"AFLUEI‘ACE 0F TEE AEREEAL GLAIAu S, G3’3A:1F.S, FESTES AM} AGE CIA THE ”73‘ 16A :‘E EASEFEAZKC GRAEAEAATEE CEL NUMBERS OF RAT I’ALTESTEPAE Times}: {09 (Ave amt-m cf 95. D. 2§A£bu.ué. STATE UI‘AAA’F “TY V“ Srey Mae «£07121: sen ‘97 C3) Arm-£99 This is to certify that the thesis entitled INFLUENCE OF THE ADRENAL GLANDS, OVARIES, [ZED/[RY A Michigan Sta": University TESTES AND AGE ON THE EOSINOPHILIC AND BASOPHILIC GRANULATED CELL NUMBERS OF THE RAT INTESTINE presented by Shirley Mae Johnson has been accepted towards fulfillment of the requirements for Ph . D . degree 211.2thng Major prof sor Date October 26. 1970 0-169 . 9 .15. “or. .: 2’ n . . A» , J. . h . .. i I . 3.: 3 u .. A, :. ., . ... IL‘. . ., O r“ o . a . . ... . Haw . . .3 A x .; '1 2 v .. .. . . . . .’ b .9 .K I. t ‘ 3 l- .h 3 - A. Ellill'iji t . .. . v . . : 1.. ._. . . . . . ,. , > " ‘1 22 4 ’ ~. 6 i - - v: I Writ: ' ll" 7 ‘I - ~ ‘ x .. . 2 ‘ ~th. Lnxluag.u :3 4d,", . - 3 ‘- L". ;o.":.. x 3' _.$l000cyret :. 'JL ... :3. “EL\:. - . "“5 m'“):3::\. .‘l: "'V“c-. . .‘r j ',' _. ., .‘ ‘1 Cl." T V. ‘ I. kl... .9 1‘0 and 70665312; 7. A, .C‘ 1*. .53. ‘ ‘ " (9:; $.36 flifliiolaqtcol ~'s_v- v- .22. >~J:‘ n-~:Jz£nt 2‘ ' f ‘ — ABSTRACT INFLUENCE OF THE ADRENAL GLANDS, OVARIES, TESTES AND AGE ON THE EOSINOPHILIC AND BASOPHILIC GRANULATED CELL NUMBERS OF THE RAT INTESTINE BY Shirley Mae Johnson The intestinal tract is intimately involved as a primary structure in the defense mechanisms of the body, as a physical barrier at the interface of the external and internal environment and as a producer of local and systemic antibodies. The lamina propria contains all of the tissue cells normally associated with defense reactions: fibro- blasts, lymphocytes, neutrophils, baSOphils, eosinOphils, monocytes, macrophages and plasma cells. These tissue cells have been reported to be in a dynamic state of flux and in- fluenced by a variety of stimuli. Earlier research in this laboratory on the female reproductive tract demonstrated that hormonal treatment can result in a production or destruction of a base series of cells that normally form the tissue eosinOphilic granulated cells. This present thesis concerns the influence of the gonads and the adrenal glands on tissue leucocytes in the leucocyte—rich intestinal tract, another mucous membrane, and correlates the changes in tis- sue eosinophilic and baSOphilic cells of the rat intestine under various physiological states and following endocrine ShirleynMae Johnson gland removal. Female rats were studied at different stages of the estrous cycle and during pregnancy and lactation. Male rats were studied at various ages in the intact con- dition and following castration, adrenalectomy and adrenal- ectomy-castration. I Tissue sections from the duodenum, ileum and colon were fixed in BouinéHollande fixative, dehydrated, embedded and sectioned at 6 micra. The sections were stained by an alcian blue-eosin procedure, developed during the study, that clearly allowed differentiation between the tissue eosinophils and baSOphils and other tissue components. Cell counts were made on eight random areas of a cross section of the intestine equal to 0.5 sq mm. of a tissue section. Eosindphilic and basophilic granulated cell num- bers varied with the age of the rats, eosinOphilic cells decreased after sexual maturity and baSOphilic cells in- creased with age. The stage of the estrous cycle, pregnancy and lactation influenced eosinophilic and bas0philic granu- lated cell numbers. EosinOphils were highest at estrus and progressively decreased as proestrus was approached. Late lactation was associated with a reduction in both eosin- ophils and bas0phils. 'Eosinophilic cell numbers during pregnancy were comparable to diestrus and proestrus, but bas0philic cell numbers during pregnancy were higher than at any stage of the estrous cycle. The effects of castra- tion, adrenalectomy and adrenalectomy-castration on granu- lated cell numbers varied depending on the time of autOpsy Shirley in as Johnson "surgery. Stressful sacrifice caused a reduction dnmhere of eosinophilic and basophilic cells. These results indicate that the intestinal granula- 3'13"," Jalif‘lte in a continuous state of flux and their numbers . 't _ . jhith the hormonal state of the animal. at I‘A“L‘l‘-’.‘i% I' -‘ ‘~'-..' .. 3' IX? ! 1 INFLUENCE OF THE ADRENIL GLANDS, OVARIES, TESTES AND AGE ON THE EOSINOPHILIC 'AND‘BASOPHILIC GRANULATED CELL NUMBERS OF THE RAT INTESTINE BY Shirley-Mae Johnson A THESIS “""‘," u I. 3 Submitted to ~ Michigan State University ‘. in partial fulfillment of the requirements ' for the degree of DOCTOR or REILOSOPHY Department of Physiology 1970 ACKNOWLEDGMENTS Dr. John E. Nellor, my advisor, has had a tremendous influence upon the course of my life. Words are inadequate, but I Want to try to thank him for his constant belief in me and for his continuous encouragement during the course of this study. His sensitive appreciation for life has made me value my association with him both as a teacher and as a friend. I also extend my appreciation to Dr. W. D. Collings, Dr. William L. Frantz, Dr. Walter N. Mack, Dr. Joseph.Meites, Dr..Evelyn M. Rivera and Dr. Lester F. Wolterink for their guidance in my graduate career and for their help in making this a more meaningful thesis. From the inception of my graduate studies I have been grateful for the willing help given to me by Dr. Gail D. Riegle. In the progress toward my degree help and encourage— ment have come from many sources. I would especially like to thank Starrla Cornell, James Evans, Fred Howe and Pat hzsmith for their technical help; and my fellow graduate _ ,students Abubakar Shaikh, Charles Wira, Gerald Hess and '.j Wandell Hofman for their enthusiastic support. My sincere thanks also go to my parents, Mr. and Mrs. Anders 0. Johnson, for their unfailing belief in their ..Idaughter. ***** ' Emma's .Buthanasia . . . . . . . . . The Normal Female Rat . . . . . . The NOrmal Male Rat . . . . . . . Male-Rat Acute Castration . . . . .Male Rat Castration Series . . . . '. Male Rat Adrenalectomy Series . . , =. -Male ,7‘ ‘Inte Immunity . . . . . . . . . . . Blood and Tissue Cells .Staining . . . . . . . . TABLE OF CONTENTS monuc'rxon............. LITERATURE REVIEW . . . . . . . . . . Definition . . . . . . . . . Phylogenetic Development . . . Thymus . . . . . . . . Bursa of Fabricius . . . . . Humoral Immunity . . . Cells Involved in the Antibody e e e e o e o e e e e e e e e e e e e 0 Response Factors that Influence the Antibody Response Blood Cells . . . . . . . . Tissue Cells . . . . . . . . . Gastrointestinal Tract . . . . . . Histology . . . . . . . . . Cell Types . . . . . . . . . . Intestinal Immunity . . . . . .iRAEIONALE FOR THIS THESIS RESEARCH . . -irnamrms AND METHODS . . . . . . . . -Rat.Adrenalectomy--Castration stinal Lengths . . . . . . . . .AND CONCLUSIONS . . . . . . . Series 0 e I e e U s e e e . “N _ LAMI-n A“. I '«W 1".1' COUNTING PROCEDURE . . . . . . . . . . STATISTICAL METHODS . . . . . . . . . 1‘ £1”.— gran: 0! :.. n ;-, ', - (in H.151", rat (3: my, -. duck: , i: Q- Ma 2 g . Gr :7 u'. v ', 611136»; nets Gram“. :c_;‘ . duudn.«.-Z v." C“ ttlJt't I' ‘- ’A ‘9“ Baffler ; " . -.-CranL10vgi . h g, ., 6001*!“ :11 ,. ' - , . a NH: £WDI! 6N ': .‘ ' 1~sr ‘IL “.33 “'39'1'7' . 131‘» -.‘ '. ”Wha'a 7...)“; (‘1 ; -“- .‘ccowf’k‘ 7 "-.‘ .-' c. v, (H .n' Page 167 168 173 174 A Lb LIST OF TABLES Page Influence of sacrifice method on duodenal granulocytes . . . . . . . . . . . . . . . . . 109 Granulocyte cell numbers in 0.5 sq mm of duodenal lamina propria in the female rat . . . . . . . . . . . . . . . . . . . . . . 110 Granulocyte cell numbers in 0.5 sq mm of duodenal lamina propria in the male rat . . . . 111 Age response to castration in male rats . . . . 112 Granulocyte cell numbers in 0.5 sq mm of duodenal lamina propria of mature castrated male rats at various times post-surgery . . . . 113 Granulocyte cell numbers in 0.5 sq mm of duodenal lamina propria of mature adrenal- ectomized male rats at various times post- surgery . . . . . . . . . . . . . . . . . . . . 114 Granulocyte cell numbers in 0.5 sq mm of duodenal lamina propria of mature adrenal- ectomized-castrated male rats at various times post-surgery . . . . . . . . . . . . . . 115 Mean intestinal lengths in 6 month old Long-Evans hooded rats . . . . . . . . . . . . 116 Figure 1. 10. 11. LIST OF FIGURES Influence of sacrifice method on duodenal granulocyte cell numbers in 6 month old male rats . . . . . . . . . . . . . . . . . Duodenal eosinophilic cell numbers in female rats . . . . . . . . . . . . . . . Duodenal basophilic cell numbers in female rats . . . . . . . . . . . . . . . Influence of age on duodenal eosinophilic cell numbers in male rats . . . . . . . . . Influence of age on duodenal basophilic cell numbers in male rats . . . . . . . . . Intestinal eosinophilic cell numbers in male rats, autopsied 9 days following castration . . . . . . . . . . . . . . . . Intestinal basophilic cell numbers in male rats, autopsied 9 days following castration Total intestinal granulocyte cell numbers in male rats, autOpsied 9 days following castration . . . . . . . . . . . . . . . . Effect of castration, adrenalectomy or adrenalectomy—castration on duodenal eosin- ophilic cell numbers in male rats . . . . . Percent of basophilic cells of the total granulocyte cell numbers in male and female rats . . . . . . . . . . . . . . . . Photomicrographs of rat tissue eosinophilic and basophilic granulated cells . . . . . . vi Page 117 118 119 120 121 122 123 124 126 126 128 INTRODUCTION Previous studies in our laboratory demonstrated that the hormonally controlled differentiation of basophilic tis- ‘sue cells into eosinophilic plasma cells in the reproductive tract was associated with an increase in bactericidal agents, ipresumably secreted by degranulating plasma cells. The similarity of cell forms within the genital tract and the lintestinal tract suggests that this phenomenon might be lv characteristic of mucous membranes in general. - This thesis deals primarily with changes that occur :' in basophilic and eosinophilic cell numbers in the intesti- ' nal tract under the influence of various physiological states and following endocrine gland ablation. These changes, as those seen in the reproductive tract, may relate to the immune capability of an animal and circumstantially E5 Ears indicative of hormonally controlled immune competence. .fiagbhe:far reaching implications of such a tissue developed :Sjigfense system justifies, in fact demands, that the rela- '3~£Egonship of the intestinal tract to the entire immune system sig:{considered in detail. All aspects of this complex and 3{1y understood entity will be reviewed in order to clar- 1' cellular transitions and the eosinOphilic and baSOphilic cells can be demonstrated. This treatment of acknowledged defense mechanisms in the review of literature was justified since it suggests that the more familiar portions of the body defense mechanisms, the bone marrow, blood and lymph nodes may be secondary to the less completely understood, but more dynamic primary mucous membrane defense mechanisms. The mucous membranes of the body are constantly exposed to foreign materials and may represent the body's first line of defense. Understanding the dynamic inter— relationships of the immunocompetent cells is essential for elucidating the defensive mechanisms of these membranes. Information on eosinophilic and baSOphilic cells in various control states should indicate what sort of controls exist for these cells and what parameters need to be considered in studies involving hormonal control of the body's defenses. LITERATURE REVIEW Immunity Definition The ability of an organism to resist foreign invad- ers is essential for the survival of a species. The ques- tion of how organisms actually resist invasion has been chronically pondered by immunologists for years. From the early history of immunology two conflicting theories emerged. .In the Cellular Theory proposed by Eli Metchnikoff about 1884, immunity was based on a system of cellular "phagocytes" (Greek, phagein-—to eat; kutos--cell) which engulfed and removed invading particles (Metchnikoff, 1905). In the Humoral Theory of George Henry Falkiner Nuttall proposed around 1888, immunity was based on chemicals called anti- dotes or antibodies which neutralized foreign invaders f”'(flutta11, 1904). These two lines of thought, each widely --:{ investigated and proponed have now been fused and both LSthems are part of the current knowledge on body defense. f. Throughout their existence, animal bodies are I~é§posed to a variety of stresses which stimulate nonspecific i .;16 specific immunological responses known respectively as ifstance and vauired immunity (Gray, 1967). "Immunity" originally referred to complete freedom or exemption. Boyd (1966) viewed immunity as a weapon in the basic struggle for life which imparted degrees of refractoriness against aggression. 1 Many factors contribute to the defense of organisms against foreign invaders. Carpenter (1965) has categorized these in the following manner: Normal Protection Against Infectious Disease 1. Nonsusceptibility provided absolute protec- tion against particular diseases and was associated with species physiological and anatomical characteristics, e.g., normal body temperature, diet, etc. Antibodies were not involved. 2. Natural immunity_varied in the degree of pro— tection for individuals. This protection depended on the presence of "natural“ anti— bodies without obvious external stimuli. The concentration of these antibodies Was low and the origin uncertain, e.g., antibodies against foreign erythrocytes. 3. Natural resistance was nonSpecific and indi- vidually variable. It was not dependent on antibodies and could be related to the integ— rity of physical (epithelium, mucous membranes or phagocytes) and chemical (digestive acidity or alkalinity, Complement, lysozymes, etc.) barriers which could be changed by environmen- tal factors, nutrition, fatigue, aging, etc. Acquired Immunity Against Infectious Disease 1. Active immunity depended on natural or artifi— cial stimulation of antibody producing mecha- nisms to combat disease. 2. Passive immunity depended on passive transfer of antibody from another individual. Phylggenetic Development Considering immunity from a phylogenetic and evolu— tionary viewpoint, millions of years elapsed before such a complex approach to body defense could be attained. Phago— cytosis and enzymatic mechanisms appeared to be the only means for invertebrates to destroy pathogens (Hildman, 1962) or nonspecific defenses (Sirotonin, 1960). These inverte- brate mechanisms and the more involved systems of antibody production and cell mediated immunity are necessary in vertebrate immunological processes. Papermaster gt al. (1964a) traced the appearance of vertebrate immunity to primitive marine vertebrates appearing about 400 million years ago. Hagfish (Eptatretidug stoutii) did not possess adaptive immunity and did not form circulating antibody or show delayed hypersensitivity; nor did they seem to differ— entiate tissue homografts from autografts. These primitive vertebrates did not possess lymphocytOpoietic or thymic tissue. The adult lamprey (Petromyzon marinus) was shown to have circulating antibodies and a primitive thymus which was a foci of lymphoid cells. It also possessed a spleen and was capable of some antibody production, immune memory, delayed hypersensitivity and homograft rejection. The guitarfish (Rhinobatas productus), a more developed verte— brate, responded to a larger variety of test antigens than the lamprey. Elasmobranchs and teleosts, arising some 250 million years ago, possessed lymphopoietic and thymic tissue, circulating lymphocytes and gamma globulin and were immuno— logically competent with respect to antibody production and homograft rejection. Their tissues contained the first well defined plasma cells. In work with poikilotherms it has been shown that the immune reaction was closely temperature dependent. At colder temperatures antibody production was slower and a longer time was needed for detectable antibody titers to develop (Hildman, 1962). The possibility has also been pro- posed that fish, even without serum gamma globulins, could respond to antigenic stimulus (Sigel t al., 1963; and Clem and Sigel, 1963). Plasma cells are abundant in the lamina propria of the intestinal tract of amphibians. Reptiles, birds and mammals possess true lymph nodes, bursa or bursa—like tis- Sues and a thymus and are fully immunologically competent (Good, 1967). Thus, adaptive immunity developed parallel with phylogenetic develOpment of the thymus and lymphoid system (Papermaster t al., 1964b). Good (1967), in retrospect, viewed this phylogenetic development and questioned whether these primitive systems originally were involved with defenses against infeCtion. He stated that lymphoid devel- opment was associated with the appearance of more elaborate species with increasingly complex tissues. He viewed the rapidly multiplying hematopoietic tissues and the dynamic 4-figastrointestinal tract as having great potential for somatic variation. Since a stable species cannot exist with vari- ation a "police system" had to develOp which could distin- guish "self“ from "nonself" cells and destroy the latter. Thymus Nevertheless, immunological competence and lymphoid development are correlated. The thymus was the first struc- ture in which lymphoid elements could be recognized (Archer _£__l,, 1963: and Kelly, 1963). Embryologically the thymus develOps as paired evaginations from the ectoderm and endo— derm of the dorsal part of the third and fourth pharyngeal pouches (Hammond, 1954). Debate has raged on the origin of thymic lymphocytes, that is, whether from mesenchymal or epithelial origin. Downey (1948), in studies on irradiated rabbit thymuses, put forth one of the last in a long series of articles claiming that the regeneration of lymphocytes occurred from mesen- chymatous tissue and not from the epithelial elements. A pseudo-compromise was affected by Auerbach (1960 and 1961) in his reports on the lfl.2£££2 development of the mouse thymus. Neither of the primary tissues alone could produce lymphocytes, but both were necessary. Lymphocytes developed from the epithelial elements but the mesenchyme served as an inducing stimulus and provided the stromal elements of the gland. At first a mystery, the implications of thymic func- tion are now becoming clarified. Removal of the thymus in adult mice caused a reduction in the lymphocyte population of the thoracic lymph duct, peripheral blood, lymph nodes and spleen (Metcalf, 1960). Removal in young rabbits neither enhanced nor inhibited antibody production. This caused Good, a later champion of the thymus in immune reac- tions, to claim that the thymus had no control over immune responses (MacLean _E._l., 1957). It was not until Miller (1961 and 1962) removed the thymus of neonatal mice that the role of the thymus became elucidated. Serious immune defects were evident. One to two months after neonatal thymectomy there was a marked depletion of the lymphocyte pOpulation of the blood and the tissues. There was also serious impairment of the matura- tion of delayed hypersensitivity and transplantation immu— nity. Within two to four months 70% of the mice died of wasting syndrome and diarrhea. Early thymectomy of mice caused 19S and 7S gamma globulin levels to fluctuate, but the total remained rela- tively unchanged (Humphrey §£_gl,, 1964). On the other hand, Miller and Osoba (1967) found a decrease in mouse immuno- globulin A (IgA) after early thymectomy. Neonatal thym- ectomy was also reported to delay maximum immunological responsiveness of 7S hemolysin in mice (Sinclair, 1967; and Takeya and Nomoto,‘l967). A humoral aSpect of thymic function has also been postulated. Implanting of thymic tissue in Millipore cham- bers in neonatally thymectomized mice alleviated all of the symptoms of immunological impairment (graft rejection and antibody production) and prevented the wasting syndrome (Levey §t__l,, 1963: and Osoba and Miller, 1964). Miller brought all these observations together and concluded that the thymus plays an early role in immunolog- ical develOpment of animals by seeding lymphoid precursors to peripheral tissues and by directing maturation of immu- nological capabilities by means of a humoral mechanism (Miller, 1964). This humoral factor endowed lymphoid cells with immunological competence (Osoba and.Miller, 1964). Bursa of Fabricius The bursa of Fabricius is a lympho—epithelial organ peculiar to birds. It is a blind pouch diverticulum from the posterior cloaca (Foebes, 1877; Calhoun, 1933; and Ackerman and Knouff, 1959). Foebes (1877) discovered that it was present in both sexes and maximally develOped in the young bird. The debate on the origin of the lymphoid cells seems to have resolved as did the thymus controversy, in favor of development from undifferentiated endodermal epi- thelial cells (Ackerman and Knouff, 1959). The bursa has been shown to influence the differen- tiation of other lymphoid cells and to influence the ability of the chicken to produce antibodies. Bursectomized chick- ens were less able than normal chickens to produce anti- bodies in response to antigen (Glick §£_§l,, 1956; and Mueller t 1., 1962). Immunoglobulin G (IgG) levels were 10 found to be normal at 12 weeks, but grossly deficient at six months (Arnason and Jankovié, 1967). The lymphoid development of the thymus and the bursa are similar, but in gyg treatment with testosterone will inhibit bursal tissue but not thymic tissue (Papermaster and Good, 1962; and Warner _£._l., 1962). Szenberg and Warner (1962) dissociated the immunological responsiveness of the fowl by attributing control over circulating antibodies and delayed hypersensitivity reactions to the bursa and control over the recognition of the histocompatibility of skin grafts to the thymus. Thus, the thymus has been shown to have a role in transplantation immunity; the bursa has been shown to have a role in the production of circulating antibodies. It is undecided whether the mammal has a counterpart to the bursa that can affect antibody levels. In rabbits thymectomy and appendectomy affected the ability of the rabbit to produce antibody to bovine serum globulin. This ability was im- paired more by thymectomy and appendectomy than by either surgery alone or by thymectomy and splenectomy. This pro— cedure caused lower lymphoid development, lower circulating antibodies and lower circulating lymphocytes (Sutherland gt al., 1964). In 1966 and 1968 Cooper t al. demonstrated that x-ray destruction of intestinal lymphoid tissue, Peyer's patches, sacculus rotundus and appendix, decreased the antibody response but left the graft rejection and the delayed hypersensitivity reaction intact. This led them to 11 postulate that these tissues were functionally homologous to the fowl bursa of Fabricius. Others have seen the plasma cell rich intestinal lymphoid tissue as bursal-like (Brandom §£._l., 1969; Good _£ al., 1969; Watson, 1969; and Schofield and Cahill, 1969). Good (1967) has aptly summed the data to date on the L bursal and thymic systems. He reported that the two branches } of the immune mechanism developed from the same lymphoid pre- cursor, a bone marrow stem cell. The thymus developed as an epithelial structure from the third and fourth embryonic pharyngeal pouches and under mesenchymal induction became a , lymphoid organ. The bursal system developed by budding of the intestinal epithelium. These central organs released cells into the blood stream to seed peripheral lymphoid tissues. With this concept, the lymphocytes of thymic origin control cellular immunity (homograft rejection, V delayed allergy and graft versus host reactivity) and the bursal dependent plasma cells control the humoral immunity (immunoglobulins G, A, M, D; specific antibodies to bacteria, viruses, toxins, etc.). Humoral Immunity The concept of humoral immunity has advanced consid- erably since Nuttall's first nebulous allusion in 1888 to antibodies or neutralizing chemicals (Nuttall, 1904). Tiselius and Kabat (1938) separated blood into fractions on the basis of electrical charges and demonstrated that gamma 12 globulins or antibodies are a distinct group of serum pro- teins. This discovery opened the door for an avalanche of chemical analyses on antibody fractions. The knowledge about antibodies has now advanced to characterization of major classes (IgG, IgM and 19A) of antibodies composed of two types of polypeptide chains distinguished by marked heterogeneity, but able to respond to antigens with immuno- logic specificity (Nisonoff and Inman, 1964). A review on the numerous studies characterizing the chemistry of antibodies and the relationship of the differ- ent types to various phases of the immune response is beyond the sc0pe of this thesis; however, the theories concerning antibody production and the cells that are involved in the generation of the humoral aSpect of immunity are pertinent to this research. Spanning years of investigation and the efforts of numerous scientists, three recurring theories have emerged to explain antibody formation. No one theory has gained universal acceptance so a brief consideration of each is necessary. Direct template hypothesis.-—The antigen is believed to be assimilated into the antibody producing cell and is itself a template or a pattern for specific antibody produc- tion. A complementary form or a globulin mirror image of the antigen is produced. Breinl and Haurowitz (1930) envi- sioned cells that possessed radicals that could attract or repell amino acids necessary for the formation of the many 13 globulin complements to antigens. An investigation of the spatial relationships of the antigen and antibody added to the literature on direct template theory (Mudd, 1932). Pauling (1940) contended that immune globulins and normal serum globulins differed only in configurations at the two ends of the polypeptide chain. This configuration would directly complement an antigen. He prOposed a simple scheme for polypeptide bending to achieve stability and specificity. Indirect template hypothesis.--In this theory it is not necessary for the antigen to be directly assimilated in- to the cell, but antigenic information must somehow be incor- porated into DNA so that a template can be formed. This indirect template could persist and be genetically repli- cated. Burnet and Fenner (1949) recognizing the phenomenon of enzyme modification in bacteria, extended this idea to antibody formation. It was suggested that antigen could cause enzyme modification for template production. The modification would recognize "self" and replicate to destroy foreign entities. Schweet and Owen (1957) explained anti- body production in terms of antigen modified DNA controlling protein synthesis. Selective hypothesis.--In this theory the antigen is merely a selective stimulus acting to increase antibody production from information present in the cell before the antigen contact. .Ehrlich and Moregenroth (1904) could be considered forefathers of this idea by proposing in 1899 that antibody producing cells possessed side chain receptors 14 (atom groups) with affinity for antigens. These could be regenerated and released into the blood to protect the body. The antigen, by this means, selected the receptor which it best fit. Jerne (1955) has modernized this idea by prOpos- ing natural circulating antibodies which react with antigens and are carried by the antigens to the cells that can pro- duce antibody. The antigen-antibody combination may be phagocytized and new antibody synthesis stimulated for molecules identical to the antibody present. The role of the antigen was solely as a selective carrier. Lederberg (1959) described antibody as having a unique sequence of amino acids that are coded for by DNA. Cells produce their own antibody but mutate at a high rate. When immature, they are hypersensitive to antigen-antibody combinations which can influence the antibody type they produce. In the mature form they are plasma cells which proliferate in clones. They can be stimulated by the antigen which influenced them at an earlier exposure, when they were immature. Burnet (1962) presented a theory on clonal selection that has lost and gained favor through the years. This clone idea is comparable to Lederberg's. In embryonic life a wide range of clones is present derived from mobile mesen- chyme cells, to cover all possible patterns of antigens. Clones for "self" antigens are destroyed in embryonic life and immunologically competent cells which produce antibodies to foreign antigens remain. 15 The concept of "self" recognition has puzzled immunologists since it is not infallible: immune tolerance and certain autoimmune reactions show this fallibility. A complex explanation of self and nonself recognition on a molecular basis, which distinguishes lymphoid mitotic effec- tors and tissue coding factors, has been prOposed by Burch and Burwell (1965). Elaborate schemes for the clonal differentiation of bone marrow cells have been expounded upon by various inves- tigators. Lymphocytic differentiation for the production of 19S and 7S antibody with steps for maturation and immunolog- ical memory has been diagramatically presented (Papermaster, 1967; and Sterzl, 1969). Cells Involved in the Antibody Response Phylogenetically, a positive relationship has been shown to exist between the development of lymphoid tissue and immunological capability. Mechanisms have been prOposed for the molecular production of antibodies, but a considera- tion of the actual cell types involved is necessary. Very early investigators, in their quest for the source of antibody production, proceeded to remove various organs, noting the changes in immunological competence. Removal of the stomach, small intestine, thyroid and pan- creas had little influence upon antibody production beyond the expected physiological disturbances commensurate with their removal. However, the removal of the spleen markedly 16 decreased antibody formation (Hektoen and Curtis, 1915). Utilizing ablation studies and phylogenetic correlation the lymphoid tissues were definitely implicated in antibody production. In 1935, McMaster and Hudack reported that lymph nodes responded to antigenic stimulus by agglutinin forma- tion. An investigation in 1955 by Harris and Harris demon— strated local lymph node antibody production in response to antigenic stimulus. Immunoglobulins have also been demOnstrated in lymphoid tissues of the lymph nodes, bone marrow, ileum and spleen (Asofsky and Thorbecke, 1961; Mellors and Korngold, 1963: and van Furth _E _l., 1966). The pioneering techniques of Carrel and Ingebrigsten (1912) in in yitgg culturing of bone and lymph nodes paved the way for experimental antibody production. Nilsson's investigations (1967), on rabbit lymphoid suspensions which had been stimulated in yiyg by human gamma globulin, demon- strated antibody production from lymphoid cells. DNA syn— thesis was observed ig_yi§gg 5 to 24 hours after spleen cultures were challenged with antigen (Cohen and Talmage, 1965). Although lymphoid tissues were related to antibody production it was not clear which cell types were actually involved. Lymphoid tissue is composed of a framework of reticular cells and free cells. The stromal cells may be primitive reticular cells and phagocytic fixed macrophages. 17 The free cells that can be distinguished floating in the lymph are the small, medium and large lymphocytes, plasma cells, eosinOphilic leucocytes, neutrOphils, mast cells and occasionally monocytes (Bloom and Fawcett, 1965; and Ham, 1965). Various investigators have implicated each of these cells and many others in the antibody response (Quinn, 1968). Significant progress was made in identifying the antibody producing cells when Bjorneboe and Gormsen (1943) detected a constant relationship between antibody production and plasma cell proliferation. The investigations of Fagraeus (1948) are considered classic in the implication of the plasma cell's role as an antibody producer. She recognized the wide distribution of plasma cells in the liver, spleen, bone marrow, kidney and lymph nodes. When nonantigenic substances were administered to rabbits no change occurred in the plasma cell numbers in these organs. However, upon a second challenge by an antigenic substance, the numbers of plasma cells increased, especially in the spleen, and this increase was correlated with globulin pro- duction. The plasma cell was reported to develOp from a reticulum cell. The numbers of immature, baSOphilic plasma cells were found to be greatest at the peak of antibody pro- duction; mature plasma cells released antibody, but were beyond the stage of their greatest functional capacity. Coons and co-workers (1955) developed a technique of fluorescent antibody staining which identified specific gamma globulins. The fluorescing cells, or the antibody 18 producers, were plasma cells of the Spleen, lymph nodes, submucosa of the ileum and the portal connective tissues of the liver. LeDuc §£_gl, (1955) in characterizing the anti- body response to antigenic stimulus found antibody to be first formed in large immature cells in the medullary areas of the lymph nodes. On primary challenge the response was low, but upon secondary challenge the response was high and colonies of plasma cells develOped. The work of Ortega and Mellors (1957), using Coons' fluorescent technique, demon— strated antibodies in plasma cells in the germinal centers of the lymph nodes, in medium and large lymphocytes and in reticular cells. Nossal (1958) worked with single lymphoid cells of regional lymph nodes and reported antibody production by these cells. Individual cells formed one species of anti- body. Research on the kinetics of plasma cell proliferation by Nossal and Makela (1962) indicated that plasma cells develOped from primitive lymphocytes to plasmablasts in 12 hours.' Immunological memory was also inherent in the stimulated lymphoid series. The involvement of the plasma cell in antibody forma- tion was further strengthened by ontogeny studies. Black and Speer (1959), studying human fetuses, could not detect gamma globulin production, secondary follicles or plasma cells in the lymph nodes. However, independent studies on immunoglobulins have shown human maternal IgG to increase during pregnancy up to 42 weeks (Papadatos t 1., 1969) 19 and to be equivalent in the fetus and the mother by 33 weeks of gestation (Gusdon, 1969). Since IgG freely passes the placental barrier (Cohen, 1950; and Tomasi, 1967) this find- ing is not necessarily in conflict with Black and Speer's statements. Van Furth g£.al. (1965) found IgG and IgM pres- ent in the lymphoid plasma cells of the spleen from the 20th week. In work with congenital syphilis, the time of fetal immunogenesis was placed at 6 months, a time when plasma cells were first observed (Silverstein, 1962; and Silver- stein and Lukes, 1962). Bridges and co-workers (1959) reported that the intestine exhibited the body's highest concentration of plasma cells, but that they were not pres- ent in the first few weeks of human life. Gamma globulin appeared at 6 weeks in the serum and antibody production at 67 days. A cell form called a Russell body has been suggested as a storage form of the plasma cell for excess antibody (White, 1954; and Thiéry, 1960). Moeschlin and c0dworkers (1951) found granules in rabbit spleen cells on the third to fifth day after typhoid challenge. Gamma globulins were shown from days two through five. On day six, when the anti- body was present in the blood, the plasma cells lost their granules. Ortega and Mellors (1957) have also related Russell bodies to antibody production. All these investigations serve to show that immuno- logical development seems to be related to plasma cell formation. The role of the lymphocytes and plasma cells 20 in the immune response has been thoroughly reviewed (Hidde, 1955: MdMaster, 1961; and Stone, 1967). The evidence for plasma cell production of anti- bodies and even antibody production by some lymphocytes seems well accepted. Studies on the blood and lymph vary. Landy gt al. (1964) reported that in the rabbit as much as 10% of the peripheral blood leucocytes are immunologically competent antibody producers. But, Hummeler and his co- investigators (1966) found evidence for antibody producing cells in the blood and lymph, but the frequency of antibody producing cells was only l/5,000 to 1/50,000. The granulo- cytic cells are not implicated in antibody liberation (Wilson and Miles, 1946). Investigations on the phagocytes of the reticulo- endothelial system have brought varied claims of immune competence. The phagocytic ability, which was so early recognized by Metchnikoff as a nonspecific immune response, is accepted. But questions exist on the sort of role, if any, the macrOphages give in specific antibody response. Nelson (1969) has reviewed the relationship of the macrOphages to immunity and concluded that they do not produce antibodies. The ability to phagocytize antigen (Thorbecke and Benacerraf, 1962) however, may be an intri- cate step in antibody formation. Fishman (1961) in his investigations of ig.yi££g antibody formation to T2 bacte- riOphage, suggested a macrOphage processing step in antibody formation. Lymphocytic cells produced antibody, but were e 21 stimulated to do so by an RNAse sensitive substance from the macrophage. This substance resulted from an interaction of the macrophages and the antigen. Further studies with labeled RNA showed greater incorporation of RNA in lymph- ocytes near macrophages than in those not in the area of macrophages. The transferred RNA was a low molecular weight form (Fishman _§__l., 1963). McFarland (1966) and his group have seen large lymphocytes making contact with macrophages through uropod—like structures. This macrophage dependent step may provide some key to immunological tolerance shown by various young animals to early administration of defined antigens (Smith and Bridges, 1958: and Miller—Ben Shaul, 1965). Rowley and Fitch (1965) found rat antibody formation varied with age, adults being more competent than the young. Work on the rat reticulo- endothelial system by Suzuki (1957) showed that the adult system was more develOped and more capable. Mitchell and Nossal (1966) found that the newborn rat handled antigen differently than the adult. Phagocytosis was poorly devel- oped. Martin (1966) viewed these two facts, tolerance in the young and poor macrophage activity, as indicating a macrophage dependent step in antibody formation. Evidence for a relationship of eosinOphilia and the antibody response has also been proposed. Eosinophilic cells have been shown to be involved in the antibody re- sponse (Speirs, 1958). Litt (1964b) has done considerable 22 work on this phenomenon and has reported that the antigen- antibody complex attracts eosinOphils and is phagocytized by them. He indicated that eosinophils were involved in the response after antibody synthesis. He found high con— centrations of eosinophils in the lungs, gastrointestinal tract and the skin. He found this natural eosinophilia significant because these were tissues that were constantly assaulted by foreign materials of the outside world. The eosinOphilia was an index of the antibody response. .Factors that Influence the AntibodyAResponse Vertebrates have develOped complex mechanisms for responding to foreign or antigenic stimuli. The antigenicity of a substance or its ability to stimulate antibody formation has been related to chemical composition (MW'10,000 or greet- er), spatial configuration (a large surface area) and degree of foreignness (antigenicity of animal proteins usually varies inversely with the degree of biological relationship between the recipient and the source) (Carpenter, 1965). Despite this mechanistic view of antibody response, the antibody response varies considerably in different test systems depending on the investigator and the exact nature of the physiological state of the animal; the response is by no means simple or completely understood. Pituitary hormones.—-The role of the pituitary gland in immune competence has been primarily related to the role of somatotropin (STH) and adrenocorticotrOpin (ACTH). 23 Hypophysectomy and hypopituitary function are associated with immunological deficiencies. Keefe and co-workers (1967) found a progressive reduction in the rat phagocytic index for colloidal carbon 10 weeks post-hypOphysectomy. In hypo- pituitary dwarf mice STH and thyroxin returned antibody formation to levels of normal mice (Pierpaoli _Eigl., 1969). The role of STH in antibody formation is controversial. STH was reported to increase thymus weight (Dougherty, 1952). STH is an anabolic hormone and Kalter and co—workers (1950) related this fact to the increased rate of antibody formation with STH administration. Similar results were obtained with testosterone, another anabolic hormone. Hoene gt a1. (1954) reported STH in rats to be an antagonist of cortisone. STH administered alone caused a decrease in serum antibodies but a splenic hyperplasia. STH treatment in conjunction with hydrocortisone acetate resulted in a production of Optimal levels of antibody. STH treatment also counteracted ACTH induced depression of serum antibody levels (Hayashida and Li, 1957). Selye (1952) demonstrated that STH could assist rats in withstanding fatal doses of cortisone. In work combating tuberculosis, Lemonde gt _1. (1955) indicated that STH did not have any beneficial effects. Dougherty, Chase and White (1945) reported that an early rise in circulating antibody occurred concomitant with a marked lymphocytopenia following ACTH administration. This paper stimulated many investigations on this "non-specific" release of antibodies (de Vries, 1949: Bj¢rneboe gt al., 24 1951: Moeschlin _t _l., 1952; and van der Slikke and Keuring, 1953). The lymphopenia following ACTH treatment was con— firmed, but the bulk of the evidence so strongly refuted the claim for an early rise in antibody levels following ACTH treatment that Dougherty, himself, (1954) agreed that the "non-specific" release of antibody did not occur. After cessation of ACTH treatment the depressed levels of antibody rose and antibodies were rapidly synthesized. The depressing action of ACTH on antibody titers was due to the stimulation of the adrenal cortex and resultant glucocorticoid secretion (Recant _t _l., 1950; and Kass and Finland, 1953). Baroni gt a1. (1967) suggested a hypo- physeal control of the thymus, perhaps of the primary immune response, which influenced the balance of the antibody pro- ducing system. Adrenal hormones.--Cortisone decreased plasma cell numbers in lymphatic tissue and decreased gamma globulin content in the blood (Teilum §£__l., 1950). Cortisone and hydrocortisone also decreased antibody production (Kass gt al., 1955). The appearance of serum antibodies was delayed by cortisone and the titer peak reduced (Hoene gt al., 1954). Berglund (1952 and 1962) reported that cortisone acetate and prednisolone were very effective in decreasing agglutinin titers in the initial phase of the antibody response. This depressing action of glucocorticoids was also found to extend to complement production and granulocyte numbers, as well as antibody levels (Stavitsky, 1952). W' 25 In its use as an anti-inflammatory treatment for dermatitis, cortisone was found to decrease the patient's resistance to infection (Dougherty, 1954). Robinson and Smith (1953) reported that cortisone inhibited formation of hyaluronic acid, the ground substance of connective tissue, and by this mechanism decreased the animal's resistance to infection by allowing microorganisms to spread from the place of induction. Hayes (1953) demonstrated that local antibody pro— duction to antigen was delayed by cortisone administration. Due to cortisone's inhibition of macrophage activity the antigen was not contained locally, but escaped to the spleen and there stimulated systemic antibody production. A review by Kass and Finland (1953) on the role of the adrenal hormones in infection and immunity stated that evidence to date suggested that ACTH and glucocorticoids depressed mechanisms of body resistance. Large doses of cortisone administered to mice increased their susceptibil- ity to virus infections whereas adrenalectomy or desoxycor- ticosterone administration had no observable effects on resistance (Southam and Babcock, 1951). The actions of the adrenal hormones vary with the type of disease and the level of adrenal activity. Adrenal- ectomy in rats and rabbits increased antibody formation (Perla and Marmorston—Gottesman, 1928; and Criep gt al., 1951). However, adrenalectomy in rats decreased their 26 resistance to typhoid infection (Jaffe, 1926). Adrenalectomy in the rabbit, however, did not reverse the course of tuber- culosis (Favour, 1954). Clarke and his co-investigators (1954) showed that low urine 17-ketosteroid levels were associated with extensive tuberculosis in humans. Chronic emotional stress was related to decreased resistance. The authors reported that hyperadrenal activity was related to increased resistance. Perla and Marmorston—Gottesman (1928) related adre- nalectomy in rats to decreased hemolysin formation, 1 week after surgery. Two weeks later there was an increased anti— body titer which paralleled lymphoid hyperplasia. Selye's (1937) famous "alarm reaction" stated that stress was related to adrenal hyperplasia and lymphatic involution. Adrenalectomy caused lymphoid and thymic hypertrophy which could be counteracted by lymphoid destroying cortisone treatment (weaver, 1955). ACTH or cortisone administered during the maximal antibody response caused a decrease in normal lymph node histiocytes, adrenal cortical mass, germinal reaction centers and mitotic figures (Craig, 1952). Thus cortisone and ACTH, acting through the adrenal system, have a depressing effect on lymphoid tissue which can be reflected in serum antibody titers. The molecular action on the lymph node is associated with inhibited DNA synthesis, decreased glucose utilization, decreased protein synthesis and decreased numbers of cells able to incorporate thymidine (Lang gt gl.. 1967)- 27 This adrenal-antibody system is very dynamic. Handling of animals during bleeding or for injections can result in lymphopenia and decreased complement in noncondi- tioned rabbits (Stavitsky, 1952). Antibody formation is also related to the integrity of the reticulo—endothelial system. Adrenalectomy and hypo- physectomy decreased phagocytosis. The adrenal therefore appeared to have a reticulo-endothelial maintenance activity (Gordon and Katch, 1949). Corticoids, however are reported phagocytic activity (Germuth t 1., 1952; Robinson and t I l to have a depressing effect on mesenchymal tissue and on a I Smith, 1953; Gell and Hinde, 1953; and Essellier _£.§L-: 1955). The adrenal medullary hormone epinephrine, which is released in large amounts during stress reactions, depresses antibody levels. This depression in rats is dependent upon an intact adrenal-pituitary system (Recant _t _t., 1950). The common use of glucocorticoids as anti-inflamma- matory agents may have far reaching effects on the entire immune picture by their depressing effect on antibody production and phagocytic activity. Sex hormones.--The gonadal sex hormones have also been investigated in relation to the immune responses. This relationship is immediately seen at sexual maturity when thymus involution is accelerated (Dougherty, 1952). Gonad- } ectomy, like adrenalectomy, has been shown to increase the amounts of lymphatic tissue (Marine t al., 1924). 28 Estradiol administration can result in thymic and lymphoid weight decreases in newborn mice (Kappas and Palmer, 1963; and Thompson and Russe, 1965). Yet, despite the abil- ity to decrease the amount of lymphoid tissue, the majority of the reports depict estrogens as beneficial to the immune response. Diethylstilbesterol (DES) was found to stimulate the reticulo-endothelial system, increase serum gamma glob- ulins twofold and increase specific antibody production eightfold (Cordingley and Nicol, 1961). Estrogen adminis- tration to female castrates increased the resistance to viruses (Sprunt and McDearman, 1940). Von Haam and Rosenfeld (1942c) reported that estrone increased resistance to pneumo- coccus infection in mice. The antibody response started earlier, peaked higher and lasted longer following estrone treatment (von Haam and Rosenfeld, 1942a). DES administra- tion did not produce a significant effect. Von Haam and Rosenfeld (1942b) also noted a "non-specific" increase in hemagglutinin titers following estrone administration alone, but after pneumococcus vaccine estrone administration resulted in the stimulation of more specific antibodies. DES treated mice were highly resistant for 8 to 10 days to a virulent dose of hemolytic streptococcus (Foley and Aycock, 1944). On the other hand, Southam and Babcock (1951) could not demonstrate any protective influence of estradiol admin- istration on virus infections. 29 Estrogens seemed to exert no detectable effect on tuberculosis immunity in female guinea pigs (Gray and Black, 1939), but 1220 and Cicado (1947) reported that estradiol diproprionate administration decreased survival in tubercu— 1ar guinea pigs, perhaps through a pituitary mechanism pro- ducing hypothyroid animals. If the animals were castrated their survival rate increased. Prolonged testosterone administration can also result in thymic involution (Weaver, 1955). Testosterone's beneficial effects seem to be less than estrogen. Crabtree ._t gt. (1939) reported that male rats were more resistant to the raticide red squill than females. The presence of the testes or testosterone was reported to be responsible for this resistance. However, testosterone administration had no influence on survival rate with viral infections (Southam and Babcock, 1951) and decreased the survival rate in tuber- cular guinea pigs (1220 and Cicado, 1947). Testosterone is an anabolic hormone and Kalter _t__l, (1951) related this effect on protein metabolism to an influ- ence on the increased proliferation of influenza virus. Castration resulted in decreased viral growth. Progesterone administration seems to have little effect on immune responses. There was no apparent influence on virus infections (Southam and Babcock, 1951) or on the course of tuberculosis in guinea pigs (Greene and Morgan, 1938). However, in poliomyelitis, progesterone administration 3O offered the most complete protection of the hormones tested (Anderson and Bolin, 1946). The levels of IgG and IgA immunoglobulins are not different in human adult males and females, but IgM is sig- nificantly higher in females (Butterworth gt gt., 1967) The examination of the influence of various hormones on a single disease, poliomyelitis, will serve to illustrate the fact that while hormones improve the resistance of an animal this may not necessarily be related to immune mech— anisms. Teodoru and Schwartzman (1954) reported that in the hamster there was a seasonal increase in adrenal gland activ— ity. With increased adrenal gland activity the mortality to poliomyelitis increased. The adrenal hormones, desoxycorti- sone acetate and cortisone, also increased the susceptibility to infection, but adrenalectomy decreased mortality. The work of Schwartzman gt_g;, (1955) served to cast new evidence on this phenomenon. They noted that brown fat was the prin- ciple extraneural proliferative site for the virus. Brown fat is an extremely labile connective tissue mass that responds to seasonal and temperature changes, stress and endocrine influences. Cortisone increased the weight of brown fat with a parallel virus proliferation. Adrenalectomy decreased the weight of brown fat with parallel decrease in viral numbers. Thyroid.--Dougherty's work (1952) on hormones in immune responses indicated that thyroidectomy could decrease thymus weight and that thyroxin administration could 31 counteract this change. Dunn (1954) agreed that hypothy- roidism led to slight thymic involution. Thyroidectomy could decrease the immune related complement concentration in rabbit blood serum. Thyroxin administration increased the serum complement titer. The response to vaccinia virus was not altered by thyroidectomy or thyroxin administration (Cope and Kopnick, 1940). In hypopituitary immune deficient mice thyroxin and somatotropic hormone injections could restore immune efficiency (Pierpaoli gt gt., 1969). The involvement of thyroxin in the total body metab- olism complicates clear cut definition of a direct involve— ment in the immune response. The total involvement of hormones in the immune response has been further reviewed (Dougherty, 1952; Dunn, 1954; and Melnotte _t gt., 1958). Pregnancy.--The relative resistances of the pregnant versus the nonpregnant animal has been a matter of conten- tion. Early work by Jungeblut and Engle (1933) on the virucidal activity of human sera suggested that body resis- tance increased during pregnancy; B sera had four times the activity of A, and O was intermediate. Studies on serum precipitin titers in anaphylaxis in the rabbit during preg- nancy indicated that antibody titers increased during preg- nancy (Jackson, 1935). In studies on tuberculosis, Burke (1940) reported that pregnancy had no apparent effect on the course of tuberculosis in the rabbit. But Wade's studies in 1942 indicated the opposite effect—-that pregnancy did ex- tend the life of the infected rabbit and even decreased the N‘h EV“ 32 severity of the disease. Greene and Morgan (1938) also found improvement in tuberculosis in women in the early stages of pregnancy. Knox (1950) in his investigation on poliomyelitis found that pregnancy increased susceptibility twofold over controls. In humans, Mitchell gt gt. (1966) reported that leucocytes from pregnant women were more phagocytic and that the sera was more bactericidal than the sera of the nonpreg- nant women. Pregnant patients had a high hemagglutinin titer to Esherichia coli. The women were also particularly resistant to gram negative organisms. The hormonal state of pregnancy is complex, and may vary from one animal species to another and definitely varies during the course of pregnancy. The action of all the hormones involved, superimposed on the immune mechanisms, makes analysis of the immune response in pregnancy very intricate. Germfree state.--The concept of the germfree animal in research was eXpounded upon by Gordon in 1965. The many distinguishing characteristics were described in detail. The relationship of the intestinal tract to the immune response is worthy of special notice. Gustafsson and Laurell (1958) reported that 5th gen- eration germfree rats had significantly lower serum concen- trations of beta and gamma globulins than control animals. The normal rat was also able to produce gamma globulins 33 three times more rapidly than the germfree counterpart. The authors suggested that the normal intestinal flora or microorganisms, which the germfree animal lacked, were an important stimulus for gamma globulin producing cells. This was further complemented by Gordon and co-workers (1966) when they suggested that the intestinal flora induced a mild inflammation in the intestinal wall. The germfree animal, therefore, had an underdevelOped intestinal defense system. Bosma's group (1967) refuted this in mice tested for hemag- glutinin and hemolysin titers. The conventional and germ- free animals were similar, so they concluded that natural microbial flora and maternal antibodies were not needed for the normal develOpment of progenitors of hemagglutinin and hemolysin producing cells. Recent work has again questioned the relationship of microbial flora to the immune response. The intestinal lamina propria is rich in IgA producing plasma cells. The germfree animal has a paucity of intestinal plasma cells. Conventional mice have a high content of IgA which decreases from the duodenum to the colon. It has been suggested that the IgA difference between conventional and germfree animals may relate to the microbial flora (Crabbé gt_gt,, 1969). Reference to the phagocytic function by Perkins gt_gt. (1966) indicates that the activity is similar in germfree and con- ventional mice. 34 Agg,—-The importance of the macrophage system to antibody production has been discussed and the importance of its development with age has already been related to the phenomenon of immune tolerance. Culbertson's investigation (1939) exemplifies this research. Rats from 6 to 60 days of age were compared for their phagocytic ability; the ability increased with age. Stern (1963) has reviewed the activity of the reticulo-endothelial system in old age. Initial investigations by Baumgartner (1937) showed that the precipitin reaction increased with age when rabbits 7 to 8 weeks of age were compared to 2 year old rabbits. Sprunt (1939) also demonstrated that the ability to produce antibodies increased with age. Subsequent studies on humans are not conclusive. Sabin's investigation (1947) with antibody develOpment to Japanese B encephalitis indicated that the age group from 15 to 35 years of age had higher antibody levels than the group from 35 to 56 years of age. In reSponse to Salmonella Itypht, human agglutinin titers in ages 15 to 65 and 66 to 78 years of age showed no age related differences (Brenner gt gt., 1951). Acheson and JeSSOp (1962) studied serum gamma globulins in human males aged 65 to 85 years. A titer curve peaked at 75 years of age and then declined. Recent investigations on rats and mice have shown that Optimal antibody forming capacity progressively de- creased with age. Studies by'Makinodan and Peterson (1962) with mice 1 week to 29 months of age showed increased 35 antibody capacity up to 8 months and a gradual decrease over the subsequent 21 months. Goullett and Kaufmann's (1965) study confirmed this trend in rats. Studies were conducted on rats from 3 to 32 months in age. YOung animals had inferior antibody production. This improved and stabilized in mature animals and decreased in the aged animal. The vigor of homograft rejection in old mice of 15 to 20 months of age was as strong as in young animals (Krohn, 1961). Other factors.--Some very basic physiological param- eters are important in immune evaluations. The nutritional state of the animal is known to be important in immune reac- tivity. Cannon (1942) demonstrated that the formation of antibodies, modified serum globulins, was dependent upon the intake of amino acids. Low protein decreased the capacity to form gamma globulins. Vitamin deficient states were shown to be concomitant with the antibody response and reticulo-endothelial activity (Wertman gt_gt., 1954; Axelrod and Pruzansky, 1955; Cordingley and Nicol, 1961; and Harmon _t__t,, 1963). Antibodies, in addition to their obvious role of deactivating antigens, have also been shown to be instrumen— tal in a feedback system which regulated the antibody levels (Wigzell, 1966). Antibodies reacted during the macrOphage dependent stage of the primary immune response to combine with antigens to surpress the immune response (Pierce, 1969). The ratio of the antigen-antibody combination directed the 36 outcome of the antigenic stimulus. Antibody provided recog- nition and regulated the response level (Myers, 1969). The study of circadian rhythms or biological clocks relate to many aSpects of man's existence. ,Mills (1966) has reviewed the many investigations on ramifications of this phenomenon. Of particular interest was Pincus's observation (1943) that a diurnal rhythm existed in adrenal function and Doe gt gt. (1956) observation that blood eosinOphil numbers followed this rhythm. Circadian rhythms also have been found to influence births and deaths (Halberg, 1960), para- sites (Hawking, 1963), toxicity (Ertel _t__t,, 1963), body temperatures (Miles, 1962) and potassium and sodium excre- tion (Lewis and Lobban, 1957), etc. Each of these factors relate to physiological states of the animal which may in- fluence the components of the immunological equilibrium. Recent work by Stein _t_gt, (1969) with hypothalamic lesions suggested that psycosocial phenomena may modify immune responses by a central nervous system mechanism. This may relate to the central nervous system's influence on endocrine function, the autonomic nervous system and other intermediate processes. Earlier allusions were made to the central nervous system's involvement by Berezhnaya (1959) When he showed that conditioned or nonconditioned stimuli influenced nonspecific mechanisms which influenced antibody formations. Finally, surgical procedures and anesthesia affect iWUmunity. Surgical stress depressed immunity (Bruinauskas 37 _t_gt,, 1965; and Riddle and Berenbaum, 1967). In the rat surgical depression of the immune response was related to decreased peripheral leucocytes, decreased numbers of anti- body cells in the spleen as well as decreased serum antibody titers (Wingard gt_gt,, 1967). COppinger and Goldner (1950) have shown extensive age related blood eosinOphil changes with surgery. Blood and Tissue Cells Blood Cells Investigations concerning the circulating forms of blood cells overwhelm the reviewer. For example, the study of eosinOphils began in the 1870's, but by the time of Schwarz's study in 1914, he cited over 2700 bibliography references. Information concerning the origin, life span, function and elimination of blood cells has mushroomed since that time. The commonly found circulating formed elements are the erythrocytes, thrombocytes, neutrophils, lymphocytes, bas0phils, eosinOphils and monocytes (Ham, 1965). They vary in relative numbers in the various animal species. Typical control values for the rat are not really possible; the cells vary with the experimental conditions and with the investigator (Schalm, 1965), the area of the sample (Quimby ,_t_gt., 1948), the diurnal patterns (Jakobson and Hortling, 1954), and the age of the animal (Reich and Dunning, 1943). 38 Origin.--The involvement of the thymus and periph- eral lymphoid tissue in lymphocyte production has already been described in detail. However, the origin of the other cell forms has been the focal point of numerous investiga- tions. The bone marrow is commonly accepted as the prolif- erative site for all the blood forms (Wintrobe, 1967), including some lymphocyte production (Yoffey, 1966). This by no means indicates that the bone marrow is the only site of production; on the contrary, continuing debates of long standing exist on whether extra-medullary hematOpoiesis does indeed occur. Human erythrOpoiesis begins in the yolk sac as shown by Bloom and Bartelmaz (1940). It will suffice here to trace hematopoietic development as shown by Wayt (1964) in a recent paper on the developing guinea pig embryo. This paper typifies other work reported in this area. In the guinea pig erythrOpoiesis began at 13 days in the yolk sac, in the liver at 24 days, in the spleen at 27 days and then in the bone marrow. Erythropoiesis decreased in other areas, but the bone marrow continued production throughout life. The granulocytic series production first began in the liver at 27 days, in the spleen at 35 days, the bone marrow then produced and maintained the granulocytic numbers. Lymphocyte cell production began in the thymus at 24 days, lymph nodes at 37 days and 40 days in the spleen. Experiments on the embryological develOpment of hemOpoiesis showed the bone marrow as the main structure producing blood forms in the adult (Barnes t al., 1964). 39 Experiments indicate that the potential ability to form cell lines is not confined to the marrow, but under certain con- ditions can exist in the liver and spleen (Barnes gt gt., 1964; Goodman, 1964; and Thompson, 1967), the peripheral blood and cells of the peritoneal cavity (Goodman and Hodgson, 1962; and Cole, 1963) and the tail of the mouse (Robinson _t__t,, 1965). These potential cells were able to restore hemOpoiesis to animals given normally lethal doses of irradiation. The ability to restore hemOpoietic potentials to inhibited animals has been extensively investigated for applicability to transplantation and leukemic therapies. The search here, now evolves on the elusive stem cell. Con— troversy again emerges. Wintrobe (1967) classified the con— troversy into three schools: the monOphyletic (lymphatic tissue can form any type of blood cell); the neo-unitarian (some other cell has the potential, not the lymphocyte); and the polyphyletic (many blast cells form blood cells). Is there only one pluri-potential stem cell for both the erythroid and myelocyte series (Diggs t g;,, 1954; Gurney gt_gt,, 1962; and Hellman and Grate, 1967)? Are there separate stem cells for the erythroid, granulocyte and lymphoid series (Cronkite, 1964; and Killmann gt_gt,, 1964)? Or are there normally erythroid stem cells and myelOpoietic stem cells (Hulse, 1964; Moffatt gt_gl,, 1967; and Korst and Quirk, 1968)? The controversy on whether a 40 single multipotent cell or several types are present has not been resolved (Korst and Quirk, 1968). Function.--The various blood cells are commonly said to be functional in the following states: red blood cells (oxygen and carbon dioxide transport); neutrophils (phago— cytosis); eosinophils (phagocytosis, allergy and delayed hypersensitivity); basophils (not phagocytosis, perhaps anti- coagulation; monocytes (phagocytosis); and lymphocytes (anti- body production) (Thompson, 1967; and Wintrobe, 1967). Yet, it is commonly accepted that, despite the multitude of research conducted on these "common" cell forms, we have much to learn about their origin, life span, function and elimination. Many reviews have attempted to tabulate what is currently accepted (Garrey and Bryan, 1935; Sturgis and Bethell, 1943; Ficktelius, 1953; Tullis, 1953; and Yoffey and Courtice, 1956). In complex organisms the ability to maintain homeo- stasis is essential for life. The bone marrow and its progeny are intricately involved in this life struggle, and yet this tissue is not inherently stable. It is subject to many changes in internal environment (hormones, anoxia, nutrition, anesthesia, parasites, stress, irradiation, drugs, and chemicals, disease, sex, age, etc.) and it responds to these changes. Factors that affect blood cells.--Anesthetic agents influence blood cells. Green and Eastwood (1963) found that nitrous oxide decreased peripheral counts of white blood 41 cells and depressed bone marrow production. Halothane proved to have granulocytopenic effects which seemed to act through a cytoplasmic disruption rather than an effect on DNA (Bruce and Koepke, 1966). Bruce (1966) further demon- strated that halothane inhibited extra-vascular mobilization of neutrophils which he postulated as resulting from either a change in the pliability of the leucocyte or the permea- bility of the blood vessels. Hubler and his co-workers (1952) subjected rats to hyperthermia. The elevated temperature produced lymphOpenia, eosinOpenia and neutrophilia. Adrenalectomy abolished the lymphocyte and eosinOphil reduction. This resulted in the suggestion that the mechanism was pituitary-adrenal depen- dent; however, the neutrOphilia was unaffected by adrenal- ectomy. Handling untrained rats for bleeding and injection resulted in a decrease in circulating eosinOphils (Recant, 1950), and in lymphocytes (Stavitsky, 1952). Speirs and Meyers (1949) reported that factors that produce stress responses (cold, ACTH, hemorrhage, handling, etc.) decreased eosinophils. Essential amino acid deficiencies can cause eosino-. penia (Aschkenasy, 1964). Starvation decreased eosinOphils (Opie, 1904). The exposure of rats to irradiation showed that the destructive influence had a predilection for the endocrine organs, the intestinal tract and the urinary-genital organs 42 (Berdjis, 1963). The bone marrow cells were also markedly reduced and their mean generation time reduced (Lord, 1964). This phenomenon of reduced generation time allowed erythro- cyte numbers to remain stable despite reduced marrow eryth- rocyte numbers (Lamerton and Lord, 1964). Humans subjected to pelvic x-ray showed eosinOphilia. If x—rayed in the thorax or abdomen, eosinOpenia occurred, but x-rays of the head produced no change in eosinOphils (Kurohara t al., 1964). Lott (1967) showed decreased eosinOphil numbers in x-rayed rats. Allergic reactions produced increases in eosinophil levels (Connell, 1968). Antigens increased eosinophils (Chapman, 1963; and Litt, 1964a and 1964b); the increase was related to the number of molecules and not to their size (Cohen and Sapp, 1967). Allergic eosinophilia was produced in female genital organs with tissue breakdowns (Divak and Janovski, 1962). Penicillin has revolutionized medicine and helped to conquer some diseases, but it is a two-edged sword, for it can cause significant bone marrow depression (Levitt gt_g;,, 1964). Opie in 1904 showed that guinea pigs infected with Trichina spiralis had increased blood eosinophils commensu- rate with a decreased fatty content of the bone marrow. The amounts of blood changes during disease are innumerable. Decreased lymphocytes and increased leucocytes 43 in typhoid infection are only examples of these diagnosti- cally applicable changes (Lewis and Page, 1948). In rabbits and humans females had more circulating baSOphils than males (Thonnard-Neumann, 1963). Male guinea pigs had less circulating eosinophils than females (Dworetzky _t__;,, 1950; and Schweizer, 1957). Older humans and rabbits were shown to have higher blood baSOphil numbers than younger animals (Thonnard- Neumann, 1963). However, in rats compared between 50 and 400 days of age, baSOphils, eosinOphils and monocytes were relatively unchanged, but neutrOphils were markedly in- creased and lymphocytes decreased with age (Reich and Dunn- ing, 1943). Hourly production rates of eosinOphils de- creased with age only when related to body weight (Alexander ,_t,gt., 1969). As with research on the hormonal influence on anti- body levels, the research reports on hormone influence on leucocytes is vast. Conflicting reports by investigators are often hard to interpret, but variations in experimental animals and design are often responsible. Gordon and Charip- per (1946) have reviewed the endocrine role on hemOpoiesis. HypOphysectomy resulted in a decrease in red and white blood cells, including neutrOphils and lymphocytes, concomitant with a bone marrow hypoplasia (Gordon, 1954). GonadotrOpin administration resulted in reduction in circu- lating baSOphils in rabbits (Zachariae t 1., 1958). 44 Coitus and other sexual excitement also depressed basophils (Boseila, 1959). Boseila (1958) reported that ACTH could depress circulating basophils. ACTH was also able to depress blood and tissue lymphocytes (Hill t 1., 1948). ACTH has a definite eosinOpenic effect (Thorn _t _t., 1948; Thorn, 1950; Kellgren and Janus, 1951; Boseila, 1958; Dworetzky gt_gt., 1950; and Tonelli _t__t,, 1964). Epinephrine administration resulted in a decrease in mononuclear cells. This response was unaffected by hypophys- ectomy, but abolished by adrenalectomy, indicating the mecha- nism is dependent upon adrenal cortical stimulation (Hunger- ford, 1949). The rapid influx of polymorphonuclear cells into inflammatory sites was inhibited by cortisone administration (Gell and Hinde, 1951). Cortisone was shown to have a de— pressing action on the bone marrow (Cardinali t gt., 1964). Cortisone also depressed lymphocyte production and lymphoid tissue weights, but the adult animal seemed to be more sensi- tive to this action than the fetus (Angervall and Lundin, 1965). Hydrocortisone seemed to have more of an effect upon the short-lived small lymphocyte than upon the long-lived small lymphocyte (Esteban, 1968). Thorn (1950) found the eosinopenic effect of ACTH to be dependent on the adrenal cortex. Cortisone was shown to depress circulating eosino- phil numbers (Dworetzsky gt_gt,, 1950; and Kellgren and Janus, 1951). The daily diurnal eosinOphil blood patterns were reported to be related to circulating corticoid levels 45 (Visscher and Halberg, 1955; Panzenhagen and Speirs, 1953). Cortisone implants within the peritoneal cavity were able to depress circulating eosinophils (Panzenhagen and Speirs, 1953). Hortling 2E.§L- (1964) further showed that certain l7-hydroxycorticosteroid levels could depress circulating eosinOphil numbers. Recent work with the synthetic gluco- corticoid, dexamethasone, demonstrated its ability to depress marrow, blood and tissue eosinOphils (BlenkinSOpp and Blenkinsopp, 1967). Testosterone administration is a powerful stimulus to erythrOpoiesis and corrected the depressing effect of hypOphysectomy on red blood cell production (Gordon, 1954). However, it had no effect on either rat circulating eosino- phils or tissue mast cells (Baker _t _t., 1967). In guinea pigs testosterone administration induced blood eosinOpenia (Schweizer, 1956). Testosterone was eosinOpenic in guinea pigs with or without the presence of the pituitary (Schweizer, 1957). .Estrogen's eosinOpenic action on the vaginal smear is well known (Biggers and Claringbold, 1954). The effect of estrogen administration on the uterus resulted in a peak eosinOphilia at estrus and proestrus (Bjersing and Borglin, 1964; and Lobel gt.gt., 1967). In late estrous and post- estrous stages macrophages ingested the uterine eosinOphils (Ross and Klebanoff, 1966). Spayed rats treated with estro- gen exhibited uterine eosinOphilia (Bjersing and Borglin, 46 1964). Estrogen administration also resulted in eosinOpenia when the pituitary was removed (Schweizer, 1957). Progesterone administration decreased uterine eosino- philia (Baker gt_gt,, 1967). Progestational cyclic phases characterized eosinopenia (Bjersing and Borglin, 1964). At the stage of ovulation baSOphils were reported to leave the blood and invade the genital organs (Zachariae gt_g;,, 1958). ErythrOpoiesis is stimulated by pregnancy in the mouse (Fruhman, 1968). Jepson and Lowenstein (1964) reported that prolactin administration stimulated erythrOpoiesis in normal and cas- trated animals. Lactation was associated with pronounced t al., 1926). neutropenia and lymphopenia (Emmel ErythrOpoietin administration stimulated erythrocyte production (Erslev, 1964). Insulin treatment induced blood eosinopenia (Alvarez- Buylla and De Alvarez-Buylla, 1968). Leucocyte elimination is mediated by the lungs, intestine, liver and spleen (Bierman t 1., 1955; and Ambrus and Ambrus, 1959). Tissue Cells The blood forming elements are thus responsive to environmental influences in the marrow, in the blood stream and in the tissues. Each of the circulating blood forms has a comparable counterpart in the tissues camouflaged by a variety of names. 47 The origin of these counterparts is and has been a matter on which there are diverse views. These counterparts are enmeshed in the supportive tissues of the body. A vari— ety of cells are found: reticular cells, fibroblasts, mesen- chymal cells, adventitial cells, histiocytes, macrOphages, monocytes, eosinophils, basophils, mast cells, neutrophils, lymphocytes, plasma cells and a host of unclassified "tran- sitional" forms. At one time or another a proposed series from tissue stem cells has involved each of these forms. Fibroblasts.--Fibroblasts are elongate, basophilic staining cells which may contain fine granules. They elab- orate collagen and are reported to be differentiated cells (Ham, 1965; and Bloom and Fawcett, 1965). Estrogen in- creased fibrinolytic activity (Kwaan and Albrechtsen, 1966), and glucocorticoids (Taubenhaus and Amromin, 1949; Bhisey and Sirsat, 1966; and Berliner and Ruhmann, 1967), and progesterone (Bhisey and Sirsat, 1966) inhibited fibroblast activity. Desoxycorticosterone acetate stimulated fibro- blast activity (Taubenhaus and Amromin, 1949; and Bhisey and Sirsat, 1966). Fibroblasts have also been implicated as transition forms in cell morphogenesis. Bloom and Taliaferro (1938) claimed that fibroblasts could form large lymphocytes. Fibroblasts or undifferentiated mesenchymal cells were also reported to give rise to immature mast cells (Asboe-Hansen, 1959) and eosinOphilic plasma cells (Nellor and Brown, 1966). Another connective tissue form, the primitive mesenchymal 48 cells, have been viewed as precursors of red blood cells, white blood cells, phagocytes and connective tissue cells (Bloom, 1940). Klein and Block (1953) suggested that undif- ferentiated mesenchyme is the source of plasma cells. In inflammatory responses Libénsky (1960) claimed that the mononuclears at the site of inflammation were formed from mesenchymal rather than blood origin. 12.!lEEQ studies on the rabbit suggested that the omentum could form fibroblasts and macrOphages (Aronson and Shahar, 1965). Blood buffy coats also produced fibroblasts and connective tissue tg_ytttg_(Hjortdal and Rasmussen, 1969). Mast cells.--The mast cells are the bas0philic tis- sue form named by Ehrlich in 1879 (Ham, 1965). They contain heparin and histamine and stain metachromatically (Riley, 1959). Mast cells also contain hyaluronic acid and stain at very low pH's (McManus, 1954). A correlation has not been established between blood and bone baSOphils and mast cell numbers (Juhlin, 1963). Mast cells are also responsive to hormone administra- tion. The influence of estrogen is not clear. Johansson and westin (1957) reported a local reductive action of estrogen on uterine mast cells in mice, but not on cutaneous mast cells. Zachariae (1958) found that estrogen increased mast cell numbers in the rabbit genital tract. In Harvey's hamster study of 1964, uterine mast cells were highest at estrus and lowest at proestrus. Ovariectomy decreased mast 49 cell numbers. Estrogen influence caused mast cell produc- tion and progesterone influence caused disruption. However, treatment with 0.6 gamma of estrogen depressed mast cell numbers. Mast cells were distributed evenly along the length of the uterus. Work in cattle demonstrated the presence of large numbers of mast cells at proestrus and estrus, fewer at met- estrus and a moderate number at diestrus (Likar and Likar, 1964). Levier and Spaziani in 1966 reported mast cell numbers to be highest at diestrus and lowest during estrus or in ovariectomized animals. Baker gt gt. (1967) could demonstrate no estrogen influence on the mast cell numbers in the rat uterus. Cortisone and cortisol administration decreased mast cell numbers (AsboeeHansen, 1950), and cortisone treatment resulted in the degranulation of the mast cells (Cavallero and Braccini, 1951). Résanen (1960b) showed that ACTH admin- istration in the intact rat resulted in a rapid and almost complete degranulation of gastric mucosal mast cells. This influence could be prevented by adrenalectomy. Adrenalec- tomy resulted in a slight increase in mast cell numbers in the mucosa. He also reported that prednisolone degranulated mast cells. Cortisone treatment inhibited mast cell prolifera- tion in culture systems (Paff and Stewart, 1953). Thymectomy produced no changes in mast cells in mice (Walker, 1964). 50 Testosterone treatment increased mast cell numbers in ovariectomized guinea pigs (Iversen, 1962) and in male hamsters (Kelsall, 1961); but decreased mast cell numbers in the mouse uterus (Johansson and Westin, 1957). Age does not appear to profoundly influence mast cell pOpulations (Selye, 1965). In the rat there was a progressive decrease with age in the mast cell numbers around the heart (Constantinides and Rutherdale, 1954). Padawer and Gordon (1956) reported, in the rat, that the peritoneal mast cell numbers and the number of abnormally shaped cells increased with age. As age increased mast cell turnover was less rapid (BlenkinSOpp, 1967). Mast cells are presumably derived from lymphoid cells ig_ytyg_(Riley, 1959) and tg_ytttg_(Ginsburg and Sachs, 1963). Mast cells are suggested to be eosinophilic precur- sors (Baker EE.§L-: 1967) and also to be derived from eosin- ophilic cells (Van den Hooff, 1962). Mesenchymal cells or fibroblasts are also suggested to be precursors of immature mast cells (AsboeeHansen, 1959). Lymphocytes.--The origin and early distribution of lymphocytes has been linked to immune responses. The lympho- cyte may also have a role as a pluri-potential tissue cell. Reports in the literature suggest innumerable functions. Early work reported fibroblasts giving rise to lymphocytes (Bloom and Taliaferro, 1938). Pale transitional cells were cited as precursors to lymphocytes (Moffatt t 1., 1967). 51 In thymic regeneration studies, reticular cells or large lymphocytes formed mature lymphocytes (Lundin and Schelin, 1968). Lymphocytes may form plasma cells (Braunsteiner gt_ gt,, 1953; Tompkins, 1960; Moore and Schoenberg, 1964; Sainteiharie, 1964; and Potter and MacCardle, 1964), red blood cells and granulocytes (Bloom, 1937; and Jordon, 1939), histiocytes and macrophages (Downey, 1955; and Braunsteiner ._t._t., 1953), mast cells (Riley, 1959; and Ginsburg and Sachs, 1963) and blast cells (Caron, 1967), if properly stimulated. Lymphocytes undergo nuclear polymorphism and frag- mentation (Kingsbury, 1944). In antibody responses the antibody cells, 5 to 8 days after immunization, lost their cytoplasm and released nuclei when they formed cells resem- bling small lymphocytes (LaVia and Vatter, 1969). NeutrOphils.--The neutrOphilic leucocyte forms are distributed extensively in the tissues, particularly in inflammatory reSponses (Ham, 1965). Thymic pluri-potential stem cells have been shown to migrate to other tissues and form plasma cells, heterophil granulocytes, reticular cells and macrophages (Murray and Woods, 1964). The tissue source of neutrOphils has been attributed to lymphocytes (Bloom, 1937; and Jordan, 1939). ;g_ytttg studies on leucocytes indicated that they form macrOphages and epithelioid cells (Lewis, 1925) and large baSOphilic cells (Bain QE.§L-: 1964). 52 The neutrophilic cell's relation to the estrous cycle is well known in the rat (Long and Evans, 1922; and Mandl, 1951a). The neutrophilic response is not abolished by ovariectomy in the rat (Mandl, 1951b). MacrOphages.--Tissue macrOphages are part of the reticulo-endothelial system (Bloom and Fawcett, 1965). They are comparable to blood monocyte forms and are considered tissue monocytes. Bloom (1940) claimed that mesenchymal cells gave rise to these tissue phagocytes. Macrophages appearing during inflammation were shown to originate from monocytes and thus from a hematogenous origin (Kolouch, 1939; and Cohn and Benson, 1965). Downey (1955) claimed that macrOphages originated from lymphocytes. ;g_yiggg blood cultures of leucocytes from chickens, hens, mice, guinea pigs, dogs and humans formed macrOphages (Lewis, 1925). Plasma ce11§.--P1asma cells seldom appear in the blood and have no blood counterparts. For this reason their origin poses a unique problem--from.whence their origin. Plasma cells are heavily distributed in the body connective tissues supporting wet epithelial membranes. They are usually described as oval, baSOphilic cells with an eccen- tric nucleus in which the chromatin is arranged in wedge- shaped clumps in a spokewheel pattern around the nuclear membrane. Some demonstrate a perinuclear halo (Ham, 1965; and Bloom and Fawcett, 1965). Plasma cells may also form large irregular eosinOphilic granules called Russell bodies. 53 These are condensed secretory products (White, 1954) and not simply a degenerate form of the plasma cell as once proponed by Thiéry (1960). Research on the plasma cell commenced in 1890 when Ramon Y. Cajal first accurately described this cell (Michels, 1931). The mystery on the physiological role of the plasma cell continued until Fagraeus (1948) demonstrated that plasma cells are the body antibody producers. The speculation on the origin of the plasma cell is extensive and contemporary. Klein and Block (1953) reported that undifferentiated mesenchymal cells gave rise to plasma cells. Fagraeus's extensive research suggested that retic— ular cells were precursors of the plasma cells. Lymphocytes, due to their similar morphology, have often been cited as plasma cell precursors (Thompkins, 1960; and Moore and Schoenberg, 1964). Saintefluarie (1964) reported that lympho- cytes were precursors of plasma cells, but emphasized that plasmacytOpoiesis deve10ped along different lines. Follow- ing formation, plasma cells were said to travel to other areas by perivascular channels. Thymic pluri-potential stem cells have been claimed to be the ultimate source of plasma cells (Murray and Woods, 1964). Potter and MacCardle (1964) suggested a dual plasma cell source from both mesenchymal cells and endothelial cells. Adventitial cells were also claimed to proliferate into plasma cells (Hirata-Hibi, 1967). 54 The fate of plasma cells is still debated. It is a terminal cell form? Recently, Swartzendruber (1964) re- ported that spleen cells phagocytized plasma cells and affected their removal. The degeneration or release of Russell bodies has also been suggested as a destructive phase of plasma cells (Thiéry, 1960). Plasma cells may also transform to other cell types (Bloom and Fawcett, 1965). Bueno (1950) stated that plasma cells reverted to reticular cells. Eosinophils.--Jones, in 1846, and Forster, in 1861, first described the eosinOphilic leucocytes and Ehrlich, 1878-1879, described the distinct staining of the granules by eosin. The bone marrow origin has already been described, but the extramedullary myelopoiesis also occurs (Marshall, 1956). Rytomaa (1960) in his extensive work on the organ distributions of eosinOphilic cells reviewed work claiming that eosinophilic production occurred in the sclerotic aor- tic wall, adrenal gland, renal hilus, alimentary canal, spleen, mesentery and stroma tissue of tumors. Bloom and Fawcett (1965) still maintain in this edition of their work as they have in the past that the intestinal tract has spe- cial conditions prevailing that allows local eosinOpoiesis. In the rat spleen eosinopoiesis has been reported to take place unrelated to pathological states. Cells develop with a solid nucleus which develOps a central hole that enlarges until a ring-shaped nucleus emerges (BroéRasmussen and Hendriksen, 1964). Uterine eosinOphils have been reported 55 to develOp from baSOphilic plasma cells (Nellor and Brown, 1966). Others believe that the bone marrow is the only source of eosinOphilic cells (Homma, 1921; and Sundell, 1958). Enzyme patterns of rat uterine eosinOphilic cells were reported to be similar to blood cells and this led Fischer and Schaefer (1966) to suggest that uterine eosino- philic cells were of hematogenous origin. Foot (1965) reported that eosinOphilic cells migrate to the intestinal tract from the blood. The numbers were unrelated to the age of the animal. However, Rytbmaa (1960) after his extensive study on eosinOphilic cell distribution continues to suggest that local eosinOpoiesis was possible. Many transformations to and from cell types have been proposed. Hulse (1964) described eosinophilic cell develOpment in the rat. Gansler (1956) reported that eosin— ophilic cells could be formed from uterine smooth muscle cells. The influence of estrogen on mast cells suggested to Baker and his co-workers (1967) that mast cells formed eosin- Ophils. Van den Hooff (1962) stated that eosinOphilic cells formed mast cells. Maximow (1927) reported that the alimen- tary tract eosinOphilic cells arose from lymphocytes. Braun- steiner and Zucker-Franklin (1962) reported that this did not occur. Von.Mollendorff and von Mollendorf (1926) indicated that eosinOphils originated from a connective tissue network of fibrocytes. Bloom (1937) described lymphocytes as pro- ducing eosinOphilic cells ig_vitro. Oeller (1925) cited the 56 source of eosinOphilic cells to be the endothelium of blood vessels. Godlowski (1953) suggested that any cell could become an eosinophil upon exposure to protein-antigen. Plasma cells have also been observed with eosin- Ophilic granules (Mjassojedoff, 1926; and Maximow, 1927). Despite the many investigations and the wide dis- tribution of body eosinOphilic cells, a statement on their precise function can not be agreed upon. Gastrointestinal Tract Histology The histology of the gastrointestinal tract is mor- phologically readily identifiable from the mouth to the anus. Certain basic features are common to all portions: a mucosa composed of a continuous epithelial lining with a protective mucous covering rests on an area of loose connective tissue (lamina prOpria), glands, capillaries, lymphatics and muscle fibers; a submucosa of connective tissue, blood vessels, lymphatics, glands and Meissner's nerve plexus; a muscularis area of two to three muscle layers, depending on the tissue location, separated by connective tissue and the Myenteric plexus of nerves; and an outer adventita or serosa of con- nective and adipose tissue interspersed with blood vessels. The epithelium in various parts of the tract has specialized cells described as goblet cells, paneth cells, enterochromaf— fin cells, argentaffin cells, chief cells and parietal cells, which are usually assigned digestive functions. Lymphoid 57 tissue is commonly dispersed in the digestive tract and is concentrated in ileal areas in nodes called Peyers' patches (Bevelander, 1961; Bloom and Fawcett, 1965; Hamm, 1965; and Toner, 1968). The mucous membranes of the gastrointestinal tract are constantly exposed to foreign materials from the exte- rior. The intestinal wall presents an important interface in the body's defense system. What sort of mechanisms exist in the intestine to protect against foreign invasion? Does the intestine actually possess a defense mechanism or is it simply a physical barrier? All the cell types that have already been described as active in defense mechanisms elsewhere in the body exist extensively scattered or condensed in complexes along the digestive tract. The components for an active defense system are present. Cell Types Fibroblasts.--Fibrob1asts are extensive in number in the connective and reticular tissues, but recently a dynamism of fibroblasts has been connected to the constantly renewing epithelial cells. Colonic pericryptal fibroblast sheaths have been reported to renew and migrate with the epithelium as a unit (Pascal gt_g1,, 1968; and Kaye g£_al,, 1968). Lymphocytes.--The lymphocyte pOpulation of the intestines is high. Lymphocytes were found to be mostly 58 intra-epithelial in mice (Andrew and Andrew, 1945), humans (Andrew and Collings, 1946), rats (Andrew, 1965), and ham- sters (Kelsall, 1946). The cells degenerated as they migrated to the lumen and were reported to be part of intra- epithelial defensive cell reaction centers (Andrew and Andrew, 1945; Andrew and Collings, 1946; Andrew and Sosa, 1947). The origin of the intestinal lymphocytes is not certain. Gowans and Knight (1964) reported that large lymphocytes migrated to the intestine from the blood and subsequently became plasma cells. Ackerman (1966) reported the origin of appendix and tonsil lymphocytes to be reticu- lar, mesenchymal or from blood infiltration. Darlington and .Rogers (1966) reported that lymphocytes in the epithelial cells of mice handled 35 S-sulphate and tritiated thymidine far differently than the epithelial cells or the blood lymphocytes. They concluded that there was no evidence of lymphocyte migration from the blood into the intestine and that the intestinal connective tissue was the source of the lymphocytes. Globular leucocytes.--A cell of the gastrointestinal tissues whose real significance is yet to be clarified is the globular leucocyte. It usually assumes an intra- epithelial position, stains eosinOphilic and morphologically resembles a plasma cell (Toner, 1965). The origin has been debated. -Dobson (1966b) described the globular leucocyte as a specialized plasma cell, due to its fluorescent staining. 59 The globular leucocyte seemed to fluctuate in relation to the plasma cell fluctuations. The ultimate origin was reported to be lymphoid. A positive relation to helminth infestations was reported (Dobson, 1966a). Globular leuco- cytes numbers were reported to associate with worm self— cures (Whur, 1966; and Whur, 1967). Carr (1967) studied the globular leucocyte granules and reported them to differ from eosinophilic cell granules and to represent aggregates of antibodies in plasma cells. Jarrett §£_§1, (1967) suggested that rat mast cells of connective tissue differed from lamina prOpria mast cells. They reported that intestinal mast cells formed globular leucocytes. A lymphoid origin has also been stressed (Kent §£_gl,, 1954; Toner, 1965). Kent and c0dworkers (1954, and 1956) reported globular leucocytes to be decreased by administra- tion of adrenal hormones as blood lymphocytes and eosino- phils are decreased. Irradiation and hypOphysectomy resulted in decreased numbers of intestinal globular leucocytes. .EosinOphilic cells and mast cells.-€Rytomaa's (1960) organ distribution study of eosinOphilic cells in the rat indicated that the intestinal tract had a high content of eosinophilic cells. The ileal and gastric cell numbers increased with age up to 6 months and then plateaued. A variety of factors have been shown to affect the eosino- philic cell concentrations. Fasting decreased intestinal eosinOphilic cell numbers (Josey and Lawrence, 1932; and 60 Teir _£_gl,, 1955). A meat diet increased tissue eosino- philic cells more than carbohydrates or fats (Rous, 1908). Helminth infestation induced a fivefold eosinophilia with altered cell granules (Casley-Smith, 1968). Weill (1920) reported that in humans the eosinOphilic cell concentration increased from the stomach to the colon. Newborn animals have less eosinOphilic cells than adults (Rytomaa, 1960), but Rasanen (1958) reported that women before 50 years of age exhibited more gastric eosinophilic cells than after 50 years of age. In contrast, Siurala and co-workers (1959) reported mast cell numbers in men to be less before 50 than after 50 years of age. Teir and c0dworkers (1963) suggested that intestinal eosinOphilic cell concentrations reflected the main site of elimination for the body's eosinOphils. Hormones have been reported to influence numbers of eosinOphilic cells and mast cells. ACTH increased lamina propria eosinOphilic cells and decreased eosinophilic blood levels (Godlowski, 1952). .Essellier _§_gl, (1954) reported that if the reticulo-endothelial system was blocked that the eosinOpenic response to ACTH was abolished. Cortisone injec- tion increased intestinal lamina prOpria concentrations of eosinOphilic cells in the rat (Sundell, 1958). Repeated doses resulted in eosinopenia. ACTH was not eosinOpenic in the hypOphysectomized animal. Apparently adrenal involution following hypOphysectomy prevented the adrenal response. Cor' end to 61 Cortisone administration had no effect if the reticulo- endothelial system was blocked. Chronic corticotropin and cortisone treatment of 10 to 12 days in rats and humans caused significant decreases in gastric eosinophilic cell numbers (Rasanen, 1960b; and Siurala $3.91., 1969). In 1962, Rasanen indicated that the degree of eosinOphilic response depended on the biological activity of the glucocorticoid administered. Dexamethasone administration tremendously decreased gastric eosinOphilic and mast cell concentrations. Histamine, food and insulin were reported to in— crease eosinOphils within 2 to 4 hours in the rat stomach with a concomitant decrease in mast cell numbers (Rasanen, 1964). In subsequent work insulin administration resulted in a biphasic response with an initial decrease in mast cells up to 2.5 hours, then an increase at 3 to 4 hours and again a decrease at 5 hours. The initial response was re- ported to be related to vagal acetylcholine action on hista— mine release and the later response to the hypoglycemic effect of insulin on glucocorticoids (Rasanen, 1969). The origin of eosinOphilic cells appearing in the tissues is a subject of debate. Arguments have previously been presented for tissue eosinOphilic cell production. The situation in the intestinal tract can be added to by a few further references. Duran-Jorda (1943) indicated that large lymphocytes entered the intestine, transformed and passed 62 through a paneth cell stage and eventually formed eosino- philic cells in the intestinal mucosa. Sundell (1958) reported that the blood supplied most of the eosinOphilic cells in response to glucocorticoids because the pro- eosinOphilic cells were only 8% of the intestinal eosino— philic cells and could not produce the large numbers appearing in the tissues. However, Rytomaa's (1960) extensive work on the organ distribution of eosinOphilic cells was summarized by suggesting that local eosinOpoiesis may be a normal event. The concentrations of these cells in the lamina prOpria of the intestinal tract is suggestive of defensive function. The juxtaposition of plasma cells, macrOphages and eosinOphils suggested to Deane (1964) that defensive reactions were occurring at sites of antigen-antibody complexes, and that this was a chronic action in the intestinal tract. This review illustrates that there are many reports in the literature on the influence of the endocrine glands on wandering cells of the intestinal tract. Some are com- plementary and others contradictory, but it must be agreed that a well-defined physiological role of the adrenal glands or gonads on the mobilization, modification or function of the tissue granulocytes has not been develOped. With the recognized role of the endocrine system in embryogenesis and in the develOpment of motility and diges- tive functions it is not unreasonable to assume that hormones 63 might also be responsible for the regulation of an intes- tinal defense mechanism. Particular emphasis should be placed on the role of the adrenal glands and the gonads, since their hormones are already clearly implicated in influencing the blood cells and the lymph nodes in general body defense. Intestinal Immunity The described cell interrelationships are static, mute references to the potential for defensive action. Substantial evidence now exists for an active first line defense mechanism for the tissues associated with the mucous membranes. The relationship of the lympho-epithelial cells of the intestine to antibody production appears established. But lymphoid removal (appendix, sacculus rotundus and Peyer's patches) drastically impaired antibody production in the rabbit but did not impair delayed allergy or homo- graft rejection (Good, 1967; Perey gt al., 1968; and Good _£.gl,, 1969). The plasma cell rich intestinal lymphoid tissue has been equated to a bursa-like immune system (Brandom gt 1;, 1969; Good §£_§l,, 1969; Watson, 1969; and Schofield and Cahill, 1969). The involvement of the intestine in allergic and delayed hypersensitive reactions has been implicated in numerous intestinal disorders (Taylor, 1966). The intestine 64 has been shown to exhibit true delayed hypersensitive reactions that are not related to circulating antibody levels (Bicks t al., 1969; and Bicks and Rosenberg, 1969). Brandom.§£_§l, (1969) demonstrated in primates that local intestinal defense to cholera is paramount and is not related to circulating antibody levels. Thus, the complexity of the intestinal tract emerges. Allusions to the bursa as our previous discussion has shown related to circulating antibody levels which are predomi- nantly IgG. Allusions to delayed reactions and allergy refer to cell bound IgA antibody. As early as 1953, Koshland reported IgA antibody to cholera in rabbit feces. The antibody levels were indepen- dent of serum levels. He reported that the antibodies in the feces were produced at local sites along the intestinal tract and were carried to the lumen by the lymphocytes. Cell bound antibody was produced in rabbits in response to cholera which caused Sanyal and co-investigators (1969) to suggest that local immune mechanisms of the intes- tinal tract were responsible. Local antibody production of cell bound IgA, separate from systemic circulating antibody, has been demonstrated in external secretions of saliva Chomasi g£_gl,, 1965; South t 1., 1966; and Tomasi, 1967), colostrum (Tomasi gt al., 1965), the intestine (Crabbé 1. 1965; South §£_§l,, 1966, Gelzayd §£_gl,, 1967; Crabbé m m n n m H 1968; Plaut and Keonil, 1969; and Crabbé t 1., 1969), and 65 the reproductive tissues (Bell and Wolf, 1967; and Hulka and Omran, 1969). The occurrence of cell bound antibody has been directly related to plasma cell numbers in the human intes- tine in the following ratios: 181,000/cu mm, gamma A type; 18,000/cu mm, gamma G type; and 30,000/cu mm, gamma M type (Crabbé _t_ _l_., 1965) . A relationship of IgA to plasma cell numbers and microbial flora has also been reported (Crabbé __§.gl., 1968). The plasma cells of the intestinal lamina propria can therefore be linked to gamma globulin production. Oral immunization in mice to horse ferritin produced a high intestinal IgA response in the plasma cells. Subcutaneous or intraperitoneal administration produced plasma cells in the lymph nodes and spleen which produced IgM after one challenge and IgG after the second challenge. The predom- inant circulating form was IgG, but immunization by any route resulted in only IgA production in the intestine (Crabbé __t_a_l_, 1969). Crabbé and Heremans (1967) have also shown that the tonsils react like the spleen and lymph nodes with predominant IgG production in contrast to predominant IgA production in the intestine. Tomasi (1967) has further looked at this first line of gamma A defense. He repOrted that secretory IgA of mucous membranes possessed a unique tail piece that lends it stability. The piece may be a transport phenomenon added 66 by the epithelial cells. The piece distinguished secretory IgA of saliva, tears, colostrum, gastrointestinal secretions and mucoid tissues of the genito-urinary tracts from circu- latory IgA of extra-intestinal lymphoid tissues. With these mechanisms in mind the local mechanism of oral immunization seen in regard to poliomyelitis (Keller _§__l,, 1969), cholera (Brandom gt_gl,, 1969), and test antigens (Levanon §£.§L-: 1968) can be more clearly under- stood. RATIONALE FOR THIS THESIS RESEARCH The relationship between tissue cells and the immu- nological capabilities of mucous membranes has been of great interest to our laboratory for years. The recognition that the highest bactericidal activ- ity in the genital tract was coincident with the follicular 1., 1953; and phase under estrogenic influence (Black gt Hawk §£_gl,, 1957) has led to a close examination of the cells of the reproductive tissues at various stages of the estrous cycle. The recognition that a cellular transition, related to hormone influence, existed for the development of tissue leucocytes from baSOphilic fibroblasts through the plasma cell stage to eosinOphilic tissue cells in the fallOpian tubes, the vagina, the uterus and the ovary presented the possibility of a relationship of these cells to the immu- nological competence of reproductive tissues (Nellor, 1963; Nellor, 1965; and Nellor and Brown, 1966). This transi- tional scheme has been verified in cattle, sheep, swine, rabbits, rats and guinea pigs (Montakahabolayaleh, 1964), and has been reported to be concomitant with increased estrogen secretion by the ovary (Brown and Nellor, 1968). 67 inm int tre cle a c tis if Phy: rele tenc trol fUtu 68 This cellular series has also been related to genital tract bactericidal activity in the rabbit. At estrus or during the estrogenic state, the highest bacte— ricidal activity was coincident with the marked mobilization of tissue leucocytes. A tissue transitional series was observed to be activated in the early stages of Eschericia _ggli infection. Highest bactericidal activity was present at a time when eosinOphilic plasma cells were degranulating. Later, leucocytes of blood origin were observed in the tis- sues, but subsequent to the peak of bactericidal activity (Wira, 1966; and Wira and Nellor, 1966). The poignant awareness of these phenomena in the reproductive tissues lead to a natural question about the immune capacity of the extensive mucous membrane of the intestinal tract. The evidence of IgA secretion in the tract is strong. High plasma cell concentrations have been clearly shown. This thesis research was undertaken to determine if a cell series similar to that shown in the reproductive tissues also existed in the intestinal tract; to determine if hormone related cellular changes were evident in control physiological states and if these could be circumstantially related to factors that affect known immunological compe- tence; and if these changes were evident, to determine con- trol values for eosinophilic and baSOphilic cells to which future pharmacological manipulations could be compared. The ten COD! pIOE Stai the red, time! to Va or fr cells After titat 69 Considerable effort was devoted to the development of a suitable procedure that would clearly identify intes- tinal eosinOphilic and baSOphilic tissue granulocytes without intensely staining the remainder of the tissue. A combination of tissue fixation and staining was needed that eliminated intense background staining in order to facili- tate counting procedures. Harris hematoxylin and eosin stain, Mallory's stain, Papanicolaou's stain, Dominci's stain, May-Grunwald's stain, methyl green-pyronin stain and various experimental stains in which eosin, orange G, picric acid, alcian blue, light green and toluidine blue were uti- lized at a variety of pH's, dilutions and staining times. These procedures (Appendix II) gave variations in the in- tensity and range of acidOphilia and baSOphilia but also stained the majority of the tissue components, resulting in considerable subjectivity being introduced during counting procedures. In the process of experimenting with the various stains many cytochemical variable elements were observed in the tissues. Granulated cells were observed to stain purple, red, orange, or yellow, depending on the procedure. Dis- tinctly different nuclear morphological forms were observed to vary from solid to compact, wedge-shaped, ring—shaped, or fragmented. Nongranulated plasma cells and neutrophilic cells also were observed with a variety of nuclear forms. After considerable experimentation it was decided that quan- titation of mononuclear eosinOphilic and finely granulated 70 baSOphilic cells were most pertinent to this research. An alcian blue-distilled water-eosin procedure was develOped (Appendix II) that enabled clear differentiation of these eosinOphilic and baSOphilic tissue granulocytes (Figure 11). Tissues were fixed in Carnoy's, Bouin's, 10% forma- lin, Helly's and BouineHollande fixatives. For the quanti- tative purpose of this study the fixative Bouin-Hollande was selected because it effectively enhanced eosinOphilia. Accurately quantitating cell counts on tissue sec- tions the size of rat intestinal tissues presented many problems. Counts were attempted with 2.5, 10, 25, 40, and 100 X objectives and 12.5 X eyepieces. With the 40 x oil objective it was possible to clearly differentiate cells in a fairly large field. Initial attempts were made to count the entire cross section of the tissue, but errors were induced due to eye fatigue and the difficulty of identifying areas that had or had not been counted. A projecting microscope was used to project the image of the field on a screen, but accurate counts could not be made due to the loss of resolution in projection. Photomicrographs were also prepared of the tissue cross sections, but clear resolution at lower magni- fications and the excessive time and expense involved made such a procedure prohibitive. A reduced transparent photo- graph of a hemocytometer was placed on the microscope field diaphragm and reflected on the bottom of the microsc0pe slide. This procedure divided the tissue sections into 71 squares that were easy to count. Difficulties were encoun- tered, however, when the slide was shifted to a new area, since it was difficult to know where the last counts had been made. Because of the numerous errors induced by these procedures, cell counts on total cross sections by these methods varied tremendously. It was decided to count the tissue cells in only a representative portion of the tissue to determine if a sta- tistically repeatable quantitation could be obtained. A hemocytometer grid was placed in the eyepiece and four or eight areas of tissue lamina prOpria were counted. With this procedure cell counts were obtained that were statis- tically similar as measured by the chi-square goodness of fit test. Tests were done on counts of 20 to 30 serial sections of lamina propria l8 micra apart. These cell counts, as analyzed by chi-square, indicated that the pro- cedure was repeatable and that values obtained were within the same population. Thus, although marked differences were observed among the major areas of the intestine, that is the duodenum, ileum and colon, there was no significant differ- ence in the numbers of granulated cells in the adjacent tissue sections. Less variation in cell counts was obtained if eight different areas were counted as compared to four areas. Cell counts by this procedure were conducted on variously treated and control animals to determine if dif- ferences in cell counts were significantly different. 72 These values were analyzed by Student's T test. The values were shown to be from statistically different populations. Thus, cell count repeatability and significant differences between counts were indicated by these tests. The selection of the Long-Evans hooded rat was apprOpriate since not only is the rat the most resistant of the conventional laboratory species, but the Long-Evans hooded rat excells among the rat breeds as the most hardy. Preliminary experiments were conducted to determine if the level of feeding, body weight gain or the presence of intestinal parasites influenced granulocyte cell numbers in the intestine. No significant differences were observed due to these parameters. It should be emphasized that there is no evidence develOped in this thesis that the observed fluctuations of the tissue granulocytes are directly related to increased intestinal bactericidal activity or involved in immune competencies. The inference that the granulocytes may be related to these phenomena is based on the morphological and histochemical similarity of the intestinal granulocytes to the genital tract granulocytes where a direct relationship of eosinOphilic cell numbers and increased bactericidal activity has been reported. ha: yea ani stL ten Ani fen .MATERIALS AND METHODS* A stable, healthy colony of Long-Evans hooded rats has been develOped at the Endocrine Research Unit by ten years of genetic isolation and elimination of unhealthy animals. These uniform assay animals were utilized in this study. All animals were housed under conditions of constant temperature and humidity in a dark-light ratio of 12:12. Animals received food ad libitum. The following male and female rats were included in the studies: Normal Female Rats (n==55): l. Immature (8 to 28 days of age) . . . . . . (9) 2. Estrous cycle stages (estrus, metestrus, diestrus and proestrus) . . . . . . . . . (24) 3. Pregnant . . . . . . . . . . . . . . . . . (2) 4. Lactation . . . . . . . . . . . . . . . . (15) 5. Anomalies (infertile pseudOpregnant, constant estrus and constant diestrus) . . (5) Normal Male Rats (n==40): l. Immature (8 to 28 days of age) . . . . . . (10) 72 days of age . . . . . . . . . . . . . . (2) 3 months of age . . . . . . . . . . . . . (3) 6 months of age . . . . . . . . . . . . . (3) 6.5 months of age . . . . . . . . . . . . (7) 7 months of age . . . . . . . . . . . . . (5) Aged (over 2 years) . . . . . . . . . . . (2) .Euthanasia (chloroform versus decapitation) . . . . . . . . . . . . . . (8) (Dwain-boom *See Appendices for all procedures. 73 74 Malngat Ablation Studies (n==96): 1. Castration (70 day old animals, 9 days post-surgery) . . . . . . . . . . (18) 2. Castration (6 month old animals, 9 days post-surgery) . . . . . . . . . . . (l8) 3. Castration (post-surgery, 12 hours, 1, 2, 3, 5, 7, 9, 11 and 13 days post-surgery) . ... . . . . . . . . . . (20) 4. Adrenalectomy (post- surgery, 12 hours, 1, 2, 3, 5, 7, 9, 11 and 13 days post- surgery) . . . . . . . . . . . . . . . . . (20) 5. Adrenalectomy-castration (post-surgery, 12 hours, 1, 2, 3, 5, 7, 9, 11 and 13 days post-surgery) . . . . . . . . . . (20) Female rats were selected by daily vaginal smears for the four cycle stages. Vaginal smears were obtained with a metal spatula, spread on a clean glass slide, fixed with Clay Adams spraycyte fixative and stained with Papani- colaou's procedure for vaginal smears. The female rats were followed through two normal 4 day cycles before being select- ed for inclusion in this study. A clean but not sterile technique was used in all rat surgical procedures. Orchidectomy or castration of male rats was performed while the animals were under ether anes- thesia. An incision was made in the tip of the scrotum through which the testicles were drawn. A suture was made around the internal spermatic blood vessels and the sper— matic ducts. The testis, along with the epididymes, was excised. The incision was closed by a surgical wound clip. Animals were adrenalectomized while anesthetized with a 2.5% solution of sodium pentobarbital at a dose rate of 4 to 5 mg/100 grams of body weight. Animals were shaved 75 on both sides in the lumbo-dorsal region and incisions were made on both sides at the level of the center of the kidney in the angle made by the lowest rib. The adrenals and the surrounding fat were removed by pulling them free of their connecting vessels with a curved forceps, taking care not to rupture the adrenal capsule. The incisions were closed with wound clips. The animals were kept warm and free from drafts until they had completely recovered from the anes- thesia. Upon recovery they were maintained under normal colony conditions, but supplemented by a 1% sodium chloride solution. Animals were adrenalectomized and castrated with a combination of the two procedures. Sodium pentobarbital was used for anesthesia and the bilateral adrenalectomy was per- formed first followed by castration. AutOpsies were conducted between 9 and 11 a.m. Except for the euthanasia experiment all sacrifices were made by stunning the animal with a blow on the head followed by quick decapitation. In the euthanasia experiment compar- ison was made between sacrifice by chloroform inhalation until respiration ceased and the quick decapitation method just described. After decapitation tissue pieces were immediately removed from the pre-established areas of the duodenum, ileum and colon; in addition to these, a piece of the uterus was also removed from the females. Tissues were fixed in 76 Carnoy's,Bouin's, 10% formalin, buffered formalin, Helly's and BouinéHollande fixatives. .For the quantitative purpose of this study the fixative Bouin-Hollande was selected as the fixative of choice to demonstrate eosinOphilic cells. The tissues remained in Bouin-Hollande for 48 hours, were transferred to 50% ethyl alcohol for 24 hours and then to 70% ethyl alcohol for at least 24 hours before processing by the autotechnicon through a 10 hour series of 70%, 80%, 95%, 10m%, 100% and methyl salicylate, methyl salicylate, paraffin with a 54 to 56° c melting point, and paraffin with a 56 to 580 C melting point. Tissue pieces were embedded in tissue-mat paraffin with a 56 to 580 C melting point. Tissue sections were routinely cut at 6 micra on a rotary microtome. Stains employed on the processed tissues were Harris hematoxylin and eosin, Mallory's aniline blue, Papanicolaou's, Dominci's, May-Grunwald's, alcian blue-methyl green-pyronin and a develOped alcian blue-eosin combination. The devel- Oped alcian blue-eosin stain clearly demonstrated eosino- philic and bas0philic cells. In order to establish the methods of cellular quan- titation reported in the results, 20 to 30 serial sections were prepared on test tissues 18 micra apart. A Leitz hemo- cytometer grid placed in a 12.5 X eyepiece was utilized in the counting procedure. Counts of eosinOphilic and baso- philic granulated cells were made through a Zeiss microsc0pe with a 40 X oil objective at a total magnification of 500 77 times. Eight random areas of eosinOphilia were counted, 4 at the villi bases and 4 at the villi tips for a total area of 0.5 sq mm. Two cross sections were counted for each animal. Chi-square, standard error and Student's T test analyses were performed on the values obtained. RESULTS Staining The use of an alcian blue-distilled water-eosin stain, develOped during this study, enabled a clear demon- stration of eosinOphilic and baSOphilic tissue cells. Treatment of the tissue with a 10% hydrochloric acid rinse greatly reduced nonspecific staining of tissue components such as nucleic acids and cytoplasm.which normally stain ‘weakly with cationic dyes at pH 3 to 4.: The use of the cat- ionic dye alcian blue (k% alcian blue in 70% alcohol at pH less than 0.1 induced staining of only mucins and some con- nective tissue components. Dilute eosin (0.25%.at pH 6.8) is on the alkaline side of neutrality for the majority of the tissue proteins and so they would have a net negative charge at this pH. The use of the anionic dye eosin at this pH implies that the majority of the proteins would not be stained, since eosin would be attracted to positive or cat- ionic tissue components. Utilization of this procedure in- sured that a limited pOpulation of protein containing cells would be stained: acidic sulphated muc0polysaccharides staining baSOphilic and proteins with an ioelectric point above pH 6.8 staining acidOphilic. 78 79 The majority of the cells with cytoplasmic granules staining eosinOphilic by this procedure were not the typical ring-shaped blood eosinophils of the rat, but were primarily mononuclear forms with the chromatin arranged in clumps around the nuclear membrane, appearing similar to an eosin- ophilic form of the plasma cells. The baSOphilic staining cells were mononuclear, finely granulated large cells that were easily distinguished from the classical mast cell (small, oval cells with large granules found in areas around the blood vessels). All eosinOphilic and baSOphilic tissue cells were included in the counts, although typical mast cells were rarely observed. .Euthanasia The method of euthanasia markedly affected the num- bers of duodenal baSOphilic and eosinOphilic cells as demon- strated in Table l and Figure l. Chloroform inhalation until respiration ceased resulted in a decrease, significant at the .05 level, in duodenal eosinophils, baSOphils and total granulocytes when compared to sacrifice by quick stunning and decapitation. After this phenomenon was evi- dent, all sacrifices were carried out by decapitation. The Normal Female Rat Experiments on changes in cell numbers within the various normal physiological female conditions (Table 2) demonstrated an ovarian cyclic hormonal influence on the 80 duodenal granulocytes. Counts on immature (8 to 28 days) male and female duodenums were not significantly different, but the influence of maturity on cell numbers was signif- icant. EosinOphilic cells (Figure 2) were at the greatest number during estrus and significantly different (P <.05) from diestrous or proestrous cell numbers, but not different from eosinOphilic cells observed during metestrus. The num- ber of eosinOphilic cells observed during metestrus was also significantly different from cells observed during diestrus and proestrus. Bas0philic cell numbers (Figure 3) were at the greatest number during proestrus, but the numbers were not significantly different from the baSOphilic cell numbers during other stages of the estrous cycle. The percentage of basophilic or alcian blue staining cells of the total granu— 1ocytic cells (Table 2) was lowest (19.10%;to 19.15%) during estrus and metestrus, intermediate (24.79%) during diestrus and highest (28.58%) during proestrus. During pregnancy (10 and 18 days) duodenal eosino- philic cell numbers were not significantly different from metestrus, diestrus or proestrus, but were significantly different from estrus. Duodenal baSOphilic cell numbers in pregnancy were significantly higher than those observed dur- ing estrus and metestrus, but not higher than those observed during proestrus and diestrus. The baSOphilic percentage of total granulocytes was 32.68%s higher than any stage of the estrous cycle. 81 During early lactation (only one animal at 8 days) duodenal eosinophilic cell numbers were comparable to the numbers in the diestrous and proestrous stages of the estrous cycle, but basophilic cell numbers were higher than pregnancy or any stage of the estrous cycle. The percentage of baSOphilic staining cells (42.64%) was the highest ob- served in the female rat. Late lactation (21 to 25 days) was associated with significant decreases in the numbers of duodenal eosinOphilic cells, different from those observed during pregnancy and all stages of the estrous cycle. Duo- denal bas0philic cell counts were comparable in numbers in late lactation, estrus, metestrus and diestrus, but were significantly different from proestrus and pregnancy. Five females were examined that represented the hormonal conditions of pseudopregnancy, constant estrus, constant diestrus and infertility over several cycle lengths despite the constant exposure to a.fertile male. Although the absolute number of the duodenal eosinOphilic and baso- philic cells varied over a wide range, the notable feature of this group was that the percentage of basophilic cells of the total cells counted, 25.87% to 37.97%, was higher than the mean estrous cycle percentage of 22.98%. The Normal Male Rat The granulocyte cell distribution in the duodenal tissue of various aged male rats (Table 3) demonstrated that age does affect the numbers of these cells. The numbers of 82 duodenal eosinOphilic cells (Figure 4) increased signifi- cantly from 8 days to 15 through 28 days of age, from 15 through 28 days to 72 days, and decreased significantly from 72 days to 3 months of age. There were no significant dif— ferences in cell numbers from 3 months to 7 months of age, but the cell numbers in this group (3 months, 6 months, 6.5 months and 7 months) were significantly different from the 2 year old age group. A significant change in the duodenal basophilic cell numbers (Figure 5) was seen between 3 months and 6 months of age in the male rat. The numbers were also significantly different between 6 months and 6.5 months, 6.5 months and 7 months, and 7 months and 2 years of age. The percentage of basophilic staining cells progressively in— creased with age. Comparisons of the mean numbers of duodenal granulo- cytes of the female rats during the estrous cycle and mean numbers of duodenal granulocytes of the mature male rats demonstrated no significant differences between the sexes when means of cell numbers were considered. Male Rat Acute Castration Nine—day castration (Table 4) had profound effects on the numbers of intestinal eosinOphilic cells. Castration of 72 day and 6 month old male animals resulted in a signif- icant reduction in eosinophilic cell numbers (Figure 6) com- pared to control values. The duodenal tissue was the most responsive to this change. The ileum was less responsive 83 and the colon did not demonstrate a change in cell numbers following castration. Duodenal eosinOphilic cell numbers were higher in the younger animals. Castration also in- fluenced intestinal baSOphilic cell numbers (Figure 7). Castration significantly reduced baSOphilic cell numbers in the duodenum, ileum and colon. BaSOphilic cell numbers were significantly higher in the older animals than in the younger animals. When the total granulocytes were consid- ered (Figure 8) the variations in eosinOphilic and baso- philic cell numbers in the duodenum and the ileum of control animals balanced each other. Therefore, the total granulo- cyte numbers were not significantly different. At nine days post-castration there was a reduction in the percent- age of bas0philic cells in both age groups compared to the controls-—15.20% at 72 days changed to 11.06%; 25.lr% at 6 months changed to 21.69%. Male Rat Castration Series The influence of castration upon duodenal granulo- cytes was studied at various autOpsy times post-surgery (Table 5). Fluctuations in eosinOphilic cell numbers are noted (Figure 9) that are significantly different between control and immediately post-surgery, post-surgery and 12 hours, 1 day and 2 days, 2 days and 3 days, 2 days and 5 days, 7 days and 9 days, and 9 days and 11 days. The meancell values for all groups are not significantly different from the mean cell values for the control mature males. The mean 84 percentage of the alcian blue baSOphilic staining cells (26.87%) was slightly higher than the mean percentage for the male controls (23.02%). However, percentage at 9 days post-surgery (23.87%) is comparable to the percentage for the mature males and approximates the percentage from the previous 9 day castration experiment. Male Rat Adrenalectomy Series The influence of adrenalectomy upon duodenal gran- ulocyte cell numbers was studied at various autopsy times post-surgery (Table 6). Fluctuations in eosinophilic cell numbers are noted (Figure 9) that are significantly differ— ent between control and immediately post-surgery, post- surgery—l4 hours and day 1, days 1-2 and day 3, day 3 and days 5, 7, 9, 11 and 13 following adrenalectomy. The mean cell values for all groups were significantly higher than those found in the castrated or the mature male rats. The mean percentage of baSOphilic cells (21.94%) was less than that found in the castrated male rat (26.87%). Male Rat Adrenalectomy-- Castration.8erie§ The influence of adrenalectomy-castration upon duo- denal granulocyte numbers was studied at various autopsy times post-surgery (Table 7). Fluctuations in eosinOphilic cell numbers are noted (Figure 9) that are significantly different between controls 12 hours, 1 day and 3 days, and 85 9 days and 13 days post-adrenalectomy-castration. The mean cell values for all groups were significantly higher than those found in rats adrenalectomized or castrated or those found in mature males. The mean percentage of basophilic cells was decreased to 18.75%.of the total granulocytes. Intestinaquengths The measurement of the mean intestinal lengths in mature male and female rats demonstrated that the lengths are significantly different between the sexes (Table 8). The combination of these data and the mean eosinOphilic cell number data indicates the tremendous potential of these animals to produce tissue eosinOphilia under normal physio- logical conditions. DISCUSSION Histological examination of the intestinal tract reveals a complex tissue composed of numerous cell types, many of whose exact functions are yet to be elucidated. Cells emphasized in this investigation have been previously referred to as migrating blood cell forms, and the signifi- cance of their numbers in the intestinal tract has not been clearly defined. The intestinal tract is a very dynamic tissue, easily renewing its epithelial cells every 24 hours (Toner, 1968). The number of tissue forms of eosinOphilic cells, basophilic cells, neutrophilic cells, lymphocytes, macrOphages and plasma cells are also known to vary consid- erably. In this histologically complex and continually changing tissue, cell quantitation is a formidable task. In this present study statistically repeatable methods of quantitation were not obtained with conventional Harris hematoxylin and eosin stain, Mallory's stain, Papanicolaou's stain, Dominci's stain, May-Grunwald's stain, methyl green- pyronin stain and various experimental stains in which eosin, orange G, picric acid, alcian blue, light green and tolu- idine blue were utilized at a variety of pH's, dilutions and staining times. These procedures (Appendix II) stained the 86 87 majority of the tissue components and made counts on indi- vidual tissues quite variable. In the process of experimenting with the various stains a morphological variation became evident among the eosinOphilic cells. Staining with Papanicolaou's procedure clearly demonstrated this variation (Figure 11). Nuclear morphology varied from solid, mononuclear forms resembling plasma cells to ring-shaped structures resembling the rat eosinophilic blood cell. Papanicolaou's stain contains two acidOphilic dyes--orange G and eosin, and both selectively stained morphologically different cells. Mononuclear cell forms were normally stained red by eosin and ring-shaped or fragmented forms were usually stained yellow to bright orange by orange G. Intermediate or transitional forms were noted between these two morphological extremes. Cell forms resembling the develOpmental stages of the rat eosinOphil in the bone marrow were observed, in which a solid nucleus becomes notched with chromatin wedges around the nuclear membrane, and the nucleus becomes slightly annular, even- tually enlarging and resulting in the formation of the typical ring-shaped rat circulating blood eosinOphil (Hulse, 1964). The presence of transitional forms strongly suggests that local eosinOpoiesis occurs in the rat intestine as sug- gested by other investigators (Cooke, 1932; Duran-Jorda, 1943; Godlowski, 1952; and Bloom and Fawcett, 1965). The forms are very similar to those described by BroéRasmussen 88 and Hendriksen's (1964) investigation on local tissue eosinOpoiesis in the rat spleen. In 1902, Howard and Perkins, described in detail similar develOpmental stages in human tissues for the formation of polymorphonuclear cells of varying degrees of eosinOphilia from either a baSOphilic and eosinOphilic plasma cell series or a mono- nuclear leucocyte series. The alcian blue-distilled water-eosin procedure develOped during this study (Appendix II) enabled a clear differentiation of eosinOphilic and bas0philic tissue gran- ulocytes (Figure LU. The pH's of this procedure, < 0.1 for the alcian blue and 6.8 for eosin, limited the cells acquir- ing the stain to very specific pOpulations. This limited staining allowed cell counts to be made (Appendix III) that were statistically repeatable. At pH < 0.1 the cationic dye alcian blue is a sensitive stain for sulphated muc0polysac- charides and eliminates staining of nucleic acids and other cytoplasmic components which normally stain with alcian blue around pH 3 to 4 (Thompson, 1966). At pH 6.8 the eosin solu- tion is on the alkaline side of isoelectric points for most tissue proteins (Sober, 1968). Since some of the gamma globulins have been reported to have isoelectric pH ranges from 5.8 to 7.3 (Sober, 1968) and Russell bodies, which are secretory products in plasma cells (White, 1954), have been reported to have isoelectric pH ranges from 6 to 7.8 (Thomp- son, 1966), it is conceivable that the eosinOphilic cells staining at pH 6.8, which is on the acidic side of some of 89 these isoelectric points, contain immunologically competent cell products. With this procedure it was difficult to accurately determine nuclear morphology but it appeared that the pre- dominance of cells staining eosinOphilic by this procedure were morphologically related to eosinOphilic plasma cell forms. The bas0philic cells were finely granulated, large mononuclear cells. A few baSOphilic cells localized around basal blood vessels, were heavily granulated, oval cells typical of the classical definition of mast cells. Blood eosinOphils were not stained either eosinOphilic or baso- philic by this procedure. Results of the current study demonstrate that intes- tinal eosinOphilia can be influenced by the ovaries, the testes and the adrenal glands. There is a correlation between the eosinOphilic and baSOphilic cell numbers and ovarian cyclic activity. The greatest number of eosino- philic cells were found during the estrogenic phase, and minimal numbers were found during the progestational phase. EosinOphilic cell number fluctuations in the intestine par- alleled changes in eosinOphilic cell numbers in the uterus of the rat as reported by Rytomaa (1960) and as noted in uterine counts in this study. The appearance of intestine baSOphilic cells was inversely related to this eosinOphilia—- greatest baSOphilic cell numbers appeared in the progesta- tional phase of the cycle, and minimal numbers in the estrogenic phase. 90 Table l and Figure 1 demonstrate that not only cyclic ovarian activity but also stress can influence eosin- ophilic and baSOphilic cell numbers in intestinal tissue. This factor was of considerable importance to the remainder of the studies. Six month old male rats were used to test two methods of sacrificing that reflected varying degrees of stress. Chloroform inhalation until respiration had ceased represented a very stressful sacrificial method (fluorometric assays of corticosterone levels averaged 50 ug/100 ml plasma)* and stunning and rapid decapitation (fluorometric assays of corticosterone levels averaged 5.8 ug/100 ml plasma)* represented a less stressful sacrifice method. Glucocorticoid levels in the chloroformed rats were comparable to those determined in 154 day old rats exposed to ether stress (Hess, 1970), whereas the glucocorticoid levels in the rats sacrificed by decapitation were consid- ered to be comparable to a "non—stressed" state, that is less than 10 ug/100 ml of plasma (Riegle, 1970). Chloroform sacrifice resulted in significant reductions in duodenal eosinOphilic and baSOphilic cells compared to decapitation cell numbers. This variation demonstrated an influence of stress on numbers of tissue eosinOphilic and basophilic cells, where extreme stress associated with high circu- lating glucocorticoid levels resulted in eosinOphilic and *Through the courtesy of Dr. Gail D. Riegle, Endo- crine Research Unit, Michigan State University, E. Lansing, Michigan. 91 baSOphilic granulocyte cell disruption and degranulation. Because of this response the sacrifice of choice for further studies was the quick decapitation method. Circulating glucocorticoid levels were also assayed in relation to the order of sacrifice. The order of han— dling animals prior to sacrifice did not result in modifi- cation of circulating glucocorticoid levels that affected the numbers of tissue granulocytes, despite reports in the literature that handling rats for bleeding or injections does result in a decrease in circulating eosinOphils (Speirs and.Meyers, 1949; and Recant, 1950), lymphocytes (Stavitsky, 1952) and peritoneal eosinOphils (Panzenhagen and Speirs, 1953). Fluctuations in blood granulocyte numbers have not been shown to necessarily reflect changes in the number of tissue granulocytes (Cook, 1932; Godlowski, 1952; Panzen- hagen and Speirs, 1953; and Teir gt al., 1955). Analysis of female rat intestinal tissues obtained at different stages of the estrous cycle (Table 2, Figures 2 and 3) demonstrated that the numbers of rat intestinal eosinOphilic and baSOphilic granulocytes were affected by female hormonal changes. Duodenal eosinOphilia was observed to be highest at estrus and to progressively decrease through the ensuing stages of the estrous cycle. Pregnancy (10 and 18 days) was associated with eosinOphil cell counts compara- ble to proestrus and diestrus. One animal autOpsied on the 8th day of lactation exhibited eosinOphilia comparable to diestrus and proestrus, but in later stages of lactation 92 (21 to 25 days) eosinOphilic cell numbers were significantly different from either pregnancy or any stage of the estrous cycle. During the estrous cycle bas0philic cell numbers were highest during proestrus and comparable during late lactation or during estrus, metestrus and diestrus. Preg— nancy was associated with significantly higher baSOphilic cell numbers than values found during the estrous cycle and the numbers of baSOphilic granulated cells during early lactation were higher than during the estrous cycle or pregnancy. Recent reports on changes in circulating hormone levels during the estrous cycle in the rat indicated that highest estrogen and progesterone levels occur at or after ovulation in the proestrous stage of the cycle. Progester- one is high during estrus and progressively decreases during metestrus and diestrus. Estrogen levels are at the lowest during estrus and progressively increase during metestrus and diestrus (Schwartz, 1969; and YOshinaga.;§._l., 1969). At 10 and 18 days of pregnancy progesterone values are extremely high and estradiol values are comparable to metes- trous and diestrous phases of the cycle (Grota and Eik-Nes, 1967; and Yoshinaga _§._l., 1969). In lactation up to 8 days progesterone values are high, comparable to late pregnancy, and high estrogen values are comparable to dies- trous and proestrous stages of the normal cycle (Grota and Eik-Nes, 1967). 93 These hormone values when considered in light of the changes in the numbers of eosinOphilic and bas0philic cells in the rat intestinal tract suggested a definite influence of hormones on this cell series. Eosinophilic cell values in the intestinal tract were highest at estrus a time shortly after peak blood levels of estrogen and progesterone. It is obvious that the appearance of maximal eosinOphilic cell concentrations was not coincident with, but slightly lagged behind reported peaks in blood estrogen and progesterone. It is of interest in this study that peak eosinophilic cell counts in the intestinal tissue occurred at the same time as peak eosinOphilic cell counts in the genital tissue of the same animal. This would suggest that if estrogens are re- sponsible for the production of these cells in the intestine and in the genital tract that there is some delay between the increase of circulating levels of estrogen and their actions on these cells. This does not appear to be the case with increased levels of blood glucocorticoids and the depression of intestinal eosinOphilic cell numbers, since this occurred within a 5 minute period from the introduction of the chloroform stress. Estrogen administration to spayed female rats results in uterine eosinOphilia (Bjersing and Borglin, 1964). Progesterone administration can decrease uterine eosinOphilia (Baker _g._l., 1967). The highest number of baSOphilic cells were seen at proestrus, but the values were not significantly different fr bl th in ti ho in di is we r) 94 from the other stages of the estrous cycle. Progesterone blood levels peaked after ovulation but were rather constant throughout the remainder of the cycle. Progesterone may influence the baSOphilic origin of the cell series and con- tinually supply base forms upon which the influence of other hormone combinations may be superimposed. The circulating levels of estrogen during pregnancy in the rat were comparable to levels reported during the diestrous and proestrous stages of the estrous cycle. It is of interest that the intestinal eosinOphilic cell numbers were similar in the pregnant, diestrous and proestrous rat. The progesterone blood levels were reported to be extremely high during pregnancy. The numbers of intestinal baSOphilic granulocytes were significantly higher during pregnancy than during any stage of the estrous cycle. The hormone levels of lactation up to 8 days showed high progesterone values comparable to late pregnancy and estrogen values comparable to diestrous and proestrous stages of the normal cycle. EosinOphilia of early lactation was comparable to diestrus and proestrus; the baSOphilic cell values were significantly higher than those found in other animals. The eosinOphilic and baSOphilic values of late lactation were both depressed and this may indicate prolonged stress depletion of cell forms. Therefore, during the stages of the estrous cycle when estrogens have had the most influence, eosinOphilic cells were at their maximum in the intestinal tract and the 95 uterus. At the stages of the estrous cycle when estrogen influence was minimal, the eosinOphilic cell numbers in the intestine and genital tract were also minimal. In contrast, during the stage of the estrous cycle dominated by progesterone, the numbers of baSOphilic cells in the intestine were the highest. BaSOphilia was not associated with the estrogenic phases, but with the proges- tational phases of the estrous cycle. Although it is fairly well established that proges- terone administration is associated with the production of baSOphilic plasma cells in the genital tract and that estro- gen administration is associated with granulation and degran- ulation of plasma cells (Nellor and Brown, 1966), which is associated with increased bactericidal activity in the uterus (Wira, 1966; and Wira and Nellor, 1966), no reports in the literature suggest that cycling ovarian activity might increase or decrease bactericidal activity in the intestine. The gastrointestinal tract is reported to have a high content of immunologically competent plasma cells (Crabbé g §_1_., 1965) . If cells of the genital tract and the intestine assume the same function, it is not unreason- able to assume that the fluctuations of the cells of the intestinal lamina prOpria during the cycle stages, pregnancy and lactation and hormone treatment may relate to the vary- ing resistances to disease shown by many investigators dur— ing these times (Jungeblut and Engle, 1933; Jackson, 1935; Sprunt and McDearman, 1940; Anderson and Bolin, 1946; Black 96 t 1., 1953; Hawk t 1., 1957; Cordingley and Nicol, 1961; Mitchell _£__1,, 1966; and Wira and Nellor, 1966). Differ- ent cell titers, as influenced by hormones, could account for the changes. A few animals with abnormal reproductive activities were examined; the constant estrus, constant diestrus, pseudOpregnant and infertile female rat. These animals varied tremendously in the absolute number of eosinOphilic and baSOphilic cells in the intestinal tissues. Constant estrous animals were reported to display constant estrous smears, absence of ovulation, the maintenance of a large proestrous uterus and high plasma LH concentrations (Schwartz, 1969). Ovaries of constant diestrous animals have an imma- ture appearance (Takewaki, 1964). PseudOpregnant rats have functional corpora lutea for two weeks. Obviously the hormonal balance of these animals varied tremendously, but the percentage of basophilic cells of the total granulocytes in the tissues may be indicative of the types of hormones being secreted. The constant diestrous (26.99%) and pseudo- pregnant (25.87%) animals resembled the proestrous (28.58%) and diestrous (24.79%) animals in their percent of baSOphilic cells. These animals may be progesterone dominated. The complex and unknown hormonal balance in the infertile (31.77%) and the constant estrous (37.97%) animals seemed comparable to the pregnant (32.68%) and the late lactation (30.66%). The high eosinophilic cell numbers in the constant estrous 97 animals may be indicative of continued high estrogen secre- tion causing eosinOphilia. The early lactation (42.6¢%) animal appears to be in a class by itself. High basophilic cell numbers may reflect increased demand for base form pro- duction and the lowered eosinophilic cell count may reflect immediate degranulation--a state of constant cell turnover. The studies described demonstrated a marked influ- ence of ovarian activity and stress in the female rat. It is reasonable to assume that the ovaries and the adrenal glands in the female rat are only enhancing or inhibiting a basic physiological phenomenon characteristic of mucous mem- branes, and certainly a characteristic common to mucous mem- branes of the male rat. It was of interest to establish the influence of several physiological conditions on the numbers of baSOphiliC and eosinophilic granulocytes in the intestine of the male rat. Investigations on normal male rats (Table 3, Figures 4 and 5) demonstrated that eosinOphilic granulated cells increased with age up to 72 days of age and progressively decreased after 3 months to 2 years of age. Rytomaa (1960) also found that eosinophilic cell numbers in the ileum decreased after 6 months of age in Sprague-Dawley rats. Basophilic cell numbers and the percent of baSOphilic cells progressively increased with age. Significant changes in both baSOphilic and eosino- philic granulated cells seemed to occur with age, partic- ularly around sexual maturity, and became compounded with 98 age. Antibody levels in rats and mice increased up to 8 months of age and then decreased with age (Makinodan and Peterson, 1962; and Goullet and Kaufmann, 1965). Older humans and rabbits have higher circulating baSOphils than younger animals (Thonnard-Neumann, 1963). Rasanen (1958) reported that women had more gastric eosinOphils before 50 than after 50 years of age. Siurala _£__l, (1959) reported mast cells increased in men after 59 years of age. Thus, some changes occur with age that affect cells associated with immunity and the immune response. At sexual maturity testosterone secretion increases. This may be the hormone responsible for the eosinopenic and baSOphilic response that ensues in the tissues. But the continuing increases and decreases of cell numbers with age, when testosterone would be waning, confuses a clear cut relationship. Testosterone may simply be a moderator on the continued production of this cell series and as gonadal func- tion decreases with age granulocytes are released from this control. Testosterone has been shown to be the hormone responsible for male guinea pigs having less circulating eosinOphils than female animals (Dworetzky §£_gl, 1950; and Schweizer, 1957). Testosterone has also been reported to increase mast cells in the spayed rat uterus (Iversen, 1962), but to have no effect on eosinOphils or baSOphils in male rat accessory tissues (Baker gt al., 1967) or the female uterus (Bjersing and Borglin, 1964). 99 These age related changes in eosinOphilia and baso- philia must also be considered in regard to the hormones secreted by the adrenal glands (estrogen, progesterone, testosterone and glucocorticoids). The influence of gluco- corticoids on eosinOphils has already been mentioned in relation to stress effects. ACTH itself has been found to increase with age in cattle, but glucocorticoid levels were relatively unchanged (Riegle, 1963). Recent work has shown that "resting" levels of glucocorticoids may be higher in rats over 2 years of age than in younger animals (Riegle, 1970). The ability of the rat adrenal to respond to ether stress was shown to decrease significantly at 44 days of age and plateau at this level (Hess, 1970). Hydrocortisone has been shown to have no effect on uterine eosinOphils (Bjersing and Borglin, 1964). The levels of adrenal progesterone or estrogen may also be increasing or decreasing with age to contribute to the decrease in eosinophilic cell numbers and the increase in baSOphilic cell numbers with age. Romanoff gt.§l, (1969) have shown that human male progesterone excretion is de- creased after 65 years of age. Obviously, both the male and the female are immuno- logically competent. This competence may be regulated by different hormones. Of interest is the comparison of mean values for "mature male" and mean values for "mature female" eosinOphilic, baSOphilic and total granulocyte numbers. No significant differences between the sexes are demonstrated 100 when mean cell numbers are considered. Mean percentiles of baSOphilic cells are also comparable. The effects of castration demonstrated the influence of the male gonad on eosinophilic and baSOphilic cell num— bers. An initial eXperiment (Table 4, Figures 6, 7 and 8) compared the effect of acute castration (checked 9 days post-surgery) on granulocyte numbers in 72 days and 6 month old animals. The duodenum was the most responsive of the three intestinal parts examined to the effects of castration. At autOpsy 9 days post-surgery the numbers of eosinophilic and baSOphilic cells were significantly reduced. The per- cent of baSOphilic cells was also reduced. This decrease in the numbers of both types of granu- locytes may represent a continued stress related to recovery from surgery and may be caused by increased glucocorticoid levels. Or, perhaps the removal of the testes has released the cells from a moderating influence. Testosterone may also be eosinophilic, the lack of which results in eosin- Openia. The effect of castration itself is not clear. Cas- tration appeared to increase adrenal weight (Hausberger and Hausberger, 1966) but whether this increase was functionally related to increased circulating glucocorticoid levels was uncertain (Kitay, 1969) or to increased androgens and estro- gens (Lipshutz, 1950). Castration will elevate gonadotropin levels and Zachariae t 1. (1958) found that increased 101 gonadotrOpin levels increased tissue mast cells and decreased blood baSOphils. Other experiments on the male rat intestine demonstrated a more complex influence of the testes on eosinOphilic and basophilic intestinal cell numbers than was originally evident. The removal of the testes and the adrenal glands profoundly upset the hormonal homeostatic mechanisms regulating these cells. The granulocyte cell numbers varied at different autopsy times post-surgery, but the mean values of eosinophilic cells in the castrated animals were comparable to control values for mature males; mean values in the adrenalectomized animals were signifi— cantly higher than control or castrated values; and mean values in the adrenalectomized-castrated animals were significantly higher than all other groups. Tables 5, 6 and 7, and Figure 9 demonstrated the effects of castration, adrenalectomy and adrenalectomy- castration at various autopsy times, immediately post-surgery, 12 hours, 1, 2, 3, 5, 7, 9, 11 and 13 days post-surgery. ‘Varied post-surgical fluctuations are evident in all surgical ;preparations. The immediate effect post-surgery seemed logi- cral. Castration, a less stressful surgical procedure caused iruitial eosinOphilia and adrenalectomy a more stressful Stargical procedure caused eosinOpenia. The effect 12 hours Post-surgery also seemed clear, a parallel depression due to stcressful recovery from surgery. Sodium pentobarbital is 102 known to cause blood eosinOpenia (Jakobson and Hortling, 1954). Perhaps it also affects tissue eosinOphilic cells. The adrenalectomized-castrated animals do not seem to fit this stress pattern. Eosinophilic cell numbers in- creased immediately after surgery and remained above the control values for the duration of the period examined. The eosinophilic and baSOphilic cell numbers in the castrated and in the adrenalectomized animals also fluctuated during the test period. Cell numbers plateaued in the adrenalecto- mized animal at 5, 7, 9, 11 and 13 days post-adrenalectomy. The changes in the tissue granulocyte cell numbers seen in these three surgical preparations appeared to rep- resent systems released from control or systems more respon- sive or unstable to the mechanisms trying to control the cell numbers. The castrated animal appeared to have estab— lished some cyclic system that may be related to the intact adrenal responses. The adrenalectomized animal, lacking this mechanism of control, recovered a precarious level of control, and the adrenalectomized-castrated animal, devoid of steroid hormones or perhaps lacking a braking effect of testosterone on the adrenal showed no obvious pattern. Two animals castrated at 1 month of age were also examined at 6 months of age. The eosinOphilic cell numbers had recovered from the initial depression seen in the acute castration experiment and were comparable to younger control animals. The early removal of the testes seemed to prevent age related eosinOphilic cell depression. Other mechanisms 103 must be able to control or compensate for these hormonal imbalances. Another factor may be involved here that is diffi- cult to assess. Lisk (1969) has shown that male rats have cyclic fluctuations of sexual activity that are not related to androgen levels or treatment. He postulated unknown neural mechanisms for the regulation of this behavior. Whatever this unknown, that can cause cyclic breeding behav- ior in 79% of the males for only 1 hour per day for 3.5 to 5.5 day periods, perhaps it is contributing to the variabil— ity of our cell responses. The added possibility of the development of adrenal rest tissue after adrenalectomy may also be changing the total hormonal climate to which these cells are exposed. Lack of hormonal controls seemed to affect the absolute numbers of eosinophilic cells more than the baso- philic cell numbers. This may relate to the precarious immunological state imposed on the animal due to progressive degeneration caused by adrenalectomy. The intestinal tract may be called upon to respond to antigens that in the normal healthy animal would be denied access to the intestinal lamina prOpria plasma cells by the integrity of the physical barrier. The eosinOphilia observed may be a protective reSponse to increased antigenic challenge. These studies demonstrated that a variety of control mechanisms regulated the appearance of eosinOphilic and baso- philic cells in the mucous membranes of the genital tract 104 and the intestine. In the female genital tract strong evidence exists that suggests that the cells are cyclically associated with bactericidal activity. Circumstantially the cells of the intestine are cyclically related to body de- fense. In the male the appearance of intestinal granulo- cytes and presumed bactericidal activity may be profoundly influenced by testes secretion and adrenal influence. Since stressful conditions can influence cell numbers it must be assumed that the increase or decrease in cell numbers, what- ever their relation to body defense, is a defined homeo- static mechanism of the gonads and the adrenal glands in the male and female. The tremendous potential of the intestinal tract for the production of eosinOphilic cells can be shown by considering average numbers of eosinOphilic cells in a total cross section of normal 6 month old male rat intestinal tis- sue (X for the total section of ileum, colon and duodenum = 1470) and the total intestine length (43.3 inches) (Table 8). The eosinophilic cell numbers in the rat intestine are calcu— lated to be around 159,127,500. The total eosinOphils in the blood of a normal 250 gm male rat is only about 1.8% of this total. Thus, the tissue eosinOphilic cells represent the majority of the body eosinOphils. Looking to future studies, in so far as the develop- ment of a stable preparation for testing the administration of exogenous hormones is concerned, the data demonstrated 105 that the adrenalectomized animal between 5 and 13 days post- surgery had a plateau in eosinOphilic cell numbers. The animal seemed to be a preparation of choice in which to test the effects of various exogenous hormone regimes. The abso- lute numbers of eosinophils may vary because of many factors. Age, adrenal responses, female cyclic changes and testes influences have been shown to cause variations in granulo- cyte numbers. Sundell (1958) found his control groups for his rat studies on intestinal eosinophils to vary from 419 i 35 to 665 :_30 for supposedly the same type control animals. Because of these variations in controls, adrenal- ectomized animals between 5 and 13 days post-surgery may present an ideal, stable situation. A final area of discussion concerns the importance of a primary mucous membrane defense mechanism, if it is indeed dependent upon the participation of tissue eosino- philic granulocytes described in this thesis. It is reason- able that animal life shoulddisplay, at each portal of entry to the body systems, the most efficient and eXpedient means of neutralizing organized (living) and disorganized (particulate) invasion from the environment. It suffices to say that the human body is subjected to a variety of bacte- ria, viruses and toxins from inhalation, ingestion and intercourse that are not accompanied by marked shifts in blood leucocyte counts, increased circulating levels of antibodies or any of the overt changes that signal success- ful penetration into the tissues or blood stream. The daily 106 insults to the mucous membranes of the body obviously are handled by some mechanism that does not need the assistance of the systemic alarm centers of the body. Indeed it might be suggested that only when the capacities of the mucous membrane body defense mechanisms are exceeded are the more obvious participation of the blood cells, immune centers and lymph nodes called to battle. If we could signify a single cell capable of con- fronting any variety of antigens at the mucous membranes it must be the plasma cell. If the clonal theory is accepted, then each plasma cell is a memory cell that is capable upon mobilization of producing antibodies against one specific antigen that has previously invaded the body, and the plasma cells of the intestine of the rat represent different cells capable of responding and neutralizing most of the antigens characteristic of the present and past environment of an individual. The presence of the plasma cell in the mucous membranes of an animal, therefore, should define his eXpe- riences as well as his adaptability and interestingly enough, could at the same time be minimal and exceptional. If the endocrine system is accepted as a means of modifying physiological capabilities rather than originating them, a role of the endocrine organs can be recognized. Certainly at physiological estrus the estrogen induced degranulation of genital tract eosinOphilic plasma cells, and ensuing antibody released should neutralize the majority of antigens introduced into the genital tract at coitus, 107 since the plasma cells carry competences against prior exposures. This might even explain why the first several matings may upset the cycle: the memory cells have not been develOped. It was demonstrated in this present study that stress or various endocrine influences at different stages of the estrous cycle markedly influenced the numbers of eosinOphilic and baSOphilic granulated cells in the intes— tine. An important consideration is that possibly what has been described for the intestine is typical of each mucous membrane of the body. Prior to the selection of the intes— tine as the mucous membrane of choice, it had already been demonstrated that the mucous membranes of the conjunctiva, lungs, etc. contained large numbers of eosinophilic and bas0philic cells typical in morphology and histological staining characteristics to genital granulated leucocytes (Nellor and Johnson, 1968). It is not unreasonable if one accepts the concept that the "alarm reaction" or "fight or flight reaction" is "non-specific," that the concomitant release of glucocorti- coids and degranulation of "memory plasma cells" at each mucous membrane of the body might also herald a most for- midable variety of antigens to whatever the insult may be presented. It is recognized in the current studies that the degranulation products of intestinal plasma cells were not isolated, no specific hormone treatment was administered to 108 further elucidate the role of progesterone, estrogen or adrenal hormones. Nevertheless, the circumstantial evidence presented suggests that an understanding of the dynamics of this mucous membrane defense mechanism and the specific methods of hormonal control, might dictate a pharmacological regime that could eliminate a variety of body insults, detri— mental to the well-being of the individual. The mucous membranes of the body, including the genital tract and the intestine, are constantly exposed to foreign materials and represent the body's first line of defense. 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E : : 0 § fij R \1 \ \ M \\\ . x \s , g _ “HR .59 SHBBWON "1130 L CON 120 ~E§§x<< Vi \\\\\\\:: Pkg; "\\\\\‘\‘ \ . ' 0 0 \ x \ L \\ *fx\\ \ >\\:\\: \ x x \ \ \ \ \ l\%:\ \\ \\\ \\\ \ L ~ \x ‘ \ \ “X \: \\\ N‘Qx \\\\\"\\* ;\\\\\: 4? \\\\\ S E 1 C) C) 400- 300k IOO~ SHBBWDN 1130 I5-28 72 3mo Gm/o 6/.5//mo 7mo Zyrs DflflS FEEN. 8 AGE Influence of age on duodenal eosinOphilic cell Figure 4. numbers in male rats (values + standard errors). 121 .0000000 00008000 + 00000>v 0000 0008 80 0000808 0000 0000000000 00800000 80 000 00 008000080 .m 000000 mod. 003 00 95. 8.00 9:0 can 00. 00.0. m .50... CCU ,. , a x \ _\ N. . . - \xw \ \ - N n. n n u xxx >\\ H . . \\\\ 1 \f w . r05 «.0. \\\.\ \X 0 \ . \ [0.0 . oo. 1 ,_ N \i n x - m 0T a S 0000 122 .0000000 00008000 H 00000>v 8000000000 080300000 0000 0 000000000 ~0000 0008 80 0000808 0000 000000080000 0080000080 .0 000000 kzm20mm :_<0 Ch 00. 00m 00m 00¢ 82503 was a n. .0 090028 E 08 SHHGWON 1130 ‘35. + g 23 “’3 g3- + se§ IU ma; H: ‘2’ $5 139’ 32 E saaewnu 'nao INTESTINAL SEGMENT autopsied 9 days Figure 7. Intestinal basophilic ce rats, ers). in male : standard err 11 numbers tration (values following cas D 9 DAYS CASTRATED m CONTROL 0' 124 rF FF , F 'F W F SHEBWFIN 'I'IBO 6M0 70 DAYS DUODENUM INTESTINAL SEGMENT in ma rats, autopsied 9 days standard errors cell numbers yte following castration 125 .0000 0008 80 0000858 0000 000000080000 00000050 80 8000000000|>EO0000080000 00 >800000080000 .8000000000 00 000mmm .m 00sm0m >mw0m2m-._.moa m>m~401thcpnaH 10. ll. 12. l3. 14. 15. l6. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Solution .Xylene Xylene 100% alcohol 95% alcohol 80% alcohol 70% alcohol 50%.alcohol 30% alcohol Distilled water Harris hematoxylin Distilled water Distilled water Acid alcohol Running tap water Distilled water 30% alcohol 50% alcohol 70% alcohol .Eosin 100% alcohol 100% alcohol 100% alcohol 100% alcohol Xylene .Xylene Permount Alcion Blue-Methyl Green-Pyronin** (DQOWAUJNH Solution Xylene .Xylene 100% alcohol 95% alcohol 80% alcohol 70% alcohol Acid alcohol Acid alcohol *Modification of: *9 g. (D minutes minutes minutes minutes minutes minutes minutes minutes minutes minute Rinse Rinse Rinse 5 minutes (check in sc0pe for blueness) minutes minutes minutes minutes wawwwwmmm wwww 15 seconds to 3 minutes Rinse Rinse Rinse 3 minutes 3 minutes 5 minutes 8 g. (D minutes minutes minutes minutes minutes minutes minutes minutes wwwwwwmm Manual of Histologic and Special Technics Armed Forces Institute of Pathology, washington, D.C., .1957. **D. Bower, Simultaneous demonstration of mast cells and plasma cells, g, Clin. Path. 19:298-299, 1966. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 171 Solution Acid alcohol Alcian Blue solution Acid alcohol ,Running tap water Methyl Green-Pyronin Distilled water 100% alcohol 100%.alcohol 100% alcohol ~Xylene Permount Modified Dominci Technic* \OCDQONm-D-UJNH -Solution Xylene .Xylene 100% alcohol 95% alcohol 80% alcohol 70% alcohol 50% alcohol 30% alcohol Distilled water Eosin-Orange G Acid alcohol 0.5% Distilled water Toluidine blue .Distilled water 100% alcohol 100%.alcohol 100% alcohol 100% alcohol + Xylene (1:1) »Xylene Permount Time 2 minutes 10 minutes Rinse 10 minutes 10 minutes Rinse Rinse Rinse Rinse 5 minutes 1-3 ime minutes minutes minutes minutes minutes minutes minutes minutes minutes 10 minutes Differentiate Rinse 10 seconds Rinse Rinse Rinse Rinse Rinse wwwwwwwmm 5 minutes *A. N. Roberts and J. S. Hunt, A modified Dominci technic for autoradiography: acidified eosin-orange and alcoholic toluidine blue, Stain Technology 42:7-10, 1967. 172 Alcian Blue—D. HOH Eosin* Solution Time 1. Xylene 5 minutes 2. Xylene 5 minutes 3. 100% alcohol 3 minutes 4. 95%1a1cohol 3 minutes 5. 80% alcohol 3 minutes 6. 70% alcohol 3 minutes 7. Acid alcohol (10%.HC1) 2 minutes 8. Acid alcohol 5 minutes 9. Alcian Blue (I%--pH .l)**10 minutes 10. Acid alcohol Rinse 11. Running tap water 10 minutes 12. D.HOH Eosin (.25%) **1 minute 13. D.HOH Rinse 14. 60% alcohol **Differentiate 15. 95% alcohol Rinse 16. 100% alcohol Rinse 17. Xylene 5 minutes 18. Xylene 5 minutes 19. Coverslip Solutions Acid Alcohol 70% alcohol . . . . . . . 90 m1 Concentrated HCl . . . . . 10 ml Alcian Blue Alcian Blue . . . . . . . 1 gm 70% alcohol .'. . . . . . 90 m1 Concentrated HCl . . . . . 10 m1 Eosin Eosin Y . . . . .. . . . 0.5 gm Distilled water . . . . 200 cc *Laboratory stain developed for this study. **Critical steps. APPENDIX III COUNTING PROCEDURE = 0.25 mm ll 8 AREAS: (TOTAL = 0.5 sq mm) 4 villi bases 4 villi tips 173 APPENDIX IV STATISTICAL METHODS Chiggguare X2= iii—€12 Standard Error 2 2X2-2 SE— n ' n(n - 1) Student's T Test T-(X _§)/_1_+_L/SSX1+SS§_2_ — l 2 n1 n2 n + n - 2 1 2 174 93 03062 2983 m4|("111(11vamum\Iwuwuuumw