NATURE AND BIOLOGICAL ACTIVITIES OF THYMUS HORMONES: PROSPECTS FOR THE FUTURE

NATURE AND BIOLOGICAL ACTIVITIES OF THYMUS HORMONES: PROSPECTS FOR THE FUTURE

Abraham White

Syntex Research Palo Alto, California 94304

The title designated for this presentation by those individuals who planned this conference provides the opportunity to consider three aspects of thymic hormones: ( 1 ) to summarize briefly the origins of some of the present concepts of the nature and role of thymic hormones in immunity; (2) to indicate the present day status of these concepts, as derived from pertinent literature and from the data provided by other participants in this conference; and (3) to project a cautious assessment of prospects for the future.

This presentation is dedicated to the memory of Dr. Thomas F. Dougherty, whose recent untimely death has resulted in the loss of an inspiring teacher, a brilliant investigator, and an irreplaceable friend. Indeed, my own several decades of involvement in studies of the chemistry and metabolism of lymphoid tissue were initiated by Dr. Dougherty who came to Yale in 1942 as a Post- doctoral Fellow, having just completed his Ph.D. training at the University of Minnesota with Dr. Hal Downey. Dr. Downey had transmitted to Tom Dougherty an interest in lymphoid cells, as he had to many students, including some participants in this Conference.

In the course of discussions with Dr. Dougherty about his then current studies with Dr. William Gardner of lymphoid tissue hyperplasia induced by estrogen administration to mice,’ our attention was directed to the marked involution of lymphoid tissue, notably the thymus, that had been described by Selye? as due to a stress stimulus that was mediated by the adrenal glands. Inasmuch as Dr. George Sayers, then a graduate student in our laboratory at Yale, had just isolated highly purified adrenocorticotropic hormone from beef pituitaries,:’ we embarked with Dr. Dougherty on a detailed study of the effects of adrenocorticotropic hormone on lymphoid tissue. A single injection of this hormone in the mouse produced a marked involution of lymphoid tissue, preceded by an acute, dramatic absolute decrease in the numbers of circulating lymphocytes.z These results were extended with the then available Upjohn preparation of total adrenal cortical steroids in oiL6 The synthesis of certain of these hormones, as a result of the brilliant efforts of Kendall, Sarett and their associates, provided us with the opportunity to demonstrate that the effects of adrenal cortical steroids on lymphoid tissue were unique for the 1 I-hydroxylated compounds. Moreover, the thymus gland, among the lymphoid structures, was the most sensitive to the involutionary effects of the pituitary- adrenal cortical axis.“ Of particular significance for more recent studies of the role of the thymus as an endocrine gland, the question of cortisol-resistant versus cortisol-sensitive thymocytes, and the probable secretory role of the endodermal reticular cells of the thymus, was the statement by Dougherty and White that due to hormonal treatment, “The disappearance of numerous lymphocytes ordinarily present in the medulla of the thymus left the endodermal reticular cells more exposed, thus giving the impression that an increase in these cells had occurred.”

523

524 Annals New York Academy of Sciences

It is fitting also, in a conference devoted to the role of the thymus in immunity, to note an additional contribution in which Dr. Dougherty partici- pated. Using the newly introduced Tiselius moving boundary electrophoretic technique, we demonstrated that normal lymphoid tissue contains a protein with an electrophoretic mobility identical to that of the 7-globulin of the blood 5 , * and that lymphoid cells from the nodes of immunized mice contain demonstrable O The continuing synthesis of antibody globulin by lymphoid cells, and their release under the involutionary effects of adrenal steroids, were among early indications of the role of lymphoid tissue in immune phenomena and demonstrated the role of one endocrine system on host immunity. The dissolution of lymphoid tissue by adrenal steroids, which destroy the site of manufacture of antibody globulin, laid the basis for the demonstration by Heilman and Kendall lo of the effectiveness of cortisone in restraining the growth of a transplantable lymphosarcoma in the mouse and for the subsequent use of adrenal steroids as immunosuppressive agents."

The dramatic involution of lymphoid tissue by adrenal steroids led to our initial interest in thymic extracts and the possible role of the thymus as an endocrine gland. If the adrenal steroids cause involution of lymphoid tissue, did the latter, on dissolution, yield products that might suppress pituitary- adrenal cortical secretory activity in the manner of a feedback mechanism? Pursuing this question with Dr. Sidney Roberts, and utilizing the well-established parameters of lymphoid tissue hypertrophy and lymphocytosis as an index of adrenal insufficiency, we reported in 1949 that specific, partially purified fractions of calf thymus produced lymphoid tissue hypertrophy and a lymphocytosis on injection into rats.'? The specificity of this response was inferred from the absence of this activity in other protein-containing fractions of calf thymus. This lymphocytopoietic activity of thymic extracts was subsequently confirmed by Gregoire and Duchateau l3 and by Metcalf in 1956, and similar observations are described elsewhere in this v01ume.~5-~~

The resurgence of interest in the thymus and in thymic extracts and their potential roles in immune phenomena stems, of course, from the almost simul- taneous and now classical observations by Miller,lx and by Archer and Pierce l!' of the deleterious consequences of neonatal thymectomy, observations that were made initially in the mouse and the rabbit, respectively, and subsequently confirmed in a variety of experimental animal species.2n This basic information, and the recognition that primary and secondary immunodeficiencies in the human were due to thymic aplasia or dysplasias,2n. together with subsequent demonstrations that thymic tissue in Millipore@ chambers ?* and cell-free extracts of the thymus 23 may function in lieu of the thymus in neonatally thymec- tomized animals, rekindled our interest, as well as that of many in other laboratories, in the chemical nature of thymic extracts and the possible role of the thymus as an endocrine gland. In addition, stimulus was provided by the arrival in our laboratory of a Postdoctoral Fellow, Dr. Jerome J. Klein, who had completed clinical training in medicine at Mt. Sinai Hospital and had come under the influence of Dr. K. E. Osserman, and the latter's interest in myas- thenia gravis. Dr. Klein suggested repetition of Dr. Robert's earlier studies on thymic extracts, and, with Dr. Allan L. Goldstein, who had also joined the program as a Postdoctoral Fellow, developed an assay based upon incorporation of [3H]thymidine into lymph nodes as a means of assessing the activity of potential lymphocytopoietic fractions of calf thymus extracts.?' This marked the initiation of a most productive collaboration with Dr. Goldstein that, over

White: Nature and Activities of Thymus Hormones 525

a 10-year period, has contributed to establishing the thymus as an endocrine gland and confirming its hormonal role in immunity.25 In 1966, with the help of Mrs. Florence Slater and Miss Norma Robert, we described the preparation of an active, partially purified, thymic lymphocytopoietic fraction, and suggested that this fraction contained a hormone to which was given the name, thymosin.'"

The early, partially purified preparations of thymosin, albeit not meeting the criteria of physical-chemical homogeneity, were shown to be effective in a number of models accepted as reflecting acceleration of the maturation of cell-mediated immunological 27 In these efforts, particular ac- knowledgment should be made of the cooperative studies with Dr. Mark Hardy of the Department of Surgery, and Dr. Jack Battisto, of the Department of Microbiology and Immunology, at the Albert Einstein College of Medicine. With Dr. Martin Zisblatt, then a graduate student at the medical school, it was demonstrated that thymosin was also effective in restraining the progressive growth of the sarcoma induced in mice by inoculation with the Moloney Sar- coma Virus.2s The variety of experimental models, both in v ivo and in vitro, in which thymosin fractions were significantly active led us to propose that thymosin, a lymphocytopoietic factor, acts on a precursor of the immunologically competent T-cell by accelerating the rate at which selected populations of these precursor cells mature to express T-cell characteristics and functions.'!' This hypothesis has been amply supported by several of the presentations at this conference, which appear in this volume.

The later addition of Dr. Arabinda Guha to our research group facilitated achievement of the isolation of thymosin in purified form.30 No small contribution to our progress was provided by the development in the laboratory of Dr. J-F. Bach in Paris of a modified in vitro rosette assay. With Dr. Bach's first demonstration that calf-thymosin-containing fractions we sent him could he quantitatively evaluated by the rosette assay, it became possible for the first time to assay rapidly for thymosin activity the many fractions being prepared by Drs. Goldstein and Guha. Dr. Goldstein and I are indebted to Dr. Bach and his colleagues, Dr. Marie-Ann Bach and Dr. Mirielle Dardenne, for the hospitality of their laboratory extended to us, and later to our associate, Miss Norma Robert, as well as for the numerous rosette assays they have conducted of our fractions.

Thymosin was demonstrated to be a protein with an MW of approximately 12,200, free of carbohydrate, lipid, and polynucleotides. The initial description of the amino acid composition of thymosin was presented at the Federation meetings in April, 1973, and aspects of a new method of isolation and charac- terization are presented elsewhere in this

Through the courtesy of Dr. Milton Helperin, in charge of the Coroner's office of the City of New York, we succeeded in obtaining human thymus for the preparation of thymosin from the human species. The product isolated appears to have similar biological properties and certain of the chemical properties of calf thymosin. The development of a radioimmunoassay by Mr. Richard Schulof 32 established the presence of circulating thymosin in a variety of species, including Homo sapiens. These data, together with those for a circulating thymic humoral factor described by Bach and Da~denne ,~" strongly support the role of the thymus gland as an endocrine gland, influencing several parameters of host immunological competence.

Elsewhere in this volume, Dr. Allan L. Goldstein and his colleagues have presented some of the data derived from the more recent studies of the research


group Dr. Goldstein has assembled following his move to Galveston, supple- mented with the participation of a number of investigators to whom thymosin preparations have been provided.R1

In initiating laboratory activities in Palo Alto, we have sought to improve and simplify the initial steps in the isolation of thymosin from calf thymus. This has been achieved through the efforts of Dr. Clarke Brooks, Mrs. Susan Haag, and Mrs. Kathleen Mitchell. Although this methodology has not yet advanced to a final stage of purification, we have succeeded in increasing sub- stantially the total yield of thymosin, both in terms of weight of material and its biological activity.

In preparing the second aspect of this presentation, namely, the nature and activities of thymic extracts, my task has been greatly facilitated by responses to my request, made prior to these sessions to a number of the participants in this conference, who kindly provided me with both published and unpublished information pertinent to the summation task assigned to me. Most of the data sent to me appear elsewhere in this volume. I shall summarize the more significant of these by comparing and contrasting certain of the chemical properties and biological activities of selected products prepared from thymus tissue in the laboratories of other speakers at this conference.

TABLE 1 lists the physical and chemical properties of some of the more extensively studied products obtained from thymus tissue. TABLE 2 lists some of the biological activities reported for these individual preparations. Each of these products is described in other contributions in this volume.

It is apparent from TABLE 1 that there is at present a significant lack of concensus in the area of the physical and chemical properties of the preparations isolated from thymus tissue as well as, by one laboratory, from pig serum. This lack of agreement may reflect the diverse methods of isolation of products or the existence of more than a single hormonal factor, each with single or multiple biological activities, or both. The preparation from thymus of a hypocalcemic factor, albeit one apparently differing in chemical nature from calcitonin, is perhaps not too surprising in view of the common embryo- logical origin of the thymus, and the parathyroid and thyroid glands, all of which contain the characteristic C-cells indicated to be the site of synthesis of calcitonin. Indeed, the latter hormone has been characterized in partially purified extracts of human thymus and thymoma tissue.31

With regard to the biological activities of the various described thymus factors, it may be concluded that there is rather good agreement among the several laboratories that lymphoid cells, of primary significance in selected immune phenomena, are influenced in numbers and certain manifestations of immunological function by the diverse thymus products. The molecular basis of this influence, including the elucidation of the nature of the cell types upon which thymic hormones exert their actions, remain to be delineated. Suggestive data regarding a possible basis for the action of thymus factors on lymphoid cells have been presented by both Bach and co-workers and by Trainin and his collaborators at this conference indicating that, as in the case of many other polypeptide hormones, cyclic AMP may be a modulator in the process of T-cell maturation. In addition, the increased intracellular concentration of cyclic AMP produced in lymphoid cells by prostaglandin E2 and the ability of this latter compound to enhance graft-versus-host reactivity indicate many ap- proaches to the elucidation of the mechanism of action of thymic factors.

Caution is indicated in attempting to dissect further the differences in


chemical and biological properties of active thymus preparations that have been only partially characterized from a physical-chemical standpoint. Addi- tional studies are required to ascertain whether more than one of these may be a thymic hormone. Electrostatic and hydrophobic interaction of proteins and polypeptides with other molecules present in tissues from which initial extraction is conducted, as well as in the blood, are not unknown and may lead to erroneous conclusions with regard to the state of purity of a product whose

TABLE 1

PHYSICAL AND CHEMICAL PROPERTIES OF SOME THYMUS PRODUCTS ~~

Chemical Molecular Product ':# Class Weight Properties

Thymosin

Thymopoietin I '.

Thymopoietin I1 3*

Thymic humoral factor (THF) '"

Serum TA i 40

Lymphocyte stimulating hormone (LSHh)"

Lymphocyte stimulating hormone (LSH,)"

Homeostatic thymus hormone (HTH)'?

Thymic hypocalcemic factor (TI)"

Thymic hypocalcemic factor (T?)"

protein 7,000

protein 12,000 heat stable no lipid, CHO or

polynucleotide

N-terminus: glycine

C-terminus: lysine

heat stable N-terminus:

glycine C-terminus:

lysine

protein 7,000 heat stable

polypeptide < 5,000 heat stable

polypeptide - 1,000 heat labile

protein - 17,000 heat labile

> 700

pHr -7.5

protein - 80,000 heat stable

glycopeptide 1,800-2,500 heat labile

protein 68.000 ( lO0 ,OOO) pH1=5.65

protein 57,000 ( 170,OOO) pH,= 5.40 2.4% CHO

- ~

* The superscript numbers are to references in this paper, .: Serum TA=Serum "Thymic Activity."

isolation is being sought. Perhaps less than pure preparations of differing properties will, when studied further, prove to resemble one another more closely than presently appears to be the case. In addition, previous experience, for example with insulin and parathyroid hormone, has demonstrated that a biologically active, circulating hormonal agent may be synthesized by its endocrine gland as a molecule with physical, chemical, immunological, and biological properties differing significantly not only from that of the secreted moiety but,


TABLE 2 BIOLOGICAL ACTIVITIES OF SOME THYMUS PRODUCTS

Product

Thymosin

‘Thymopoietin I

Thymopoietin I1 Thymic humoral factor (THF)

Serum TA (Serum “thymic activity”)

Lymphocytopoiesis Restoration of immunological compe-

tence in vivo and in vitro Enhancement of expression of T-cell

characteristics and functions in vitro and in v i v o

Impairment of neuromuscular transmis- sion in vivo

Induction of expression of T-cell antigens in vitro

Same as Thymopoietin I Restoration of immunological compe-

Acceleration of generation of specifically

Prevention of sensitization against self-

Induction of expression of T-cell antigens

@-positive cells in nude mice in vivo Retardation of growth of Moloney Virus

tence in v i v o and in vitro

committed lymphocytes

antigens

iri vitro

induced tumors in vivo

Lymphocyte stimulating hormone Relativc lymphocytosis (LSHh) Augmented antibody synthesis in new-

born mice Lymphocyte stimulating hormone Leukocytosis; lymphocytosis

(LSH,) Augmented antibody synthesis in new- born mice

Antagonism to adrenocorticotropin and thyrotropin in normal resting condition of their target glands, and to thyroxine

Antagonism to gonadotropins; delayed puberty

Synergistic to growth hormone (lyrnpho- cytosis)

Chemotactic influence on lymphocytes Thymic hypocalcemic factor TI Hy pocalcemia Thymic hypocalcemic factor TC Hypocalcemia

Relative lymphocytosis

Homeostatic thymus hormone (HTH)


indeed, from that of the biologically active form responsible for target cell recognition.

Finally, in closing I come to the projection of a cautious assessment of prospects for the future. Acceptance of this last assignment is both a challenging and perhaps unwise decision, since the rate of appearance of new observations relating to the roles of the thymus in immunobiology is so rapid as to make it it possible that such predictions will be out of date by the time the volume of this conference appears in print. Nonetheless, the guidelines and signposts for the direction of continuing developments in the role of thymus factors in immunity appear to be readily discernible. As for any endocrine gland, practical utility may be predicted for thymic factors or hormones in the replacement or augmentation of the functions now delineated for the thymus in immunity. The experimental and clinical evidence clearly establishes at least two roles of the thymus gland in immunity: (1 ) the export of lymphoid cells of varying types and states of maturity with respect to their capacity to participate in diverse immunological phenomena and (2 ) the synthesis and secretion of one or more hormonal factors essential for the continuing, and perhaps final, maturation of immunologically competent thymus-derived or T-cells. In this maturation process, thymosin and other active thymus preparations may function in two ways: (a) to induce the appearance of specific types of T-cells, and (b) to influence the reversion of aberrant T-cells to more normal modes of func- ti~ning.~O, 3:

The indicated biological functions of the several diverse subpopulations of T-cells permits optimism concerning the possible practical applications of one or more highly purified thymic factors. These applications range from obvious utility in replacement of a lack of il normal functioning thymus gland at birth, to the possible alleviation of primary or secondary immunodeficiencies not restricted to specific age groups, for example, aberrant, unrestrained cellular proliferation, as well as to the later years of aging characterized by declining host immunological competence, including the ability to discern between self and non-self.

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NATURE AND BIOLOGICAL ACTIVITIES OF THYMUS HORMONES: PROSPECTS FOR THE FUTURE