravel the secrets of tumor graft rejection, then perhaps humans could be induced to reject their own tumors as well. Thus, at the turn of the century, a massive effort began to study the rejection of tumor grafts. These studies generally employed normal tissues such as skin as a control, to establish that it was the host and not some peculiarity of cancer tissues that accounted for rejection.

In not much more than a decade, the general rules of graft rejection had been worked out and were summarized in 1912 in a remarkable book, Heteroplastic and Homoplastic Transplantation, by Georg Sch鰊e: (a) Transplantation into a foreign species (heteroplastic = xenogeneic) invariably fails; (b) grafts to unrelated members of the same species (homoplastic = allogeneic) usually fail; (c) autografts almost invariably succeed; (d) in an allogeneic recipient, there is a primary take and delayed rejection of a first graft; (e) rejection of a second graft from the same donor is accelerated, as is that in a recipient preimmunized with other donor material; (f) the closer the 揵lood relationship?between donor and recipient, the more likely is successful transplantation; and (g) these rules apply to normal as well as tumor tissues. There is no question that Sch鰊e viewed rejection as an active response on the part of the host抯 immune system; indeed, he coined the term transplantation immunity.

These early findings were reconfirmed and extended in a 1916 review, Tumor Immunity, by E. E. Tyzzer, who pointed out that studies with inbred mice showed the genetic nature of donor杛ecipient incompatibility, and that this was not inherited as 揳 single Mendelizing factor.?Moreover, morphologic data showed that the inflammatory infiltrate was predominantly lymphocytic, and not merely exudative, but proliferative as well. James Murphy, who worked initially with Peyton Rous, wrote a monograph in 1926朤he Lymphocyte in Resistance to Tissue Grafting, Malignant Disease, and Tuberculous Infection. Murphy showed that immune rejection did not function in embryos; that while a tumor might grow in a 損rivileged site?like the brain, cotransplantation of lymphoid tissue would induce rejection even there; and that X-irradiation would inhibit the rejection process.

It was a similar interest in the tumor problem that stimulated Clarence C. Little to found the Jackson Laboratories in Bar Harbor, Maine, devoted to genetic studies of tumor transplantation. Here, in the 1930s and 1940s, George Snell 搃nvented?the congenic mouse and helped to define the major histocompatibility complex (MHC), based originally on the demonstration by Peter Gorer that graft rejection in mice was accompanied by the production of antibodies specific for an (erythrocyte) antigen, which he labeled antigen-II. But while much was learned about the immunology of tissue graft rejection in all of these studies, it appeared increasingly to have little to offer toward a solution of the problem of human tumors. It is interesting that all of this immunologic activity on the part of the surgeons, tumor biologists, and geneticists went substantially unnoticed by the immunologists of the day. In this era of chemically oriented activity in immunology, investigators were little interested in the more biological aspects of autoimmunity and transplantation, but worked primarily on antibody and antigen chemistry, on the specificity of serologic reactions, and on quantitative immunochemistry.

Of the three groups that had worked on the problem of transplantation, the surgeons left the field first, due to the apparent impracticability of tissue and organ grafting in the human. Similarly, the tumor biologists found no way to apply the knowledge derived from animal experiments to humans, and they too moved on to other aspects of the cancer problem. The shift in interest of the geneticists was more subtle. At the outset, they had viewed the inbred mouse as the perfect tool to study human tumor susceptibility and, ultimately, therapeutic approaches. But if the early work to identify the histocompatibility loci in mice was aimed at understanding the immune response to tumor grafts, it quickly lost its oncologic and even immunologic motivations and became a study in pure genetics. They now devoted themselves to establishing the size and polymorphism of the MHC and the rules of segregation. Only later, when their practical applicability to tissue typing and histocompatibility matching was established, would the investigations return to the realm of transplantation biology.

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Chapter 2 The History of Immunology

IMMUNOLOGY IN TRANSITION, 1912?950s

The Fate of the Early Immunological Research Program

We have seen that during the period 1880 to about 1910, the young and highly productive field of immunology had organized itself predominantly in terms of a number of major areas of interest. By the beginning of the First World War, while most of its practitioners might not yet have called themselves 搃mmunologists,?institutionalization of the discipline had begun in earnest. An institute devoted to its aims had been established for Paul Ehrlich in Frankfurt, and departments and services dedicated to the discipline had been formed within many of the leading research institutions around the world. Sections devoted to one or another component of the immunologic program were to be found at International Congresses of Medicine or Hygiene, and an 搃nvisible college?existed, involving informal exchange among its practitioners. While the pages of the Annales de l扞nstitut Pasteur had long been devoted to immunological reports, the discipline was more formally recognized by the founding in 1908 of the Zeitschrift f黵 Immunit鋞sforschung and of the American Journal of Immunology in 1916. The commonality of interest of this subgroup of scientists and practitioners was recognized, at least in America, by the founding of the American Association of Immunologists in 1913.

Let us now look at developments within each of the components that composed the early immunological research program. Preventive immunization had seen its great victories in the case of chicken cholera, anthrax, rabies, plague, and several other important diseases. But increasingly, pathogenic organisms were being described for which it was proving impossible to prepare efficacious vaccines. These included not only such important agents as the tubercle and leprous bacilli, the cholera vibrio, and the spirochete of syphilis, but also the important group of disease-producing gram-positive organisms, to say nothing of a number of newly described diseases due to viruses and parasites that so ravaged humans and animals. Thus, by 1910 the great early promise of pasteurian immunization was no longer being fulfilled; new successes would thenceforth be few and far between and achieved only with great difficulty. Work in this area very rapidly left the 揷lassical?immunology laboratory and was taken over by bacteriologists, virologists, and parasitologists interested more in organisms than in immunologic mechanisms.

The study of cellular immunology and of Metchnikoff抯 phagocytic theory, as we have seen, went into decline early in the century at the hands of proponents of humoralist theories. Cells were much more difficult to work with than were humoral antibodies, and no such antibody techniques as agglutination, the antigen朼ntibody precipitin reaction, immune hemolysis, and the ability to transfer antibody passively from one animal to another existed in the field of cell studies. Indeed, the cell was still considered something of a mystery, whereas Ehrlich抯 pictures of antibodies and their specific combining sites could almost convince one that the antibody was a 搑eal?entity whose structure and properties were readily understood.

The techniques of serotherapy for the prevention or cure of disease suffered a fate similar to that of preventive immunization. After the remarkable demonstration of the efficacy of horse antidiphtheria and antitetanus sera in the treatment of these diseases, no significant further victories were recorded in this area. While laboratories throughout the world continued to produce these two antisera (the Pasteur Institute helped support itself with its stable of immunized horses), interest in this approach waned, since there were so few other significant diseases that were caused by exotoxins and thus amenable to this approach. When, much later, passive transfer of antibody would be employed, it would be by hematologists using human gamma-globulin to prevent erythroblastosis fetalis or by pediatricians employing convalescent sera to deal with poliomyelitis.

As for the interest in cytotoxic antibodies and autoimmunity, this proved to be ephemeral. Despite all attempts to implicate antitissue and antiorgan antibodies in the pathogenesis of disease, with the exception of antierythrocyte antibodies responsible for hemolytic anemias, no convincing demonstrations were forthcoming, and immunologists even forgot about Donath and Landsteiner抯 demonstration of the pathogenesis of paroxysmal cold hemoglobinuria as the possible tip of an autoimmune disease iceberg. By 1912, the study of immune cytotoxic phenomena had left the immunology laboratory to be pursued only within essentially unrelated clinical specialty areas such as ophthalmology, with its interest in sympathic ophthalmia and autoimmune disease of the lens.

Developments within the area of serodiagnosis represent a more typical example of disciplinary differentiation for the sociologist of science. These techniques had developed within the very heart of an immunologic enterprise interested in immunity in the infectious diseases, which therefore not only demanded an understanding of disease pathogenesis, but also required the ability to diagnose these diseases. Syphilis remained the mainstay of serodiagnostic laboratories, and work to perfect the technique and extend it to other diseases continued throughout the period under discussion. But very quickly, the technique became quite routine and applied, and immunologists interested in basic mechanisms soon lost interest in the area. Work in this field was taken over by classical bacteriologists, and in fact those who devoted themselves to this and other aspects of serodiagnosis soon began to call themselves 搒erologists?and worked principally in diagnostic laboratories rather than in those devoted to basic immunologic research.

Soon after their discovery, anaphylaxis and its related diseases had also become an intimate concern of immunologic experimentalists. They were interested in the nature of the antibodies responsible for these phenomena and in the basic mechanisms involved in the diseases that resulted from their action. But after a short and essentially unsuccessful struggle with the paradox of a system presumably evolved to protect now being demonstrated to cause disease, the immunologists soon deserted the field to others. In the main, those upon whom these interests devolved were clinicians interested in hayfever and asthma. Clinical allergy was established as a medical subspeciality, and it was primarily in the laboratories of allergists that further progress was realized in sorting out the mechanisms involved and in developing skin tests and therapeutic approaches to the treatment of human allergies. In addition to these, however, the study of anaphylactic and related phenomena was of great interest to physiologists such as Sir Henry Dale, who was interested in the physiologic mechanisms involved in such diseases, and also to a large group of experimental pathologists, who were interested in the comparative study of the lesions that accompanied these diseases.

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Chapter 2 The History of Immunology

IMMUNOLOGY IN TRANSITION, 1912?950s

The Rise to Dominance of Immunochemistry

Thus, the immunologic research program waned in all of its interest areas, so far as the basic scientist was concerned, and several of these areas devolved upon others. Now we shall see how the general field itself experienced a devolution into the hands of a new Denkkollektiv. The seeds of the future interest in the chemistry of antigens and antibodies can be traced back to the fertile imagination of Paul Ehrlich. Ehrlich抯 side-chain theory of antibody formation pictured antigen, antibody, and complement as true chemical molecules, and their combining sites as stereochemically complementary structures that would account for the specificity of their interactions. At the time, however, little was known of the structure and precise composition of protein molecules, and appropriate techniques were unavailable to translate Ehrlich抯 theory into laboratory experiments.

It is common in most textbooks to ascribe the paternity of the field of immunochemistry to the famous physical chemist Svante Arrhenius, because he coined the term immunochemistry in a series of lectures with that title in 1904. Like many another physical scientist, Arrhenius was attracted by the mysteries and the confusion that existed in biology and felt that he could bring some order to the chaos by the introduction of the rigorous laws of chemistry and physics. Through his Danish colleague Madsen, Arrhenius became interested in the problem of diphtheria toxin-antitoxin titration and proposed that these interactions are reversible, like the interactions that he had described for weak acids and weak bases that had contributed so much to his earlier fame. But it would probably be erroneous to attribute the fatherhood of the field to Arrhenius, because his contributions were purely theoretical, could not be adequately tested at the time, and had little immediate influence on subsequent events.

Perhaps the true turning point came in 1906, with the demonstration by Obermeyer and Pick that protein antigens could be modified chemically to alter their immunological specificity. For example, when nitrated proteins were employed to immunize animals, the specificity of the resulting antibodies appeared to be directed no longer at the original protein, but at the added nitro groups. In an encyclopedic review of this area in 1912, Pick showed that a number of different synthetic groupings (called haptens) might be joined to a carrier protein to serve as antigenic determinants. Here was a powerful new tool, with which the small molecules produced in the organic chemistry laboratory could be used to dissect intimately the nature of immunologic specificity and the character of the combining site on antibody. No one exploited this approach more assiduously or to better effect than polymath Karl Landsteiner, who in 1917 published two papers that illustrated the power of this approach and that helped to define both his own work during the next 37 years and much of the domain of immunochemistry as well. Now the biological basis of antibody formation and the biological effects of antigen朼ntibody interactions took a back seat to interest in the chemical nature of antigens and antibodies.

Another approach to the chemistry of antigens and antibodies was opened up in the 1920s by organic chemist Michael Heidelberger. Working initially in the context of a bacteriological laboratory, Heidelberger was able to show that, contrary to the classical view that antibodies could only be formed against protein antigens, the capsular polysaccharides of the pneumococcus could also stimulate a specific antibody response. This led Heidelberger to study the chemical differences among the polysaccharide antigens of different strains of pneumococcus, in pursuit of which he developed over many years an impressive set of quantitative techniques that helped establish immunology as a more exact science.

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Chapter 2 The History of Immunology

IMMUNOLOGY IN TRANSITION, 1912?950s

Theory Follows Mindset: Instruction Theories

We saw above that during the early biomedical era of immunology, the first theory of immunity advanced by Ilya Metchnikoff, trained in zoology, followed strict Darwinian evolutionary principles, and the first theory of antibody formation proposed by Paul Ehrlich, trained in medicine, was similarly based. But the chemically oriented investigators who dominated immunology after the First World War had little interest in the biological basis of immunity. They were, however, interested in antibodies and their formation, and new theories of antibody formation were not slow to appear. These new theories no longer focused on the function of antibodies, but on their chemical structure, and more specifically on the question of how such a large group of specific molecules able to interact with an ever-growing universe of potential antigens could possibly be produced within the vertebrate host. This was the rock upon which Ehrlich抯 side-chain theory had foundered: the improbability that evolution could have accounted for the spontaneous production of so many different antibodies, the greater portion of which were directed against bland and even artificial antigens of no obvious evolutionary selective force.

It is not surprising, therefore, that the new chemical theories of antibody formation were quite Lamarckian in nature; in contrast to the molecules of the biologist, those of the chemist generally have no evolutionary history. The first of the new theories to be advanced was that of biochemist Felix Haurowitz, in 1930. In this, it was proposed that only the antigen itself contains all of the information necessary for antibody formation and imposes a complementary structure on a nascent protein by acting as a template for the synthesis of a unique sequence of amino acids. This was the first so-called instruction theory of antibody formation. Here was a ready explanation not only for the tremendous diversity of different antibodies, but also for how so fine a specificity could be imparted to the antibody molecule. This instructive theory of antibody formation was further refined in 1940 by chemical physicist Linus Pauling, who proposed that the antigen serves as a template upon which the nascent amino acid chain coils to form a protein molecule.

But these chemical theories did not explain, to the satisfaction of the biologist, how antibody production might persist in the apparent absence of antigen, nor did they attempt to explain why a second exposure to the same antigen should result in an enhanced booster response. Moreover, these theories provided no explanation for newer data that showed that repeated immunization might produce changes in the quality of the antibody, in some instances sharpening specificity and in other instances considerably broadening the potential for serologic cross-reactions. It was the biological shortcomings of the direct template theories that disturbed virologist Macfarlane Burnet and caused him to advance an instructionist alternative in 1941. With the growing recognition of the importance of enzymes in synthesis as well as in digestion, Burnet suggested that the function of antigen might be to stimulate an adaptive modification of those enzymes necessary for globulin synthesis, such that a unique protein molecule with the required specificity would result. This adaptive enzyme theory had the advantage of explaining not only the large repertoire in terms of initial instruction by antigen, but also the persistence of antibody formation and the booster antibody response, by allowing replication of the adaptive enzymes within an expanding population of proliferating daughter cells, all capable of antibody formation. This latter point is especially noteworthy, since Burnet was perhaps the first to stress the important role of continuing cellular function and of cell replication in antibody production.

With an increasing understanding of the probable genetic role of nucleic acids, Burnet and Frank Fenner advanced a modification of this theory in 1949, still impelled by essentially biological considerations. They now suggested that antigen might impress the information for its specific determinant directly on the (?RNA) genome, against which indirect template a specific antibody might be formed. Not only would this new genocopy persist within the cell, but it would also be reproduced from mother to daughter cells during proliferation, thus explaining persisting antibody formation and a heightened booster response. It is interesting that so ingrained in the collective immunological psyche of the times were these chemical ideas that even biologist Burnet, in his first two theories of antibody formation, felt obliged to employ Lamarckian instructive approaches.

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Chapter 2 The History of Immunology

IMMUNOLOGY IN TRANSITION, 1912?950s

The Scope and Influence of the Immunochemical Research Program

The application of synthetic haptens to the study of antibody specificity led to progress in clarifying the size and structure of antigen and antibody combining sites and in defining the thermodynamic parameters of their interaction. These studies were facilitated by the development of quantitative techniques for the measurement of these reactions and by the identification of antibody as a gamma-globulin protein, paving the way for the development of chemical purification methods.

The scope of the field of immunology from the 1920s to the early 1960s is perhaps best epitomized by five of the leading books of the period: Well抯 The Chemical Aspects of Immunity in 1924; Marrack抯 The Chemistry of Antigens and Antibodies in 1934; Landsteiner抯 The Specificity of Serological Reactions in 1937; Boyd抯 Fundamentals of Immunology in 1943; and Kabat and Mayer抯 Quantitative Immunochemistry in 1949. These were the reference books from which a generation of young immunologists learned their trade, and little attention was paid in any of them to the biological or medical aspects of the field. If a Max Theiler developed a new vaccine in the mid-1930s against yellow fever, this was of interest only to virologists and students of infectious diseases. If a Hans Zinsser or an Arnold Rich studied allergic reactions to bacteria, or if a Louis Dienes or a Simon and Rackemann developed models of delayed hypersensitivity lesions to simple proteins in the 1920s and 1930s, this was interest only to bacteriologists and experimental pathologists. Finally, if a Thomas Rivers developed an experimental model of allergic encephalomyelitis as early as 1933, this seemed to excite no one at the time. These and other similar excursions into areas of biomedical interest lay out of the mainstream of contemporary immunology, were usually published in 搊utside?journals, and made little impression upon the governing Denkkollektiv. Only a later generation of immunologists more attuned to biological questions would identify these contributions as landmarks in immunological progress.

This is not to suggest that all work along the classical lines described above ceased during the immunochemical era. It has been pointed out that, ? . . research areas which have become well established take a long time to die out altogether. There is always some work that can be done.?Thus, as described above, the clinical allergists gave new life to the study of anaphylactic phenomena by redefining the field along new lines; continued progress was made in the preparation of better toxoids and better modes of immunization; serologists continued to improve and expand the application of serodiagnostic procedures; and from time to time, an effective vaccine would be developed against one or another disease of humans or animals.

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Chapter 2 The History of Immunology

THE IMMUNOBIOLOGICAL REVOLUTION

The research program that governed the normative science of the immunochemical era between the 1920s and 1950s produced interesting results. It had gone far to define the chemical nature of both antigens and antibodies and the precision of their specific interactions. Increasingly, however, biologists working on the fringes of immunology made observations whose explanation was not to be found in the received wisdom of instructionist theories of antibody formation. How, they asked, could antibody formation persist in the apparent absence of antigen? Why should a second exposure to antigen result in an enhanced booster response that is much more productive than is the primary response to antigenic stimulus? How can repeated exposure to antigen change the very quality of the antibody, in many instances sharpening its specificity by increasing its affinity for the antigenic determinant employed? Finally, how is it possible that immunity to some viral diseases appears to be unrelated to the presence of circulating antiviral antibodies? These and other biologically based questions began seriously to challenge the immunochemical paradigm, most notably through the pen of Macfarlane Burnet in his two books entitled The Production of Antibodies (1941 and 1949). Burnet complained repeatedly that the chemical theories, while quite elegant, failed to explain the more functional biological aspects of the immune response.

By the 1950s, the stage seemed to be set for a large-scale confrontation such as described by Thomas Kuhn in his book The Structure of Scientific Revolutions. On the one hand was the immunochemical tradition, guided by theories that could no longer satisfactorily explain all of the phenomena of the field, and employing approaches that yielded results of increasingly parochial interest and of decreasing marginal value. Challenging this classical tradition was a growing group of biomedical scientists seeking answers to a set of new and important questions that traditional immunochemical theory and practice were ill prepared to answer.

In the 1940s, Peter Medawar 搑ediscovered?the laws of transplantation, demonstrating that the rejection of tissue transplants was a purely immunologic phenomenon, but one unrelated to humoral antibody. In 1945, Ray Owen described the paradoxical situation of dizygotic twin calves that were incapable of responding to one another抯 antigens. The explanation of this phenomenon lay in the ontogeny of the immune response in the vertebrate fetus, leading Burnet and Fenner to postulate the existence of a cell-based immunological tolerance, a hypothesis that Peter Medawar (still at the time a zoologist) and colleagues confirmed experimentally, and for which Burnet and Medawar shared the Nobel Prize in 1960. Yet another observation, for which no ready explanation was available in classical theory, involved the description in the early 1950s of a group of immunological deficiency diseases in humans, the explanation of which would go to the very heart of the biological basis of the immune response. Finally, after a hiatus of some 40 years or more, interest in autoimmune diseases was reawakened by new demonstrations of autoimmune hemolytic anemias, experimental and human autoimmune thyroiditis, and allergic encephalomyelitis.

While these new phenomena provided a sufficient basis to question the old values, such questions could only be answered by the development of new methods, and these were rapidly forthcoming. The techniques of immunofluorescent staining and of hemolytic plaque assay permitted the tissue localization and quantitative enumeration of antibody-forming cells. The technique of passive cell transfer and especially that of cell culture permitted for the first time the analysis of cell朿ell interactions and immunocyte dynamics. Here was a true revolution in the offing, awaiting only the appearance of a theoretical leader to lead the charge against the old regime and its outmoded paradigm. That theoretician was Macfarlane Burnet.

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Chapter 2 The History of Immunology

THE IMMUNOBIOLOGICAL REVOLUTION

Selection Theories of Antibody Formation

Burnet and Fenner抯 indirect template theory of 1949 had pointed up another crucial biological fact that had to be dealt with by any theory of antibody formation梩he recently described phenomenon of acquired immunologic tolerance. It now became important to explain theoretically not only how antibody formation could be stimulated, but also the mechanisms whereby it might be aborted.

The first of the purely biological selection theories of antibody formation was outlined by Niels Jerne in 1955, in what he called a 搉atural selection?theory. Jerne proposed, as had Paul Ehrlich before him, that the host could indeed synthesize small quantities of each of the antibody specificities in the entire repertoire, which would appear spontaneously in the blood as 搉atural antibodies.?The function of these natural antibodies would be to interact selectively with their appropriate antigen and thereby transport that antigen into cells somewhere in the body where the antibody would signal the reproduction of molecules identical to itself; that is, it would initiate the formation of large amounts of specific antibody. The booster antibody response was thus readily explained by the presence, after initial immunization, of an increased number of antibody 揷arriers,?whose presence would also favor the selection by antigen of those antibodies with the higher affinity, thus also explaining changes in the quality of antibody following repeated immunization. The phenomenon of immunologic tolerance was also neatly dealt with for the first time by postulating that any natural antibodies formed against self-antigens would at the outset immediately be absorbed by the tissues of the body, and thenceforth would be unavailable to mediate autoantibody formation.

Although Jerne抯 natural selection theory converted few believers in instruction theories, its historical importance lies in the stimulus that it provided to biologically oriented theoreticians. This stimulus did not lie dormant very long, for within a few years Burnet gave birth to the clonal selection theory of antibody formation. Central to this concept was the postulate that antibodies are natural products that appear on the cell surface as receptors, with which antigen reacts selectively. The interaction of antigen with the surface receptors then signals a clonal proliferation of a population of cells phenotypically restricted for the given antibody specificity, some daughter cells of the clone differentiating into antibody-forming cells and others remaining as immunologic memory cells able to participate in later booster responses. Finally, the theory suggested that immunologic tolerance was due to a 揷lonal abortion,?mediated specifically by self-antigens or by those introduced from without at a critical period in the embryonic maturation of the clonal precursors. Burnet抯 theory received important support and elaboration from David Talmage and Joshua Lederberg. It was Talmage alone who addressed the questions of antibody specificity and repertoire size, pointing out that a limited number of different antibody specificities (later clonotypes) might distinguish a far greater number of antigenic determinants. Lederberg, on the other hand, considered the genetic implications of clonal selection and suggested that antibody diversity might depend upon a high rate of somatic mutation of the 搃mmunoglobulin gene.?

Within a very few years, it became clear that the clonal selection theory of antibody formation had attained wide acceptance, thanks in part to the application of newer techniques for the study of cells, and thanks also to developments in the new genetics. Once the DNA control of antibody structure was accepted, and the amino acid sequences of the immunoglobulin chains elucidated, the clonal selection theory generated its own repertoire controversy. This involved a lengthy debate by those who maintained that the entire specificity repertoire is encoded in the germ line and by others who argued that immunologic diversity is generated by the somatic mutation or recombination of a highly restricted number of germ-line genes. The resolution of this repertoire problem is one of the triumphs of twentieth century molecular biology. It involves the variable combination of a number of minigene segments, assisted by mutations, to form the large universe of antibody light and heavy chains.

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Chapter 2 The History of Immunology

THE IMMUNOBIOLOGICAL REVOLUTION

The Immunological Synthesis

We have defined three distinct eras in the 110-year history of the discipline of immunology. The first, extending from 1880 to about the First World War, centered around the new bacteriology and infectious diseases and had a distinctly medical orientation. Several of the components of the original research program in immunology failed to maintain their original momentum or to fulfill their initial high promise, and went into decline. These included the development of new vaccines, serotherapeutic approaches, the study of cellular immunity, and the study of diseases that might be mediated by cytotoxic antibodies. Two other subprograms followed a somewhat different course; the study of anaphylaxis and related diseases passed primarily into the hands of clinical allergists, while the development and adaptation of serodiagnostic techniques passed into the hands of the new discipline of serology.

As interest in the components of the old program was falling away, there developed a new area of interest in immunology. Leadership in the field devolved upon a new group of individuals with a predominantly chemical orientation to the study of antigens and antibodies, who pursued a research program and developed a theoretical base that reflected this orientation well. A science does not change its precepts and approaches spontaneously; it is moved to the new position by those who explore fertile new areas. But while the earlier immunological program had interacted extensively with many different fields of biology and medicine, the immunochemical era was characterized by a relative introversion, interacting little with other biomedical disciplines. We can date this second era from about the First World War until the late 1950s and early 1960s.

There then occurred an abrupt transition in the field of immunology, which may well be called a scientific revolution. Since the old theories and old techniques could not satisfactorily explain newer observations, the biologists took over command of the discipline from the chemists. Chemical approaches and chemically oriented theories rapidly lost ground to the new biomedical paradigm, which, guided by the clonal selection theory, now asked a markedly different set of questions involving the biological basis and biomedical implications of the immune response.

Eventually, there occurred a synthesis of the two positions. The chemists (who had approached the system by working back from the final molecular product, the antibody) and the biologists (who had worked forward from the initial cellular interactions) found that their different questions and diverse techniques were really aimed at two aspects of the same system. They reached a common ground in the chemistry and molecular biology of T- and B-cell receptors and of lymphokines; together they have clarified the major questions about antibody formation and structure, the dynamics and chemistry of cell朿ell interactions, and the mechanisms of regulation of the immune response.

Immunology once again touches many other disciplines. It has offered classical evolutionary theory the elegant model of an extremely complicated mechanism that is even able to anticipate the appearance of new pathogens, rather than merely slowly adapting its response to their presence. Indeed, this peculiarity of evolution occurred not just once, but twice梠nce for the immunoglobulin B-cell receptor and again somewhat differently for the T-cell receptor. It has offered to geneticists the unique example of an immunoglobulin gene superfamily whose components exercise a broad range of interrelated activities extending even beyond the immune response, and whose mechanism for the generation of immunologic diversity has shown how a gene product can be assembled by the variable splicing of many different DNA segments. In its study of lymphokines and cytokines, modern immunology has offered to the physiologist a variety of examples of how cells may communicate with and influence one another. Finally, the new immunology has assisted many medical subspecialities in defining the pathogenesis of some of their most important diseases, and it has pointed the way as well to the development of preventive measures or therapeutic modalities to combat these diseases, as the following chapters will demonstrate.

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Chapter 2 The History of Immunology

NOBEL PRIZE HIGHLIGHTS IN IMMUNOLOGY

1901, von Behring

The first Nobel Prize in Medicine was awarded to Emil von Behring (1854?917). von Behring studied under Robert Koch at Koch抯 Institute in Berlin. Following L鰂fler抯 isolation of the diphtheria bacillus in 1883 and the identification of diphtheria exotoxin by Roux and Yersin in 1888, von Behring, with his colleagues Kitasato and Wernicke, showed in 1890 to 1892 that diphtheria and tetanus immunity were due to the formation of circulating antitoxins. He showed that passive administration of antitoxin serum to diseased patients might effect a cure, thus opening the way for serum immunotherapy in a number of diseases. His citation read, 揊or his work on serum therapy, especially its application against diphtheria, by which he has opened a new road in the domain of medical science and thereby placed in the hands of the physician a victorious weapon against illness and death.?

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Chapter 2 The History of Immunology

NOBEL PRIZE HIGHLIGHTS IN IMMUNOLOGY

1905, Koch

The prize was awarded to Robert Koch (1843?910), 揻or his investigations and discoveries in regard to tuberculosis.?Koch had been a small-town physician in Germany when his private investigations on the life cycle of the anthrax bacillus and the etiology of anthrax excited the medical profession in 1876. He was given first a laboratory and then an institute in Berlin, and it was there, with the help of a distinguished series of students, that he made bacteriology a true science, by his development of stringent bacterial isolation and culture techniques and by his emphasis on the famous Koch postulates for proof of etiology. Koch devoted himself to the study of a number of different diseases, but it was his identification of the tubercle bacillus and of tuberculin, and his continuing devotion to the study of tuberculosis, that earned him the Nobel Prize. Both the immunodiagnostic tuberculin reaction and the 揔och phenomenon,?involving the excessive dermal reaction to tubercle bacilli in the skin of sensitized animals, played major roles in the later elucidation of the mechanisms of cellular immunity.

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Chapter 2 The History of Immunology

NOBEL PRIZE HIGHLIGHTS IN IMMUNOLOGY

1908, Metchnikoff and Ehrlich

The prize this year was shared by Elie Metchnikoff (1845?916) and Paul Ehrlich (1854?915), 搃n recognition for their work on immunity.?Metchnikoff was born in the Russian Ukraine and studied zoology with an emphasis on comparative embryology. In 1884, working in a marine biology laboratory in Italy, he made the initial observations on the phagocytic cells of starfish larvae that provided the basis for his cellular (phagocytic) theory of immunity. When Metchnikoff left Russia for political reasons, Pasteur offered him a position at his new institute in Paris, where Metchnikoff devoted the rest of his life to an impressive series of investigations in support of his phagocytic theory, and to its vigorous defense from the many attacks of those who favored the view that immunity was based upon humoral (i.e., antibody朿omplement) mechanisms.

Paul Ehrlich was born in Germany, studied medicine, and early became interested in the staining reactions of cells in tissues, devising some of the most useful stains for the tubercle bacillus and for blood leukocytes. In 1891, he became an assistant to Koch at the Institute for Infectious Diseases, where he commenced his immunologic studies. Following early work on the antibody response to the plant toxins abrin and ricin, Ehrlich made his most notable early contribution to immunology in 1897, with publication of his paper describing the first practical method for standardization of diphtheria toxin and antitoxin preparations. This same publication contained also the outline of his famous side-chain theory of antibody formation, which greatly influenced immunologic theories for several decades. With Julius Morgenroth, he published an important series of papers on the mechanism of immune hemolysis. Shortly after the turn of the century, Ehrlich gave up most of his activities in immunology to pursue his interests in the chemical treatment of disease, making important discoveries in the treatment of trypanosomiasis and syphilis (Salvarsan梩he 搈agic bullet? and helping to found scientific pharmacology.

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Chapter 2 The History of Immunology

NOBEL PRIZE HIGHLIGHTS IN IMMUNOLOGY

1913, Richet

The prize was awarded to Charles Richet (1850?935), 揻or his work on anaphylaxis.?Richet was a Parisian who studied medicine and became especially interested in physiology. These interests led him, while cruising on the yacht of the Prince of Monaco, to study the physiologic effects on mammals of marine invertebrate poisons. With his colleague Paul Portier, he discovered the phenomenon of anaphylaxis, dependent not upon the toxic properties of the substance injected, but only upon its function as an antigen in the previously sensitized animal. In so doing, he opened up a new and, at the time, surprising vista in medicine, by showing that the 損rotective?mechanisms of immunity might function also to cause disease. The later demonstration of the relationship between experimental anaphylaxis and other more familiar human allergies made this observation clinically as well as theoretically important to immunology.

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Chapter 2 The History of Immunology

NOBEL PRIZE HIGHLIGHTS IN IMMUNOLOGY

1919, Bordet

The prize was awarded to Jules Bordet (1870?961), 揻or his studies in regard to immunity.?Bordet was a Belgian physician who, at the age of 24, went to study with Metchnikoff at the Pasteur Institute in Paris. He made important early contributions to an understanding of the mechanism of complement-mediated bacteriolysis, and in 1899 he discovered the phenomenon of specific hemolysis. Shortly thereafter, in collaboration with his assistant and brother-in-law Octave Gengou, Bordet described the phenomenon of complement fixation and its diagnostic possibilities. This soon developed into a powerful tool in the diagnosis of infectious diseases, most notably in the hands of August von Wasserman and his colleagues in their complement-fixation test for syphilis. Bordet made many other important contributions to immunology, and he is known also for his famous debates with Ehrlich on the nature of antigen朼ntibody朿omplement interactions.

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Chapter 2 The History of Immunology

NOBEL PRIZE HIGHLIGHTS IN IMMUNOLOGY

1930, Landsteiner

The prize was awarded to Karl Landsteiner (1868?943), 揻or his discovery of the human blood groups.?Landsteiner was a Viennese physician who developed a keen interest in structural organic chemistry before embarking on a career in immunology. From the very outset, Landsteiner seemed always to choose important areas in which to work, or to make important those subjects to which he turned his attention. In early studies of antierythrocyte antibodies, he described in 1901 the set of human isoagglutinins that now comprise the ABO system of blood groups. In 1926, Landsteiner and Philip Levine discovered the MNP system, and with Albert Wiener in 1940 the Rh system of blood groups. He was the first to demonstrate that poliomyelitis could be produced in nonhuman primates, and he was one of the first to make the same observation for syphilis. During the First World War, he became interested in the antibody response to chemically defined haptens, and over the next quarter-century, primarily at the Rockefeller Institute in New York, he contributed impressively to an understanding of the chemical basis for antigen朼ntibody interactions, as summarized in his famous book The Specificity of Serologic Reactions. While acknowledging the importance of his discovery of blood groups, Landsteiner is said to have felt that his 1930 Nobel Prize should rather have been awarded for his work on antibody