This is to certify that the dissertation entitled ALTERATIONS IN FUNCTIONALITY 0F LYMPHOCYTE POPULATIONS INDUCED BY ZINC DEFICIENCY presented by Paula DePasquaie-Jardieu has been accepted towards fulfillment of the requirements for Ph. D. degree in MTCrObiOTOQ‘Y /2 w t? M 4 i k m a: t. k, ' M29: professor Date W2 MS U is an Affirmative Action/ Equal Opportunity Institution 0- 12771 LIRFuQEY 1.? Michigan fi’tate University 1‘ i ,J )VTESI_J RETURNING MATERIALS: Hiace in book drop to LIBRARIES remove this checkout from Jul-I‘llin. your record. FINES wiii V be charged if Boo} is returned after the date stam ed beiow. ALTERATIONS IN FUNCTIONALITY OF LYMPHOCYTE POPULATIONS INDUCED BY ZINC DEFICIENCY By Paula DePasquaTe-Jardieu A DISSERTATION Submitted to Michigan State University in partial fulfiiiment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology 1982 ABSTRACT ALTERATIONS IN FUNCTIONALITY OF LYMPHOCYTE SUBPOPULATIONS INDUCED BY ZINC DEFICIENCY By Paula DePasquale-Jardieu Dietary zinc deficiency is known to result in profound thymic atrophy and a subsequent reduction in nearly all T-cell mediated responses. In contrast, specific effects of zinc deficiency on B cell function or the consequences on distinct subsets of either class of lymphocytes have not been determined. Thus, the purpose of this research was to investigate the effects of suboptimal zinc levels on B cell responsiveness and to define in more specific terms, the outcome of the deficiency on B and T cell subsets. The results of these experiments indicate that certain subsets of B cells are clearly modified by zinc deficiency. Furthermore, mitogenic, antigenic, adoptive transfer and cell surface 19 analysis of B cells from deficient mice suggest that there is an increase in the number of splenic cells at an early or intermediate stage of maturation. For example, responses to mitogens reported to specifically stimulate immature B cells were increased as a result of the deficiency, while responses to a probe for more mature B cells remained unchanged. This pattern of mitogenic responsiveness was the same whether the lymphocytes were cultured in serum from zinc deficient or zinc adequate mice, which indicated that the level of zinc during the culture had little influence on the responsiveness of B cells from any of the dietary groups. In addition, the responses to TI-l and TI-2 antigens, also believed to stimulate less mature B cells, were elevated subsequent to zinc deficiency. Experimental evidence showed this was not a consequence of faulty T cell regulation. Moreover, the affinity and heterogeneity of the antibody produced by the deficient mice, in response to the TI-2 antigen, had the analogous characteristics to antibody made by immature B cells. In conjunction with this, the primary response following adoptive transfer, which has been proposed to be mediated by cells with surface characteristics of immature B cells (sIgM+sIgD'), was significantly elevated in hosts reconstituted with splenocytes from deficient mice compared to hosts reconstituted with control splenocytes. Preliminary fluorescent activated cell sorter (FACS) analysis also indicated that splenocyte populations from deficient mice contained an increase in both the number of sIgM+ cells and in the number of cells displaying a high frequency of sIgM, the phenotype characteristic of immature B cells. Taken together, these results strongly suggest that zinc deficiency interferes with the normal maturation of B cells. The effects of the deficiency, however, are not limited to the immature or virgin B cells since the responses of mature antigen primed cells were also impaired by the deficiency and this effect seems to be longlasting. In addition to a differential effect on B cell subpopulations, zinc deficiency also appears to have selective effects on T cell subsets. T cells from deficient mice responsive to allogeneic cells gave elevated responses compared to controls; subsets responsive to the T cell mitogen PHA exhibited altered kinetics as a result of the deficiency; while the response of the Con A stimulated population was reduced by zinc deficiency. ACKNOWLEDGEMENTS I wish to express my sincerest appreciation to Dr. Pamela Fraker for her continual support and guidance and also for her wisdom in knowing when to offer help and when it was time to try it alone. I will always be greatful to Pam for her faith in me and for helping me to have a little in myself. I would also like to thank Dr. Patterson for his advice and counseling and the other members of my guidance committee, Dr. H. Miller and Dr. G. Dodgson, for their assistance and a special thanks to Dr. w. Esselman for "piloting" the cell sorter. Lastly, thanks to my family, Pete and Panmie, for their patience, understanding, and encouragement, all of which have been above and beyond mortal limits. ii TABLE OF CONTENTS List of Tables. . . . . . . . . . . List of Figures Abbreviations . . . . . . . . . . . . IntrOduction. C O O O O O O O O O 0 Literature ReViW C O O O O O O C O O O O O O Zinc Deficiency, Nutritional, Pathological Causes 0 O O O O O O O O O O O O O O Zinc and Immunocompetence. . . . . . . Effect of Zinc on Thymic Integrit Zinc and Cell Mediated Immunity. . . . Zinc Deficiency and Thymic Hormones. . B Cells and Zinc Deficiency. . . . . . In Vitro Induction of Zinc Deficiency. Possible Mechanisms of the Effect of Zinc Deficiency Inmmne Processes . . . . . . . . . . . . . . . . . . . . B Cell Subsets . . . . . . . . . . . . . . . . . . . . . Relationship Betweeen Surface 19 Isotype and Function of Murine B Lymphocytes . . . . . . . . . . . . . . . . . B Cell Subsets Involved in the Polyclonal Response . . . TI and TD Functional Subpopulations of B Cells Antigens . . . . . . . . Immune Defect of CBA/N . . T Cell Subsets . . . . . . References . . . . . . . . CHAPTER I ASSESSMENT OF THE FUNCTIONALIT DEFICIENT MICE. AbStraCto O O O O O O O O O 0 Introduction. . . . . . . . . Materials and Methods . Mice. . . . . . . . . Diets . . . . . . . . Zinc Analysis . . . . Mitogens. . . . . . . Cell Preparation. . . Y I. IN VIVO AND OF SPLENOCYTES FROM ZINC Responding to and Genetic on IN VITRO B-CELL RESPONSES Page vi vii xi 31 33 35 35 35 36 36 CHAPTER Page Collection of Mouse Serum . . . . . . . . . . . . . . . . . 36 Cell Culture Conditions and Reagents. . . . . . . . . . . . 36 Mitogenic Stimulation . . . . . . . . . . . . . . . . . . . 37 Detection of Polyclonal Activated Immunoglobulin Secreting Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Detection of Antibody Producing Cells . . . . . . . . . . . 38 Determination of Ig Bearing Cells . . . . . . . . . . . . . 38 Determination of Thy-1 Positive Splenocytes . . . . . . . . 39 Data Evaluation . . . . . . . . . . . . . . . . . . . . . . 39 Resu‘ts O O O O O O O O O O O O O O O O O O O O O O O O O O O 40 DiSCUSSion. O O O O O O O O O O O O O O O O O O O O O O O O O 59 References. . . . . . . . . . . . . . . . . . . . . . . . . . 62 II. ASSESSMENT OF THE FUNCTIONALITY OF SPLENOCYTES FROM ZINC DEFICIENT MICE. II. IN VIVO AND IN VITRO T CELL RESPONSES Abstract. 0 O O O O O O O O O O O O O O O O O O O O O O O O O 65 IntrOdUCtiono O O O O O O O O O O O O O O O O O O O O O O O O 67 Materials and Methods Mice. . . . . . . . Diets . . . . . . . Zinc Analysis . . . Mitogens. . . . . . Collection of Mouse Serum Cell Culture. . . . . . . . Measurement of Proliferation. Mixed Lymphocyte Culture. . . Detection of Antibody Producing Data Evaluation . . . . . . . . c—l . EL. . . . . . . . . o w o . o o o o o o o . 0 0 O 9 O 0 o O o O 0 O 0 O 0 o 0 O 9 o O 0 o 0 o 0 o O o o . o 0 o o o o . o g o . o O . 0 o O o 0 o 0 O 0 o . o . o . o . o . o 0 o O o O o 0 o o g o . O 0 O 0 O 0 O 0 o O o o o o o o o o o . o . O 0 o 0 O 0 O 0 o O o . o o o o o g o o o S O n. g 0 g 0 o O Q 0 RESUItS O O O O O O O O O O O O O O 0 O O O O O O O O O O O O 72 DiSCUSSion. O O O O O O O O O O O O I O O O O O I O O O O O O 87 References. . . . . . . . . . . . . . . . . . . . . . . . . . 91 III. EFFECTS OF ZINC DEFICIENCY ON B-LYMPHOCYTE RESPONSES TO TRINITROPHENYL CONJUGATED LPS AND FICOLL IN THE A/J MOUSE Abstract. 0 O O O O O O O O O O O O O O O O O O O O O O O O O 94 IntrOdUCtiono I O O O O O O O O O O O O O O O O O O O O O O O 95 Materials and Methods . . . . . . . . . . . . . . . . . . . . 97 Mice and Diets. . . . . . . . . . . . . . . . . . . . . . . 97 Zinc Analysis . . . . . . . . . . . . . . . . . . . . . . 97 Antigens and Immunizations. . . . . . . . . . . . . . . . . 97 iv CHAPTER Cell Culture. . . . . . . . . . Collection of HMS . . . . . . . Removal of Thy-1 Bearing Cells. Haptenation of SRBC . . . . . Preparation of TNP-L-Lysine . Detection of Antibody Forming Statistical Methods . . . . . Resu‘ts O O O O O O O O O O 0 Discussion. . . . . . . . . . References. . . . . . . . . . IV. AbStraCto O O O O O O O 0 Introduction. . . . . Materials and Methods Mice and Diets. . . Zinc Analysis . . . . Immunizations . . . Detection of Antibody Restoration of the Primary and Secondary Respo EFFECTS OF ZINC DEFICIENCY ON Ce 0 —l. . O . O PRIMED AND UNPRIMED TO SRBC FOLLOWING ANDOPTIVE TRANSFER OR Forming Adoptive Transfer . . . . . . . . . Cells RESPONSES NUTRITIONAL Data Evaluation 0 O O O O O O O O O O O O O 0 Results . . . . . . Discussion. . . . . References. . . . . Summary and Conclusions Appendix I. . . . . . . . 0:00.... S ofDoOoOoO REPLETION 132 133 135 135 135 135 135 135 137 137 144 155 159 161 165 LIST OF TABLES 1.29.12 Chapter I 1 Body weight gain and diet consumption of mice maintained on zinc-deficient, restricted, or zinc-adequate diet for 28 days . . . . . . . . . . . . . 2 Zinc levels in culture components. . . . . . . . . . . . 3 Comparison of percentages of B and T cells in spleens of mice. . . . . . . . . . . . . . . . . . . . . . . . . Chapter II 1 Zinc levels in culture components. . . . . . . . . . . . Chapter III 1 Body weight gain and diet consumption of mice maintained on zinc-deficient, restricted, or zinc-adequate diet . . 2 Zinc levels in culture components. . . . . . . . . . . . Chapter IV 1 Experimental design. . . . . . . . . . . . . . . . . . . 2 Body weights of mice from each treatment group before and after the repair period. . . . . . . . . . . . . . . vi 41 48 58 81 101 121 138 139 LIST OF FIGURES Figure Page Chapter I 1 .19 vivo anti-dextran response of zinc-deficient, restricted, and control mice after 28 day feeding peri 0d 0 O O I O O O O O O O O O O O O O O O O O O O O O 42 2 Optimal mitogenic responses to PMM, LPS, and dextran $04 as measured by incorporation of H -thymidine into splenocytes from zinc- deficient, restricted, or control mice cultured in medium supplemented wih FCS . . . . . . . . . . . . . 45 3 Number of immunoglobulin producing cells induced by optimal mitogen stimulation of splenocytes from zinc-deficient, restricted, or control mice as measured by the Protein A plaque assay . 50 4 Kinetics of the in vitro response to LPS of splenocytes from zinc- deficient, restricted, or control mice cultured in medium supplemented with serum from zinc-deficient or control mice . . . . . 51 5 Kinetics of the in vitro reSponse to dextran $04 of splenocytes from zinc-deficient, restricted, or control mice cultured in medium supplemented with serum from zinc-deficient or control mice . . . . . . . . . . . . . . . . . . . . . . 53 6 Kinetics of the 1g vitro response to PNM of splenocytes from zinc-deficient, restricted, or control mice cultured in medium supplemented with serum from zinc-deficient or control mice . . . . . 55 Chapter II 1 In vivo response of zinc- deficient, restricted, and control mice after 28 day feeding period . . . . . . 73 vii Figure Chapter II Optimal mitogenic responses to Con A and PHA as measured by incorporation of H3 thymidine into DNA of splenocytes prepared from zinc-deficient, restricted, or control mice cultured in FCS supplemented medium cells. . . . . . . . . . . . . . . MLC response after 28 and 35 days feeding period . Kinetics of proliferative response to Con A of splenocytes from zinc-deficient, restricted, or control mice cultured in medium supplemented with serum from zinc-deficient or control mice. . . . . . . Kinetics of proliferative response to PHA of splenocytes from zinc-deficient, restricted, or control mice cultured in medium supplemented with serum from zinc-deficient or control mice . . . . Chapter III Dose response curves of the in vivo anti-TNP Ficoll PFC/spleen response of zinc deficient, and control mice 0 O O O O O O I O O O O O O O O O O O O O O O O 0 Dose response curves of the 13 vivo anti-TNP-LPS PFC/spleen response of zinc deficient and control mice Kinetics of the in vivo anti-TNP-Ficoll PFC/spleen response of zinc:deficient, restricted, and control mice immunized with 25 pg TNP-Ficoll . . . . . . . . . Kinetics of the ig_vivo anti-TNP-LPS PFC/spleen response of zinc-deficient, restricted, and control mice immunized with 10 pg TNP-LPS. . . . . . . . . . . .Ig vivo PFC/106 lymphocyte response of zinc- deficient, restricted, and control mice immunized with 25 ug TNP-Ficoll. . . . . . . . . . . . lfl.VlV0 PFC/106 lymphocyte response of zinc- deficient, restricted, and control mice immunized with 10 pg TNP-LPS . . . . . . . . . . . . . Concentration of TNP-lys giving 50% inhibition of TNP-LPS and TNP-Ficoll responses of mice from each treatment group 3, 4, 5, or 7 days after in vivo imnunization with appr0priate antigens. . . . . . viii 76 78 82 84 102 104 106 108 111 113 115 Figure 10 Chapter III Slope of the inhibition curve near the region of 50% hapten inhibition of the 1g vivo response of mice from each treatment group at 3, 4, 5, or 7 days after immunization with TNP-LPS or TNP-Ficoll Kinetics of the_ig vitro PFC response to TNP-LPS of splenocytes from zinc-deficient, restricted, or control mice cultured in the presence or absence of thy-1 bearing cells in medium supplemented with sera from zinc-deficient mice. . Kinetics of the 1g vitro PFC response to TNP-LPS of Splenocytes from zinc-deficient, restricted, or control mice cultured in the presence or absence of thy-1 bearing cells in medium supplemented with sera from zinc-adequate mice . . Chapter IV In vivo 1° response of zinc-deficient and 'Eantrol mice after a 28 day feeding period . . . . In vivo 2° response of zinc- -deficient, restricted, or control mice after a 28 day feed1ng per1 0d 0 O O O O O O O O O O 0 O O O O O 0 PFC responses of syngeneic irradiated hosts reconstituted with primed (2°) or unprimed (1° ) splenocytes from zinc- -deficient, restricted, or zinc- -adequate mice. . . . . . . . . . . . . . . PFC responses of syngeneic irradiated hosts reconstituted with unprimed (1°) splenocytes from zinc-deficient or zinc-adequate mice. . . . . lg vivo 2° response of zinc-deficient, restricted, or control mice after 28 days on respective diets followed by 3 weeks of feeding zinc-adequate diet to all groups. . . . . . . . . . . . . . . . . . . In vivo 1° response of zinc- -deficient or control mice after 28 days on respective diets followed by 3 weeks of feeding zinc- adequate diet to both groups . . . . . . . . . . . . . . . . . . . . . . ix 118 122 124 140 142 146 148 151 153 Figure Appendix 1 Fluorescence profiles of splenocytes from zinc deficient and control mice stained with FITC- CONJUQGtEd anti I] afltTDOdy o o o o o o o o o o o o o o o o 171 Fluorescence cytograms of splenocytes from zinc deficient, restricted, and control mice stained With FITC'COnjugatEd allti I] antibOdy o o o o o o o o o o o 173 Fluorescence cytograms of splenocytes from zinc deficient, restricted, and control mice stained with FITC-conjugated anti 5 antibody . . . . . . . . . . . 176 ADCC BSA Con A DTH EDTA FCS FITC Ig LPS MEM MLC NK PFC PHA PPD PNM sIg SRBC TI-l TI-2 TNP ABBREVIATIONS Antibody dependent cell-mediated cytotoxicity Bovine serum albumin Concanavalin A Delayed type hypersensitivity Ethylenediaminetetraacetic acid Fetal calf serum Fluorescein isothiocynate Immunoglobulin Lip0polysaccharide Minimal essential medium Mixed lymphocyte culture Natural killer cells Plaque forming cells Phytohemagglutinin Purified protein derivative Pokeweed mitogen Surface immunoglobulin Sheep red blood cells T-cell dependent antigen T-cell independent antigen - class 1 T-cell independent antigen - class 2 TrinitrOphenyl conjugate INTRODUCTION The immune system is greatly compromised by nutritional deficiencies (1). Diets deficient in specific vitamins (2), amino acids (3), protein (4), essential fatty acids (5) or trace elements have all been shown to impair immune function. Indeed, malnutrition has been suggested to be one of the most frequent causes of impaired immune function and subsequent susceptibility to disease in mankind (6). Although it is generally accepted that nutritional status is an important factor in immunological responsiveness, the relationships between dietary deficiencies and immunity remain unclear. In the case of trace element deficiencies, an obvious tool for investigation of nutrition and immune capacity is that of zinc deficiency, since this deficiency has been shown to result in severe thymic hyp0plasia and subsequent reduction in most T-cell mediated processes. Because of the detrimental effects on the thymus and thymus derived cells, most studies to date have concentrated on defects in cellular immunity resulting from zinc deficiency. The possible effects of the deficiency on B cells have, for the most part, remained undefined. Furthermore, the functional deficits due to zinc deficiency have not been characterized for individual subsets of B or T cells. Since severe lymphocytOpenia is known to be associated with zinc deficiency, it is fundamental to our understanding of the role of zinc in lymphocyte functionality to determine if suboptimal levels of zinc result in a uniform reduction in lymphocyte subsets or if preferential alterations occur in certain subclasses. Thus, the purpose of these studies was to examine the effects of zinc deficiency on the responses of subsets of T and B cells, with special emphasis on B cell suprpulation. Primary and secondary lg 1119 responses as well as lg 11339 probes of B cell function were examined to provide a general overview of the influences of suboptimal zinc levels on antibody producing cells, since little is known concerning the effects of zinc deficiency on B cells. While numerous studies have examined the consequences of zinc deficiency on in vivo T cell responses, the lg 11359 studies are in their infancy. As such, contradictory results have been reported. Part of the reason for these descrepancies may lie in an inherent problem associated with using lg 11532 assays for investigation of the effects of zinc deficiency on lymphocyte subsets. There is a possibility that the responses of lymphocytes from deficient mice might be altered during the culture period due to the zinc present in the serum used to supplement the culture medium. This is of particular concern since the results of_yh xixg_studies indicate that severely deficient weanling or adult mice can completely repair immune function and for a short time exhibit an enhanced immune capacity following refeeding with a zinc adequate diet (7,8). In the studies presented herein, an attempt was made to avoid this possibility by comparing the in vitro responses in serum containing normal or low levels of zinc. The zinc depleted serum obtained from deficient animals contained very low levels of zinc compared to the high levels of zinc found in the fetal calf serum normally used for lymphocyte cultures. This approach allowed separation of culture artifacts from the real effects of the deficiency on lymphocyte populations. Thus, this study will carefully examine the effects of zinc levels during culture along with possible differences which might exist in kinetics and dose requirement of lymphocytes from deficient mice. LITERATURE REVIEW The purpose of this study was to define the effects of dietary zinc deficiency on lymphocyte subpopulation. Since these studies are interdisciplinary in nature, this review will outline the reported effects of zinc deficiency on immune function as well as the immunological parameters which can be used to define subsets of B and T cells. This should provide a background for the experimental approach used in these studies. Zinc Deficiency - Nutritional,Pathological,and Genetic Causes. Deficiencies in zinc, an essential trace element, are among the most prevalent forms of malnutrition in mankind (9). Nutritionally induced zinc deficiency in its most severe form occurs in underdeveloped nations where diets consist primarily of cereal and grain products. These food- stuffs contain phytate, a chelator of zinc, which interferes with intestinal absorption of zinc (10). In more developed nations chronic, marginal zinc deficiency can be due to dietary insufficiency or pathological situations. Sandstead (11) reviewed the status of zinc nutrition in middle and upper income families in the United States and concluded that zinc deficiency was widespread owing to poor eating habits and low meat consumption. A study of Denver headstart children from low income families showed a number of these children to be zinc deficient as indicated by altered taste acuity, low hair zinc levels and deficient growth (12). All of these symptoms disappeared upon zinc supplementation. Those at the highest risk of zinc deficiency appear to be children and pregnant women since they are experiencing periods of rapid growth during which the zinc requirement is higher than normal (13). In addition to dietary induced zinc deficiency, there are a number of diseaSe states which can lead to zinc deficiency. These are outlined in Table 1 (14). These states usually result in malabsorption of zinc from the gut. Since there are no known zinc storage sites, failure to obtain sufficient intake of zinc on a daily basis leads to a deficient state. Flagrant cases of zinc deficiency have also been reported in patients receiving total parenteral nutrition in which zinc was completely lacking or supplied in insufficient quantities (15). A connection has also been made between zinc deficiency and a genetic defect of humans, acrodermatis enteropathica (AE) (16). These patients exhibit symptoms of thymic aplasia, deficient or absent cell—mediated responses and frequent infections attributed to Candida albicans. Moynahan noted that this disease resembled a genetic disorder of Freisian cattle which could be corrected by oral zinc supplementation. When zinc was administered to his patients with AE, all clinical manifestations of the disease disappeared (17). It is now known that both diseases result from poor zinc absorption from the upper gastrointestinal tract and it has been suggested that this is due to lack of a specific binding protein resulting from the genetic defect (18). Both diseases are fatal unless treated by large daily supplements of zinc. Table 1. Possible causes of human zinc deficiency (Adapted from Reference 14) Nutritional factors Excessive intake of phytate, fiber, alcohol Inadequate intake or poor eating habits, low meat consumption Gastrointestinal disorders Malabsorption syndrome Chronic renal disease Burns and psoriasis Chronically debilitated states, including Ialignancy Parasitic infestations Hookworm Iatrogenic causes Antimetabolites and anti- anabolic agents Penicillamine therapy Prolonged intravenous therapy Diabetes Cirrhosis of the liver Collagen diseases Pregnancy and use of oral contraceptive agents Genetic disorders Sickle cell disease Acrodermatitis enteropathica 'A) 5’ The number of instances in which zinc nutrition "my be impaired adds weight to the argument that the effects of zinc deficiency on immune function require careful examination. Zinc and Immunocompetence. A considerable body of evidence has accumulated in the past few years which implicates zinc as an important nutrient for maintenance of normal immune function. In the first study to describe zinc deficiency in humans, Prasad found indications of lymphocytopenia, atrophied thymuses and reduced capacity to respond in DTH reactions (19). Since the thymus is the site of maturation of T lymphocytes, it is not surprising that host defense mediated via the cellular immune mechanism is severely impaired. Diseases associated with depressed cellular hwnunity such as measles, smallpox, and other viral infections are leading causes of death in deficient children (20). Furthermore, attempts to vaccinate against these agents are usually unsuccessful apparently as a result of defective T cell cooperation with B cells (21). Indeed, attempts to establish a secondary response to SRBC during experimentally induced zinc deficiency in mice result in a significant depression in this response (22). This also raises the question of whether or not immune responses initiated prior to the deficiency might be impaired by intervals of zinc deficiency. One of the aims of the present study is to address this issue. Because of the consequences of zinc deficiency, it is not morally justifiable to impose the deficiency on human beings. In addition, it is much more difficult to get the controlled, reproducible conditions in human populations required to examine effects of the deficiency. For this reason, most of the work to be reviewed here has been performed with animal models. Effect of Zinc Deficiency on Thymic Integrity. As was the case with human studies, Luecke et 31, repeatedly observed that the thymus was the most profoundly atrophied organ in zinc deficient rats and pigs (23). Histological examination of the thymus of zinc deficient mice revealed that the cortex, the site of the most immature thymocytes, underwent preferential involution with sparing the medulla, the site of more mature thymocytes, until much later in the deficiency (7). To date, no mechanism has been found for the preferential involution of the thymus and subsequent impairment of T cell activity which results from zinc deficiency. It had been suggested that zinc deficiency constituted a stress on the animal which led to stimulation of the adrenal cortex and a rise in serum glucocorticoids proven thymolytic hormones for immature thymocytes (24,25). DePasquale-Jardieu and Fraker showed that markedly elevated levels of plasma corticosteroids were present in zinc deficient animals, but 50% of the immune impairment occurred prior to the rise in glucocorticoids (26). Removal of the steroid via adrenalectomy offered only a modest protection (20%) against the loss in immunity (27). However, adrenalectomy did prevent involution of the thymus. These data suggested that corticosteroids play only a minor role in the depressed immune responses resulting from zinc deficiency. Since the thymus did not atrophy in adrenalectomized mice,but the T cell helper response was still depressed, this suggests that processing of thymocytes into mature functional T cells "my be impaired by the deficiency. Zinc and Cell Mediated Immunity. Because of the detrimental effects of supOptimal zinc cells on thymus integrity it was not unexpected that functions mediated by T cells would be impaired by zinc deficiency. In an initial study by Fraker on A/J female mice made zinc deficient for 4 weeks and then hmnunized with the T dependent antigen, sheep red blood cells, the deficient mice produced only 13% as many plaque forming cells (PFC) as controls (22). If mice were reconstituted with thymocytes one day prior to a first injection of SRBC and given a second injection of SRBCs one week later, the PFC response subsequent to the final injection was 60% of the control response, rather than the 10% observed with unreconstituted deficient mice. These data implied that T helper cells were profoundly influenced by zinc deficiency. These observations have been confirmed by Fernandes (28). In addition, he showed a defective development of T-killer lymphocytes and natural killer cell activity (NK) after 1g vivo sensitization with allogeneic tumor cells in mice maintained on zinc deficient diets for 8 weeks. He also found antibody dependent cell mediated cytotoxicity (ADCC) responses of deficient mice to be normal. These results are at variance with those of Chandra who reported a significant increase in ADCC and normal NK activity to tumor cells subsequent to zinc deficiency (29). The reasons for these variations are unclear since virtually identical methods of measuring ADCC and NK were used by both investigators. The discrepancies nay reflect differences in the relative degree of zinc deficiency in one study versus the other. This is impossible to evaluate since Chandra's paper did not contain body weights or nutritional data necessary to make these comparisons. The fact that both authors independently reported differences in the effects of zinc deficiency on ADCC activity compared to effects on NK activity suggests that cell types involved in these responses might be effected in different ways. This lends support to the idea of the differential 10 sensitivity of zinc deficiency on lymphocyte populations which is a primary hypothesis of this dissertation. Decreases in delayed type hypersensitivity (DTH) have been observed in zinc deficient guinea pigs (30), mice (31), rats (32) and Fresian cattle (33). This reaction is reported to involve another subpopulation of T cells called TD cells as well as macrophages. We cannot, therefore, rule out the possibility that the decrease responses may be due in part to the effect of the deficiency on macrophages as well. Preliminary, unpublished data fran our laboratory, however, indicate that the macrOphage functions so far tested all appear to be unaltered by zinc deficiency. A number of lg gitgg studies using mitogens to test T lymphocyte functionality during zinc deficiency have also been reported. Gross, g5_ 31,, examined mitogenic responses of lymphocytes from zinc deficient rats. These authors noted diminished responses to the T cell mitogens PHA and Con A, and to PWM which requires 8 and T cell interaction in lymphocytes obtained from spleen, thymus and peripheral blood of the deficient animals (34). In patients with zinc deficiency, mitogenic stimulation of peripheral blood lymphocytes from these individuals resulted in increased responses to PHA, normal response to PWM, and diminished response to Con A (35). These results are very different than those reported by Gross. Neither of these authors describe dose or kinetic studies for the mitogens used, parameters which could easily be altered by zinc deficiency, and which might account fOr some of the discrepancies. In addition, there are no reports of the zinc level in the culture systems 11 and any effects this might have on these results was not considered. As will be seen from the results presented in this thesis, the level of zinc present during the culture period can influence responses to some of these mitogens. Zinc Deficiency and Thymic Hormones. The critical role of the thymic microenvironment in the differentation and maturation of T lymphocytes lead to a search for "thymic hormones". In the last few years considerable evidence has accumulated to indicate that the thymus is an endocrine gland responsible for the production of one or more polypeptide hormones. Many substances have been proposed as putative thymic hormones including ubiquitin, thymopoietin, factor thymic serique, thymosin and thymic humoral factor (36). Some of the effects of zinc deficiency on T cell mediated processes might be explained by the reduction in levels of these hormones resulting from the severe thymic atrOphy observed during zinc deficiency. Cunningham-Rundels, e; 31. (37) noted a decrease in serum thymopoietin levels in zinc deficient patients. Iwata (38) and Chandra (39) have reported decreases in the activity of thymus humoral factor, serum factor thymic (FTS) in zinc deficient mice. Surprisingly, Dardenne, gt g1., (40) have observed that FTS exists in two forms. One is biologically inactive and contains no zinc while the other, which they have named thymulin, has full biological activity and contains zinc. The two proteins crossreact antigenically. It appears this hormone is very much like insulin in its requirement for zinc. A decrease in the level or activity of these thymic hormones suggests that immature T cells might not be efficiently processed into mature functional T cells during zinc deficiency. Nash's observation of 12 an increase in immature T cells in the spleens of deficient mice, as . measured by autologous rosette formation, further supports this idea (41). Similar observations of increases in immature splenic T cells have been observed following adult thymectomy (42). Since zinc deficiency results in a kind of non-surgical thymectomy, this kind of effect seems plausible. B Cells and Zinc Deficiency. The effects of zinc deficiency on B cell responses are poorly characterized. Three reports do, however, indicate that zinc deficiency interferes with B cell development. Beach, e§_gl,, (43) and Hildebrandt, et 31,, (44) have shown that zinc deficiency during the lactation period interferes with maturation of normal 8 responses. In studies involving weanling mice, Zwickl, egngl., (8) have shown a 40% decrease in reSponsiveness to the T-independent antigen, dextran, after only one week of feeding a zinc deficient diet. These studies serve to demonstrate that postnatal B cell development, at least, is particularly zinc dependent. Few reports have dealt with the consequences of zinc deficiency on the B cell responses of adult animals. Many laboratories have reported diminished numbers of antibody producing cells as a result of zinc defi- ciency but in large, the responses have been measured using T dependent antigens such as SRBC (7,22,28,45,46,47). Actual antibody titers have not been well studied, however, two laboratories have reported decreases in basal serum IgG levels during zinc deficiency (33,43). Heretofore, affinity and heterogeneity of the antibody produced by zinc deficient animals has not been reported. Both of these parameters will be examined in this dissertation. In addition, specific effects of the deficiency on subpopulations of B lymphocytes remain completely undefined. 13 In Vitro Induction of Zinc Deficiency. A number of attempts have been made recently to produce zinc deficiency lg 11352 by culturing lymphocytes in medium depleted of zinc by chelation. These studies involved addition of chelators to culture medium or selective extraction prior to the culture period using chelating resins to eliminate the presence of the chelators during the actual culture. In one study by Zonzonico, e; 31. (48) in which ethylenediamine- tetraacetic acid (EDTA) was added to the medium, the mitogen response to Con A was depressed, while the mitogenic response to LPS was uneffected by culturing lymphocytes in this medium. From these results the authors concluded that T cells were sensitive to zinc deficiency while B cells were resistant to the deficiency. However, since Con A activity has been shown to be dependent on various metals (ZnTT, Mg++, Ca++) (49) and EDTA is a fairly nonspecific chelator (50) which was present during the culture, it is difficult to determine how this might have effected the results. As one example, EDTA is toxic to lymphocytes and not surprisingly viability was reduced 50% by culturing in this serum, but the authors claimed the Con A response was depressed by greater amounts than could be explained solely on the basis of cell death. In addition, the fact that the LPS response, which has not been shown to require metals for binding or activity, was uneffected in this serum suggests that alternative interpretations of the data are possible. In another study by Flynn, et 31. (51), using serum depleted of zinc by passage over a chelating resin, the generation of cytotoxic cells to allogeneic tumors was inhibited by culturing cells in this serum as was the production of T cell replacing factor (TRF). It would be important to know the viability of the tumor cell in the zinc depleted environment 14 to be sure adequate stimulation had occurred. In a similar study by Messer, gt g1. (52), in which zinc was also removed by passage over a chelating resin, a procedure which in this case reduced the zinc concentration by 90%, the PHA response of lymphocytes cultured in this medium was depressed 50% compared to the response in untreated medium. Furthermore, the addition of zinc resulted in an immediate reversal of this trend. These studies further serve to stress the importance of zinc to lymphocyte function. It should be noted that these effects may differ from the changes seen after long term dietary or pathologically induced zinc deficiency. Indeed, the results of studies to be presented in this thesis suggest that the 19 1119 and in vitro effects of zinc depletion on lymphocyte function are different. Possible Mechanisms of the Effects of Zinc Deficiency on Immunity. The biochemical mechanisms underlying the relationship of zinc and immunity remain undefined. A number of interesting observations have been made, however, which may shed light on this subject. Zinc is necessary for the activity of more than 100 metalloenzymes (53), since removal of zinc by chelation has been shown to result in complete loss of activity of these enzymes (54). Among these enzymes are thymidine kinase and DNA dependent RNA polymerase, important factors for nucleic acid synthesis. Due to the rapid proliferation and turnover of lymphocytes, defects in the activity of these enzymes could explain part of the detrimental effects of the deficiency on lymphoid cells. It is also possible that a reduction in the activity of another zinc containing polymerase, terminal deoxynucleotidal transferase, an enzyme unique to thymocytes, but not mature T cells, might explain the seemingly selective effect of the deficiency on cortical thymocytes (55). On the other hand, 15 Luecke, etugl., (56) have examined numerous zinc containing enzymes involved in metabolism during zinc deficiency. On a per cell basis their activity appeared normal, however, overall levels of activity were decreased owing to atrophy of the organ in question. It remains to be seen how enzymes important to lymphocyte proliferation and differentiation are influenced by dietary zinc depletion. Zinc itself has been reported to be mitogenic for T cells (57). Good has postulated that a deficiency in zinc might interfere with this polyclonal activation and ultimately impair immune responses in this manner. The role of zinc in membrane biochemistry is also intriguing. Zinc has been shown to be important to both plasma and lysosomal membrane stabilization (58). Since immune responses involve a complex network of receptors and membrane signaling phenomena, the effect supoptimal zinc levels might have on these parameters could be an important one. The fact that lymphocytes have receptors for transferrin bound zinc (59), and the number of these receptors increases upon mitogenic stimulation (60) suggests a critical biochemical role for zinc in lymphocyte responses. The studies presented herein have concentrated on the gross modifications of lymphocyte functionality by zinc deficiency. Presently, it is not known which, if any, of these mechanisms might be responsible for the ultimate consequences of suboptimal zinc levels on cellular responsiveness. 16 B Cell Subsets. To understand the effects of zinc deficiency on B cell function it is necessary to outline the current evidence which suggests that functionally distinct subpopulations of B cell exist. These subsets are characterized by (a) surface Ig expressed on their cell surfaces, (b) responsiveness to polyclonal activators, (c) responsiveness to different classes of antigens. (A) Relationship Between Surface 19 Isotype and Function of Murine B Lymphocytes. B cells mature from B cell precursors found in the bone marrow of adult mice. It has been demonstrated by fluorescent studies that the adult bone marrow contains large lymphocytes which have cytoplasmic IgM but lack surface immunoglobulin (19) (61). These cells are believed to give rise to smaller lymphocytes bearing surface IgM (sIgM) followed by acquisition of surface 190 ($190). This sequence is suggested by experiments with lethally irradiated mice (62). When these mice were reconstituted with stem cells from adult mice, the newly emerging B cells bore only sIgM. Cells bearing 5190 and sIgM arose sometime later. The observation that cells bearing sIgM appear first from adult bone marrow, suggests that sIgM+ cells are relatively immature while sIgM+sIgD+ cells represent a more mature B cell pupulation. This is also supported by immunofluorescent studies of B cells during gestation (63). Expression of IgM occurs on day 17 in cells from fetal liver, while surface 190 expression does not occur until 3 days postparturation. Over the next few weeks, sIgM+sIgD+ cells become the predominant cell type in all the lymphoid organs. 17 An additional line of evidence which suggests that 5190 is indicative of a more mature cell comes from the correlation between ease of tolerance induction and expression of $190. It is relatively easy to establish a state of tolerance (Specific antigenic nonresponsiveness) in populations of cells from neonatal Spleen or adult bone marrow, populations which have a preponderance of sIgM+sIgD' cells (64,65). It is much more difficult to tolerize adult Spleen cells, where the population is primarily sIgMTsIgD+ (64). This data suggests then that the relative maturity of a B cell,as related to ease of tolerance induction, is linked to surface 19. Controversy surrounds this point, however (66). The acquisition of both sIgM and $190 on virgin lymphocytes is antigen and T cell independent (67). An additional 8 cell subset, believed to be the most mature, is the memory cell. The surface characteristics of these cells appear to be sIgD+ or sIgDTSIgG+. It has been proposed by Uhrand Vitetta that the expression of 196 is antigen and possibly T cell dependent (68). (B) B Cell Subsets Involved in the Polyclonal Response. Gronowicz and Coutinho found that the mitogens, dextran $04, LPS, and PPD each activate distinct B lymphocyte subpopulations to undergo proliferation and antibody secretion (69). Furthermore, they have shown that responsiveness to these antigens develops sequentially during ontogeny in the order: Dextran $04-—9* LPS -9 PPD 18 Sequential development of responsiveness to these antigens has been corroborated by Goodman (70) who found LPS to activate a distinct subset of B cells from that activated PPD. Furthermore, the PPD responsive sub- set matures later than the LPS responsive population. That study also demonstrated that the response to 2-mercarptoethanol matures later than any mitogen response so far tested. This pattern of mitogenic responsiveness is believed to hold true for the normal maturation of B cells from bone marrow of adult mice. Gronowicz and Coutinho have shown, furthermore, that adult bone marrow cells respond to stimulation with 0x504 but not LPS or PPD (71). DxSO4 has also been shown to activate B cell precursors in adult bone marrow to become reSponsive to LPS whereas pretreatment of bone marrow with LPS could inhibit the dextran sulfate response (71). Further studies, using suicide techniques in which cells responsive to a given mitogen are killed and the residual population then evaluated, have provided additional evidence that cells responsive to LPS, dextran $04, and another mitogen, Norcardia water soluble mitogen, belong to distinct subsets (72). (C) Functional Subpopulations of B Cells Responding_to TI and TD Anti- gggg. B cells also acquire the ability to respond to antigens in a very orderly fashion as they mature during ontogeny (73). This sequence can be used to further define B cell subsets. B cell responses to the so called "TI-1" antigens (T-independent Class 1) such as TNP-LPS or TNP- Brucellosus abortus are acquired first, at one week of age. Responses to TI-2 antigens (T-independent Class 2) such as TNP-Ficoll or $5111 appear between 3 and 4 weeks of age, while responses to T0 (T-dependent antigens) such as SRBC are obtained between 4-8 weeks of age (74). 19 The ability to respond to these antigens is correlated with their surface 19 characteristics. TI-l antigens require only sIgM while TI-2 antigens and TD antigens require sIgM+sIgD+ (75). This is supported by studies of adult mice in which anti-IgD blocks responsiveness to TNP-Ficoll but not TNP-LPS. Furthermore, removal of B cells vhth antiserum to Ly 5.1, a marker found on IgMTIgD+ cells, interferes with TNP-Ficoll responses in adult mice, but not TNP-LPS responses (76). Studies in the immune defective CBA/N mouse suggest that Ly 5.1 is a marker of a late developing subset of B cells which is absent from CBA/N mice (77). Lyb 5.1 is not found on Spleen cells of normal mice before 2 weeks of age, and adult levels are not reached until 5 weeks of age (78). Immune Defect of CBA/N - A Possible Model for B Lymphocyte Maturation. As in other biological systems, the existence of a mutant strain is often useful in discerning the steps involved in a certain pathway. The CBA/N mouse has been proposed as such a model for B cell differentiation. The CBA/N mouse has an X-linked immune deficiency gene (x/id) which affects a variety of B cell traits (79). The CBA/N mouse can respond to TI-l antigens but lack the ability to reSpond to TI-2 antigens and give low responses to T0 antigens. In addition, secondary responses to T0 antigens are barely evident (80). Adult CBA/N mice do not express Lyb 3, 5 or 7 differentiation markers acquired late in B cell ontogeny (81). Surface 19 studies also indicate that they lack a population of cells found in normal mice which bears low IgM, high 190. Instead, CBA/N mice have a p0pulation of B cells with high IgM and low I90 and are Ly 5.1‘ (82). CBA/N mice are also very susceptible to tolerance induction (83). For these reasons, 20 Fidler (84) and Whitlock (85) have pr0posed that the genetic defect leads to a maturational block such that immature B cells population accumulate in these mice. Fidler was also able to demonstrate an increase in responsiveness to mature B cell mitogens such as PPD and to TNP-Ficoll with age suggesting a delay in maturation which is corrected with age. The results of studies with these mice suggest that the criteria of mitogenic responsiveness, surface antigen characteristic, and antigen responsiveness are useful for defining B cell subsets and for obtaining information as to the relative state of maturity of B cells. A diagram which suggests a possible mechanism for B cell maturation based on the experiments outlined herein, is shown in Table 2 (86). 21 C958 .0. o... 5.5% so... . =w< V8. 25.“ 283 1E0 .32 4mo 401.2 a .o $ch”. mes a a a at: +2 wfiflm : : «Hun—- <8. .22 0A a “3 0H a QC. Hoodoo! IIIIIIIIII I I I. IAI Z\_oa E 5 :3de _H.._. on»... on H a. . a 50.3 .288: H.._. ._ hmfiou 26:9: econ c9: .28 68.028 oufiwmhe m O m O =8 m eaoeé . . H m 2a om]. low: ZO_._.wm mo 2mm u come on» mucmmmeame can scam .:o_um~_::=z: emuem mxau m umezmoma mew: mmmcoamme um; .cowema m:_ummw xau mu emumm wows _ocu:oo can cmuu_eumme .u:m_u_emviu:_~ mo mmcoamme :meuxmuu_u:u o>_> mm. ._ we:m_a 43 8... w v @081 .m. m w m 8?. . m S 8?. -. wmzoamwm dekxwo 02> Z_ 29680 cN g gamma .528 eN Vk . 44 Measurement of the Mitogen Responses of Splenocytes from Zinc Deficient, Restricted,yand Control Mice in Fetal Calf Serum Supplemented Cultures. To better define effects of zinc on various B-cell subpopulations, a variety of mitogens were used to test the functionality of the residual B-cells. Mitogens were used to determine if, in general, subsets of B-cells from deficient mice were fully functional when examined on a per cell basis. The responses of equal numbers of Splenocytes from each dietary group were measured under uniform conditions in medium supplemented with 5% FCS. The optimal mitogenic responses to PWM, LPS, and dextran S04 are shown in Figure 2. Dose-response curves were determined for all mitogens employed; optimal stimulation of zinc-deficient, restricted or control cultures occurred at the same mitogen concentration in all cases. B-cells from deficient mice stimulated with dextran $04 or LPS gave two-fold higher responses to these mitogens compared to controls. These results appear to be a direct consequence of the deficiency in zinc since mice whose dietary intake was restricted gave responses statistically equivalent to controls. In contrast, the responses to PWM of lymphocytes from zinc deprived mice were statistically equivalent to control responses at this time. When maintained on the diets for an even longer period (35 days) to establish a severely deficient state, the same pattern of mitogenic responsiveness was observed with the exception that the restricted mice also gave statistically elevated responses to LPS compared to controls (data not shown). It should be noted that the differences exhibited among the dietary groups are not attributable to cell death since in all cultures viability prior to harvesting exceeded 80%. 45 Figure 2. Optimal mitogenic responses to PWM, LPS, and dextran $04 as measured by incorporation of H3-thymidine into splenocytes from zinc-deficient, restricted, or control mice cultured in medium supplemented with FCS. Each bar represents the mean 1 SEM of eight mice. Asterisk indicates significance of p<.05 or better as compared to control response. I Restricted 8 Zn deficient _ 46 .1 Zn adequate .1 - u: ....V. ..... ............ . r»\\\\\ \\\\\ ........u.u.u. .............. rm". ...................... . m A. .o. x .53 858.8... 2255 n: PWM LPS Dextran Control 47 Assessment of the Polyclonal Activated Secreting Cells from Zinc Deficient, Restricted,and Control Mice in Fetal Calf Serum Supplemented Cultures. To apply a more rigorous test of the functionality of B-cells from deficient mice, the number of polyclonally activated antibody secreting cells was determined using Protein A coated SRBC (Figure 3). LPS induced twice as many PFC/106 lymphocytes in cultures of lympho- cytes from deficient mice compared to controls. This result paralleled the increase in DNA synthesis observed in the LPS stimulated cultures. In contrast, there was no statistically significant increase in the num- ber of antibody secreting cells induced by PPD in the cultures of lympho— cytes from deficient mice compared to controls. Since dextran 504 does not induce antibody synthesis (19) it was not used in this assay. Measurement of the Mitogenic Responses of Splenocytes from the 3 Dietary Groups in Mouse Serum Supplemented Cultures. It was important to determine what effect the presence of zinc in the culture system might have had on the responses of B-cells from zinc deficient mice. To address this question, splenocytes from each dietary group were cultured in medium supplemented with 0.25% mouse serum collected from either zinc deficient or control mice. The levels of zinc in the serum and in the culture medium are shown in Table 2. Note that the zinc level in sera from the deficient mice was 22% of the control level, representing a severe depletion in zinc. Lymphocytes from the 3 dietary groups were cultured in both the deficient and control mouse serum supplemented medium. The results of stimulation with LPS, dextran $04, and PWM under these conditions are shown in Figures 4, 5, and 6, respectively. The LPS and dextran responses of lymphocytes from the deficient mice remained substantially increased compared to controls even in cultures 48 Table 2. Zinc levels in culture components Culture Component Zn level (ug/lOO ml) Zn deficient mouse serum 20.2:3+ Zn adequate mouse serum 90.6:5 Fetal calf serum 342.0:5 RPMI 1640 (supplemented) 8.216 +Mean 1 SEM 49 .mmcogmme _oep:ou oa cmeeanu mm coupon Lo mo.va ea muceuwe_cm_m mmumowccw xmwewum< .mows cm» eo 2mm H cue: mzu mbcmmmeawe Len comm .Xmmmm mzcm_a < :_muoea one xa emeammme mm more Poeucoo Lo .vmpuweumme .u:m_u_wwu o:_N see» mmuxoo:m_qm eo co_umpzswpm cmmopwe .mewuqo x5 umuzucw m_Fmo mcwosnoeq :__=no_mocsae_ eo gangs: .m mL:m_L 50 23a mnj Emma one 4 522a .28 5944mm 901/ Odd 51 .mmcoammg _oLu:oo 0p umgmasou we Lmupmn Lo mo.va mo mucmo_+wcmwm mwpmu_nc_ xmwgmpm< .ucmswgqum Lag aaogm xgmpmwu comm 50L» mu_e w mo mmuxoocm_am we mFooq mcwm: mucmewgmgxm m mo 2mm H came mcp mucmmmgamg u:_oa 20mm .muwe _ogucoo Lo pcmwuwkwu o:_~ sogm Ezgmm saw: umucmempaqzm E:_uwe cw vmgzp_:o move _ogpcoo Lo .vmpo_gummg .p:m_u_wmu ucPN 50L» mmuxuocmPQm yo ma; op mmcoammg ogpw>.mfl asp we mowpmcwx .¢ wgzmwm 52 8:2 £9680 cN :5: 2mm m c goo ”\h m 8.5. .828 cN ,8: 2mm ¢ gunxfimm ‘ .......... ‘ 29.630 5 TI... €0.0sz cN ollb u‘ ma... \ ‘ 9 8 (99' x um u0uluo mfl.mge we mewpeewx .m eeemww 54 8:2 92.88 :N Se: 98 n v «>8 mm. 8:2 Ee_e_wee :N Se: 2% 303QO 9.2.2.4 22.608 cN Oulno 2.90:3 cN I J“ ¢ (g.Ol X uLida) Wuwodmun aumufitu 2H 8 55 .emeeemmL wegpeee eu eeLeeEee we Lepuee Le mo.ve we eeeeewwwemwm wepeeweew xmwgepm< .muceeweeexe m we sum H some esp mpcemeeemg pewee seem .mews wegpcee Le peewewwee ech Eeew Eegmm new; eeuceeeweeem Eewees cw eeeeuwee mews wegueee Le .eeuewgumee .ucmwewwee eewN Eegw meexeeemwem we 22¢ ea emceemee egpw> mfl esp we mewpeewg .o meemww 56 8:2 2888 :N Eeew Sew 0 ¢ m: 8:2 286:8 cN Ee: Eem m v my Omegbemmm ‘z, .. C 8268 cN tulle E958 cN ollo \‘ V .225 ON 00. (:DI “ UL"13) uoumodmwu ammwflui EH 57 supplemented with zinc deficient serum. Further, the day 5 PWM responses of the 3 dietary groups measured in zinc deficient or zinc adequate serum were equivalent and compare with the results seen in the FCS cultures on day 5. However, analysis of the kinetics of PWM stimulation revealed that the response of lymphocytes from deficient mice was elevated compared to controls on days 3 and 4 whether zinc deficient or zinc adequate mouse sera was used for culture. Since the FCS cultures were only examined on day 5, the earlier elevation in the deficient response compared to controls was not detected. Quantitation of IQ and Thy-l Bearing Cells in the Spleen of Mice From Each of the 3 Dietary Groups. If zinc deficiency resulted in a preferential decrease in the number of T-cells compared to B-cells, it would result in an artifical elevation in B-cell number on a per lymphocyte basis. If so, this might account for the observed increase in B-cell responses in the deficient cultures. To determine if the decrease in cellularity was uniform among B and T-cells the number of Ig+ and thy-l.2 bearing cells in the spleens of mice was determined. The results are shown in Table 3. The data confirm the decrease in the number of lymphocytes in the Spleens of the deficient mice previously observed and indicate that the reduction does not alter the ratio of B and T-cells remaining in the spleens of the deficient mice. It would appear then, that the elevated B-cell response is not merely a consequence of an increased proportion of B-cells. 58 Table 3. Comparison of percentages of B and T cells in spleens of mice Dietary Total Mononuclear Percentage of surface labelled cells Group cells/spleen (xl05) Ig-bearing thy 1.2-bearing Zn deficient 23.724.1+a,b 54.5:2.3 33.61l.6 Restricted 50.9:5.4 55.3:2.7 29.5:2.6 Zn adequate 65.9:4.1 55.0:2.3 30.1:1.6 +Mean 1 SEM of 9 mice per group ap<.01 as compared to zinc adequate mice bp<.05 as compared to restricted mice DISCUSSION The present study was undertaken to examine the effects of zinc deficiency on B-cell functionality. in vivg results revealed that a decrease in B-cell response was observed as a result of zinc deficiency. Furthermore, when the status of distinct B-cell subsets from deficient mice was examined in vitro, the results indicated that B-cell populations appeared to be selectively altered by the deficiency. Of particular interest was the elevated reSponse of B-cell p0pulations from deficient mice to stimulation with LPS, dextran $04, and PWM. The reason(s) for this elevation are unknown. A number of possibilities which might have accounted for these results were tested. Since zinc deficient animals refed adequate levels of zinc have been observed to give augmented in_vivg antibody mediated responses of up to 200% of control values (8), we wondered if the rather high level of zinc in the FCS supplemented cultures might have produced the increased mitogenic response. However, when we examined the responses using zinc depleted sera, the enhanced responses to LPS, dextran $04, and PWM remained. In the latter case, the zinc content of the deficient serum was very low (20 ug/ml), a level found only in the most severely deficient mice. While it was probable that other components such as minerals, vitamins, and hormones were also altered in the deficient serun (26), this did not seem to have contributed to the results since control cells 59 60 cultured in zinc deficient serum also responded at comparable levels to those obtained in zinc adequate serum. Differences in kinetics do not account for the elevation in responses to the various mitogens. Comparison of the deficient and control responses at various time points with different doses of mitogen produced similar results. Moreover, the elevated responses did not result from an increase in the total number of B-cells since the T:B cell ratio was unchanged in deficient mice compared to controls. While the increased mitogenic responses do not appear to result from any of these more trivial explanations, other possibilities exist which might account for these observations. Since dextran sulfate and LPS are reported to stimulate B-cells in immature or intermediate stages of differentiation (20,2l), these results suggest a possible increase in the number or responsiveness of cells at these levels of maturity in the spleens of the deficient mice. This is given further support by the results of PPD stimulation of lymphocytes from each treatment group. PPD, a mitogen which has been reported to stimulate mature B-cells (22), failed to eclicit a statistically significant elevation in response in cultures of lymphocytes from deficient mice compared to controls. The differences in responses of lymphocytes from deficient and control mice might also result from alterations in the regulatory influences of T-cells (23, 24, 25). Since T-cells are known to be affected by zinc deficiency, the elevated response might be due to a lack of T-cell regulation of the response. However, augmented responses to TI-1 and TI-2 antigens have also been observed in deficient mice. Removal of T-cells was shown not to influence this elevation in response (manuscript in preparation). Therefore, while possible, 61 it is unlikely that the elevation is due to a defect in T-cell effector function. In addition to the possible modifications in cellular function mentioned, it is conceivable that a myriad of biochemical alterations such as cell surface changes, receptor alterations, or defective zinc-protein interactions might account for the observed results. Of particular interest for the understanding of the effects of zinc deficiency on B-cells was the absence of an effect of zinc levels in culture on B-cell mitogenic responses. A previous study based on the effects of in vitro zinc depletion on B-cell mitogenic responses concluded that B-cells were not sensitive to the effects of zinc deficiency (27). In their case, lymphocytes from adult mice cultured in EDTA-zinc depleted serum, were found to give unaltered responses to LPS compared to cells cultured in zinc adequate serum. The present study, however, clearly demonstrates that B-cell responses are altered by dietary zinc deficiency. Thus, attempting to induce the deficiency in vitro appears to produce different results than those obtained when the deficiency is induced ig_vivg. This is further supported by the results of T-cell susceptibility to zinc deficiency reported in the accompanying paper. T-cell responses to PHA were altered following in 1119 zinc deficiency, but were unchanged by culturing in zinc depleted serum. Since equal numbers of lymphocytes from deficient and control mice fail to give equivalent responses to a variety of B-cell probes, the data support the notion that an alteration in B-cell ratios or functionality results from zinc deficiency. Investigation is currently underway to further ellucidate the nature of the modifications in B-cell function induced by zinc deficiency. REFERENCES 1. Golden, M., B.E. Golden, P. Harland, and A. Jackson. Lancet l:l226, l978. 2. Pekarek, R., H. Sandstead, R. Jacob, and D. Barcome. Am. J. Clin. Nutr. 32:l466, l979. 3. Shanklin, S., E. Miller, D. Ullrey, J. Hoefer, and R. Luecke. J. Nutr. 96:l0l, l968. 4. Fraker, P., S. Haas, and R. Luecke. J. Nutr. 107:1889, 1977. 5. Fraker, P., C. Zwickl,and R.N. Luecke. J. of Nutr. llZ:309, l982. 6. Fernandes, G., M. Nair, K. Onoe, T. Tanaka, R. Floyd, and R. Good. Proc. Natl. Acad. Sci. 76:456, l979. 7. Fraker, P., P. DePasquale-Jardieu, C. Zwickl, and R. Luecke. Proc. Natl. Acad. Sci. 75:5660, l978. 8. Zwickl, C., and P. Fraker. Immunol. Comm. 9:6ll, l980. 9. Fraker, P., and R. Luecke. .In_Dietary Substances and Resistance to Disease. (M. Phillips, Ed) pp. l07. Plenum Publishing Corp., New York, l98l. l0. Luecke, R., and P. Fraker. J. Nutr. 109:l373, l979. ll. Luecke, R., C. Simonel, and P. Fraker. J. Nutr. l08z88l, 1978. 12. DePasquale-Jardieu, P., and P. Fraker. J. Immunol. l24:2650, l980. l3. Mishell, R.I., and R.H. Dutton. J. Exp. Med. 126:405, l967. l4. Gronowicz, E., A. Coutinho, and F. Melchers. Eur. J. Immunol. 6:588, 1976. 62 15. 16. 17. 18. 19. 20. 2]. 22. 23. 24. 25. 26. 27. 63 Bankert, R.B., G.L. Mayers, and D.J. Pressman. Immunol. ll8:lZ65, l977. Mishell, B. lfl: Selected Methods in Cellular Immunology (B. Mishell and S. Shiigi, Eds) pg. 23. w.H. Freeman and Co. San Francisco, CA, 1980. Stobo, J,and W.E. Paul,J. Immunol. ll0:362, l973. Gill, J.L., 13; Design and Analysis of Experiments in the Animal and Medical Sciences, pg. l60, Iowa State University Press, Anes, Iowa, 1978. Hammerstrom, L., A.G. Bird, and C.E. Smith. Scand. J. Immunol. llzl, 1980. Gronowicz, F., A. Coutinho, and G. Moller. Scand. J. Immunol. 3:413, l974. Gronowicz, F.,and A. Coutinho. J. Immunol. 4:429, 1975. Anderson, J., Lenhardt, H.,and F. Melchers. J. Exp. Med. 750:1339, l979. McGhee, J.R., H. Kujono, S. Michalek, J. Babb, D. Rosenstreich, and S. Mergenhagen. J. Immunol. l24:1603, l980. Tsukuda, K., Y. Tsukuda, and G. Klein. Cell. Immunol. 60:l9l, 198]. Nishikawa, S., T. Hirata, T. Nagai, M. Mayumi, and T. Izumi. J. Immunol. l22:2l43, l979. Roch, P.,and Kinchagessner, M.. In;Trace Element Metabolism In Animals-2, pg. 509 (w. Hoekstra, J. Suttie, H. Ganther, w. Mertz, Eds) University Park Press, Baltimore, l974. Zonzonico, P., G. Fernandes,and R.A. Good. Cell. Immunol. 60:203, 198]. 64 CHAPTER II ASSESSMENT OF THE FUNCTIONALITY OF SPLENOCYTES FROM ZINC DEFICIENT MICE II. IN VIVO AND IN VITRO T-CELL RESPONSES ABSTRACT Although zinc deficiency was known to cause lymphOpenia and immune dysfunction, it was not known if the various subpopulations of T-cells were uniformily affected by the deficiency or if certain subpopulations were more severely affected by the deficiency than others. To distinguish between these two alternatives, and to determine the functionality of the T—cells remaining in the deficient mice, the responses of equal numbers of splenocytes from mice fed zinc deficient, restricted, and zinc adequate diets were compared. Initially, both mitogenic and allogenic responses were examined i!.!i££2: under ordinary culture conditions containing zinc. To determine if the presence of zinc in the culture altered these responses, splenocytes were also cultured in serum from zinc deficient mice. Tests of various T-cell populations appeared to reveal a differential sensitivity to zinc deficiency. Splenocytes from zinc deficient mice cultured in FCS supplement cultures gave depressed Con A (50%), equivalent PHA, but elevated MLC (100%) responses compared to controls after 5 days in culture. Careful examination of the kinetics of these responses revealved that these differences were a product of the length of the culture period. Although the optimal Con A and PHA responses of the lymphocytes from the various dietary groups were actually equivalent, very significant differences in the kinetics and duration of the responses to the two mitogens were observed between the 65 66 treatment groups. In addition, the responses of lymphocytes from the deficient group were similar whether cultures were supplemented with sera from zinc deficient or control mice. Thus, the differences observed between the two treatment groups did not seem to result from changes induced in lymphocytes from deficient mice by the zinc present in the culture medium. Interestingly, the Con A response of lymphocytes from zinc adequate mice was influenced by the serum used for culture. When cultured in sera from deficient mice, the Con A response of control lymphocytes was decreased up to 50% compared to the response obtained in sera from zinc adequate mice. This level of responsiveness was similar to that of lymphocytes from deficient mice. In contrast, the deficient serum appeared to have a negligible effect on the PHA responsiveness of these lymphocytes. Taken together, the results suggest that a disparity in susceptibility to zinc deficiency exists among T-cell subsets. The data further suggest that the decreases in T-cell responses resulting from zinc deficiency do not result strictly from lymphocytopenia but may also be due to fundamental alterations in T-cell responsiveness. INTRODUCTION Deficiencies in dietary zinc represent a very prevalent nutritional problem (1). Since zinc is an essential trace element, it is not surprising that it has also been shown to be vital to proper function of the immune system (2,3,4). The pronounced vulnerability of T-cells to zinc deficiency has been amply demonstrated. The thymus, site of maturation of T lymphocytes, appears to be one of the most wasted organs in zinc deficient animals (5). Histological examination of the atrophying thymus indicate a preferential depletion of the cortex and loss of immature thymocytes (5,6). Significant decreases in circulating levels of the hormones produced by the thymic epithelial cells have also been reported (7). Tests of T-cell dependent functions such as delayed-type hypersensitivity, and T-cell killer and helper function all confirm the striking inhibition caused by suboptimal levels of zinc (8,9,5). While the above data indicate that many T-cell functions are impaired by zinc deficiency, little is known of the mechanism by which zinc deficiency produces these effects. For this reason, it was of interest to evaluate the status of the T-cells remaining in the spleens of the deficient mice. One would like to know if the residual lymphocytes are capable of responding to stimulation or if a significant proportion are nonfunctional due to defects resulting from the deficiency. The question also arises as to whether the observed losses 67 68 are due to proportional losses among all subpopulations of T-cells or if certain subpopulations were more severely affected by the deficiency than others. Answers to these questions would represent a first step towards understanding how zinc deficiency affects T-cell function. To this end, comparisons were made of the ability of equal numbers of viable splenocytes from zinc deficient, restricted, or adequately fed (control) mice to respond to various T-cell mitogens or to allogeneic cells. Initially, the functionality of the T-cells from each dietary group were compared in vitro in uniform culture environments containing substantial amounts of zinc. To determine if the responses of lymphocytes from the deficient mice were influenced by the presence of zinc in the culture, splenocytes from the three dietary groups were also cultured in serum prepared from zinc-deficient or control mice. In vitro tests of the responses of splenocytes from deficient mice revealed differences in the sensitivity to zinc deficiency among various subsets of T-cells regardless of the source of sera used for culture. The data support the view that the deficiency induced nonuniform alterations in functionality or cellular make up of the T-cells in the zinc deficient mice. MATERIALS AND METHODS Mice. Eight to nine week old A/J female mice (Jackson Labs, Bar Harbor, ME) weighing 19.2 1 0.9 g were used in these studies. Diets. Two groups of mice were ad libitum fed a biotin fortified egg white diet containing either deficient (6.7 pg Zn/g diet) or adequate (50 ug Zn/g diet) levels of zinc (10). The components of the diet are listed in previous publications, having been the subject of extensive investigation (l0,ll). Since inanition, or reduced dietary intake, accompanies zinc deficiency (ll), a third dietary group was included to distinguish the effects of zinc deficiency from the effects of decreased food consumption. This restricted group was fed the zinc adequate diet in amounts limited to the average daily intake of the deficient mice. Diet consumption was measured daily and the mice were weighed at least once a week. All mice had full access to deionized, distilled water ((0.2 pg Zn/g). To prevent recycling of zinc from body wastes, the mice were housed in stainless steel cages with mesh bottoms. Feed jars and water bottles were washed with 4 N HCl and rinsed with deionized water to remove all residual zinc. Zinc AnaLysis. Zinc content of the diet (4) and serum (6) was determined by atomic absorption spectrophotometry (Varian Techron, Springvale, Australia) as previously described. Mitogens. Concanavalin A (Con A) was obtained from Sigma (St. Louis, MO) and Phytohemagglutin (PHA) from Difco Laboratories (Detroit, MI). 69 70 Collection of Mouse Serum. Mouse serum was obtained from unimmunized zinc deficient or control mice as previously described (12). Cell Culture. Mice were sacrificed by cervical dislocation and their spleens were removed aseptically. Single cell suspensions were prepared from each mouse (assayed individually) as described previously (12). For the determination of proliferation, cells were cultured using a modification of the Mishell-Dutton system (12). RPMl 1640 medium was supplemented with 5% fetal calf serum (FCS) or 0.25% mouse serum collected from zinc deficient or control mice. Measurement of Proliferation. The response to each mitogen was assayed in triplicate at the following concentrations: Con A (7.5, 5.0, 2.5 ug/ml); PHA (200, 100, 50 ug/ml). After 48, 72 or 96 hours, the cultures were pulsed with 1 uCi of methyl H3-thymidine (49 Ci/mM, New England Nuclear, Boston, MA). Twelve hours later the cultures were harvested onto glass fiber filters using a multiple sample harvester (Otto Hiller Co., Madison, Wis). The amount of H3-thymidine incorporated into trichloroacetic acid washed material was measured using a Beckman scintillation counter (Beckman Instruments, Palo Alto, CA). Only optimal responses to mitogens which were the same for all treatment groups were reported. Mixed Lymphocyte Culture. According to the method of Meo (13), 2.5 x 105 responder A/J splenocytes were cultured with 5 x 105 mitomycin C treated allogeneic Balb/c splenocytes which had been determined to be the optimum concentration for stimulation. Syngeneic, mitomycin C treated A/J splenocytes were used as controls. Mitomycin treated cells did not respond to Con A stimulation. Cultures were maintained for 96 hours in 71 flat bottom microtiter plates then pulsed and harvested as described above. Detection of Antibody ProducinggCells. Mice were immunized intraperitoneally with 1 x 108 sheep red blood cells (SRBC) in sterile phosphate buffered saline. The total number of direct and indirect plaque forming cells (PFC) was determined on day 5 using a modification of the Jerne plaque assay described in detail elsewhere (5). Corrections were made for the small number of direct plaques which appear on the indirect plates. Background plaques produced by unimmunized A/J mice were negligible. Data Evaluation. Means and standard error of the mean were calculated from triplicate values for H3-thymidine incorporation in the case of cell culture, or from duplicate plates in the case of PFC responses. All data were examined by analysis of variance. Probability values were determined by Dunnett's t_test (14). RESULTS Effect of Zinc Deficiency on Food Intake and Weight Gain. The pattern of weight loss and decrease in dietary intake were outlined in the accompanying paper (12). To summarize, the deficient mice weighed 27% less than controls, while the restricted mice weighed l6% less than controls at the end of the 28 day feeding period. Based on previous experiments, this moderate degree of weight loss reduces the effects of inanition on immune capacity. Measurement of In Vivo Antibody Responses of Control, Restricted, and Zinc Deficient Mice. Prior to cell culture, it was necessary to establish the degree 0f.ifl vivo immune impairment resulting from this period of zinc deficiency. To this end, nine mice from each of the 3 dietary groups were immunized with SRBC. These results are shown in Figure 1. After 28 days of feeding the diets, the deficient mice produced 50% as many IgM and IgG PFC/spleen as did control mice (Figure 1), while the restricted mice exhibited a slight, but statistically insignificant, depression in both the IgM and IgG response compared to controls. It is important to note that when the IgM and IgG responses were considered on a per million cell basis, the responses of all 3 dietary groups were equivalent. 72 73 Figure l. In_vivo reSponse of zinc-deficient, restricted, and control mice after 28 day feeding period. PFC responses were determined 5 days after immunization with SRBC. Each bar represents mean 1 SEM of nine mice. Asterisk indicates significance of p<.05 or better as compared to control mice. PF C/Spleen 74 Restricted Zn adequate V Zn deficient 60.00 40,000 I 30,000 20,000 l0,000 V TI’IT {[411 I [47f IN vuvo 5 RBC RESPONSE PFC/I o6 Lymphocytes 75 Measurement of the Mitogeneic and Allogeneic Responses of Splenocytes from Zinc Deficient, Restricted,,and Control Mice in Fetal Calf Serum Supplemented Cultures. Various probes of T-cell function were used to determine if the T-cell populations remaining in the spleens of the deficient mice were capable of normal responses. Both mitogenic and allogeneic stimulation were used. Optimal responses to the mitogens PHA and Con A are shown in Figure 2. It should be noted that dose-response curves were determined for all mitogens employed and optimal stimulation of zinc-deficient, restricted, or control cultures occurred at the same mitogen concentration. Splenocytes from the deficient mice gave a significantly depressed response to Con A compared to restricted and ad libitum fed control lymphocytes. In contrast, the reSponses to PHA of lymphocytes from zinc deprived mice were statistically equivalent to control responses. When mice were maintained on the diets for an even longer period (35 days) to establish a severely deficient state, the same relative pattern of mitogenic responsiveness was observed (results not shown). The mixed lymphocyte response (MLC) of zinc deficient, restricted, and control mice was measured as a further test of proliferation of T-cell subsets. Splenocytes from mice fed either zinc deficient, restricted, or control diet were cultured with allogeneic cells. The results of this assay after moderate (28 days) and prolonged (35 days) zinc deficiency are shown in Figure 3. It is interesting to note that, at both times, lymphocytes from zinc deficient mice give twice the response of control splenocytes. The reSponse of the restricted mice did not differ statistically from controls. 76 Figure 2. Optimal mitogenic responses to Con A and PHA, as measured by incorporation of H3thymidine into DNA of splenocytes prepared from zinc-deficient, restricted, or control mice cultured in FCS supplemented medium cells. Each bar represents the mean 1 SEM of eight mice. Asterisk indicates significance of p<.05 or better as compared to control response. 77 p .2 d Zn adequate ‘ pm: F. _W- h _ px “v.0. x 6&8 95.58909... 05x53:- n1 78 .memceemec wecueee e» emceesee we Leupee Le mo.ve we eeeeewwwemwm meeeeweew xmweepm< .eews pgawe we 2mm H ewes esp mpeemeceec Lee seem .m xee ce eee_ELeuee we; exeue: eewewaxzuum: .mwwee e\e_em eepeecpuu eweerqu new; emcep:ee wee: eews :eeeeee Le eeuewcumec .peewewweeueewN Eecw eeeeeeee mepxee=e_em Leeeeemee e\< .eeweee meweeew mxee mm eee mm Lepwe emceemec we: .m me:m:: 2882.4 e_ee8c>w weceeeg 9.88:?- EislH//////////////////// 98 ea Oh 2...... .N 88:52 I mmzoammm wk>OOTE§b omx__2 Ee_e_w8 :N I (9.0I x quo) wuwodloom ammwflui 2H 80 Measurement of the Proliferative Responses of Splenocytes from the Three Dietarnyroups in Mouse Serum Supplemented Cultures. It was necessary to determine how the presence of zinc in the culture medium influenced the response of the deficient lymphocytes in the previous experiments. For this purpose, splenocytes from the 3 dietary groups were cultured in medium supplemented with 0.25% mouse serum collected from either zinc deficient or control mice. The levels of zinc in the serum and in the culture medium are shown in Table 1. The results of stimulation with Con A and PHA under these conditions are shown in Figures 4 and 5, respectively. The response of lymphocytes from deficient mice was seemingly unaffected by the zinc level of the serum used for culture since both the Con A and PHA responses obtained in zinc deficient serum were similar to those observed in FCS or zinc adequate mouse serum supplemented cultures. However, the responses of lymphocytes from both the restricted and control mice were reduced in the deficient serum supplemented cultures to a level equivalent to the deficient response. This sensitivity to in 21259 zinc depletion was not exhibited by the PHA responsive population since the magnitude of the PHA responses of the dietary groups were similar when cultured in serum from zinc deficient or zinc adequate mice. Analysis of the kinetics of the Con A responses in these mouse serums revealed that the deficient and control mice were equivalent on day 3. The deficient response began to decline very rapidly at this time such that on days 4 and 5, the response of lymphocytes from the deficient mice was significantly depressed compared to control and restricted mice. This decrease was similar to the results observed in FCS cultures on day 5. 81 Table l. Zinc levels in culture components Culture Component Zn level (ug/lOO ml) Zn deficient mouse serum Zn adequate mouse serum Fetal calf serum RPMl 1640 (supplemented) 24.3:3 102.015 342.0:5 8.2:6 +Mean : SEM 82 .memeeemec weeucee e» emceeeee we Leupee Le mo.ve we eeceewwwcmwm meueewecw xmwceum< .mpeesweeexe m we 2mm A ewes esp mpeemeceee pcwee seem .eews Fecpeee Le ucewewwee ech seew Eeeem new; eeueeee_eeem seweea cw eec:u_:e mews Pecueee Le .eeuewcumee .peewewweeueew~ Eeew meuxeeceFQm we < :eu ep emceemee e>wueeeww_eee we mewueew: .e mesmw: 83 8:2 2888 eN Eeew Sew 8.: 28:8 5 so: new m ¢ ¢ mu O¢ 10w um. 18W... M -8. m - w N am m. u m1 1e: m m. D (m. Ocaotumom d ........... 9 I 00. 20.680 :N Tlno 5.0. c . . woe N ell-6 < 200 84 .memseemes wesesee ep emceesee me seeues Le mo.ve we eeseewwwemwm meeeewesw sewseem< .meseswseexe m we 2mm H sees esp musemesees newee seem .ee_E wesesee Le esewewwee esz Eesw Essem sew: eepseEepeem Eeweee cw eesee_ee eewe _ecesee Le .eeeewsemec .esewewweeuee_~ sesw meexeesewem we wueseww—ese we meweeswx .m esemwm 85 8:2 £888 cN Eesw esem m e v m s 8:2 .828 5 see 28 >ea 0 ¢ n {\ 2888 eN 7!... E828 eN ole 3%. ON L 9 (gm x wda) uoswndnwl MIMI gH l I: 8 8 86 The kinetics of the PHA response was altered in cultures of lymphocytes from deficient mice whether cultured in serum from zinc deficient or control mice. In either serum, the maximum response of splenocytes from deficient mice occurred on day 3 instead of day 4 as was the case for the control cultures. By day 5, the responses of each dietary group were numerically equivalent, as would be expected from the results obtained in the fetal calf serum cultures. DISCUSSION This paper examined the effects of zinc deficiency on various T-cell populations. In 3119 examination of T-cell responses of deficient mice suggested that the observed decreases in T-cell function might simply be due to a reduction in total numbers of T-cells since, while depressed per spleen, on a per million cell basis the T-helper cell response of deficient mice was equivalent to controls. I_ 31559 examination of a number of other T-cell populations, however, revealed that lymphocytopenia is probably not the sole reason for the decline in T-cell responsiveness which results from zinc deficiency, since when lymphocytes from deficient and control mice were compared per million cells their responses to various T-cell stimuli were not equivalent. The data suggest that the Con A responsive population is especially sensitive to zinc deficiency, as both dietary zinc deficiency and a zinc depleted environment during the culture period resulted in a decreased response to this mitogen. Since refeeding of zinc to deficient mice is known to result in complete repair of all immune functions so far tested, it is of interest to note that when cells from deficient mice were cultured in FCS, which contains substantial amounts of zinc, their responses to Con A did not improve compared to the response obtained in serum from zinc deficient mice. It has been reported that the primary cell type responding to Con A is an Lyl+ cell (l5), therefore, this data suggests a decrease in the number or responsiveness of this cell 87 88 type as a result of zinc deficiency. It is also possible that the decreased response in 31559 to Con A by control cells cultured in serum from deficient mice might reflect a dependence of Con A on metals such as zinc for receptor binding or triggering. This hypothesis remains to be examined. In contrast, the PHA responsive population was seemingly unaffected by the serum used for culture since lymphocytes from each dietary group gave similar response when cultured under zinc adequate or depleted conditions. It is clear however, that T-cells responsive to PHA are altered somewhat by dietary zinc deficiency since the kinetics of the PHA response of lymphocytes from the deficient mice was shifted compared to controls. This shift may again reflect membrane alterations which could lead to different binding or signaling capacities of lymphocytes from deficient mice compared to controls. These studies also suggest that the sort of changes in T-cell subsets induced by zinc deficiency jp_viyp_may be different then those created by culturing normal splenocytes in 113:9 in a zinc depleted environment. For example Zonzonico, gt 21.,(16) observed a decrease in the Con A response of normal cells when cultured in EDTA-zinc depleted serum and concluded that T-cells were adversely effected by zinc deficiency. In the present study, the Con A response of control cells also decreased when they were cultured in zinc-depleted serum. However, the PHA responsive population was apparently unaltered by a zinc depleted environment in vitgg even though the PHA response was shown to be affected by in 1112 induced zinc deficiency. These data would suggest that caution is necessary in applying the results of zinc depletion in 89 11339 to the alterations in responsiveness resulting from dietary induced zinc deficiency. The necessity for caution is further supported by studies of MLC responses in zinc depleted environments. Fernandes (9) found that when lymphocytes from zinc deficient mice were cultured with allogenic tumor cells no decrease in the cytotoxic response was observed compared to controls. In contrast, Flynn (17) has reported a decrease in the cytotoxic response to allogeneic cells when normal lymphocytes were cultured in zinc depleted serum. In the present study, when the proliferative rather than cytotoxic phase of the MLC response was measured, the response of lymphocytes from deficient mice was actually increased compared to controls. This result might be explained by examining the cell types which respond to allogeneic stimulation. It has been reported that the Ly1+2+3+ cell is the cell type predominantly responsible for proliferation in the MLC response (19). If this is correct, it might suggest an increase in the number of more immature or Lyl+2+3+ cells in the spleens of deficient mice. This would also be in agreement with Nash's observation of an increase in immature T-cells in the spleens of the deficient mice as measured by autologous rosette formation (18). An increase in immature B-cells in the spleens of the deficient mice was also suggested as an explanation for the data reported in the accompanying paper. Regardless of the mechanism, the results indicate a change in the suprpulation of cells responsive to allogeneic cells in the deficient mice. Since equal numbers of lymphocytes from deficient, restricted, and control mice fail to give equivalent responses either in FCS or 90 autologous sera, the findings suggest that in addition to a decrease in lymphocyte number, a selective modification in lymphocyte function or alteration in responsiveness of subpopulations is induced by zinc deficiency. Further investigation is necessary to substantiate the latter supposition in more quantitative terms. Surface marker studies to investigate the phenotypes of cells remaining in the spleens of the deficient mice should allow better understanding of the cellular modifications resulting from zinc deficiency. 2. 5. 6. 7. 9. 10. 11. 12. 13. REFERENCES Prasad, A., M. Miaje, Z. Farid, H. Sandstead, A. Schulent, and W. Derby. Arch. Internal Med. lllz65, l963. Golden, M., B.E. Golden, P. Harland, and A. Jackson. Lancet l:l226, l978. Pekarek, R., H. Sandstead, R. Jacob, and D. Barcome. Nutr. 32:1466. l979. Fraker, P., S. Haas, and R. Luecke. J. Nutr. l07:l889, l977. Fraker, P., P. DePasquale-Jardieu, C. Zwickl, and R. Luecke. Proc. Natl. Acad. Sci. 75:5660, l978. DePasquale—Jardieu, P., and P. Fraker. J. hnmuwfl. l24:2650, l980. Iwata, T., G. Incefy, T. Tanaka, G. Fernandes, C. Mendez-Botet, K. Pih, and R. Good. Cell. Immunol. 47:lOO, l979. Fraker, P.J., C. Zwickl and R.W. Luecke. J. Nutr. ll2:309, l982. Fernandes, G., M. Nair, K. Onoe, T. Tanaka, R. Floyd, and R. Good. Proc. Natl. Acad. Sci. 76:457, l979. Luecke, R., and P. Fraker. J. Nutr. l09:l373, l979. Luecke, R., C. Simonel, and P. Fraker. J. Nutr. l08:881, l978. DePasquale-Jardieu, P., and P. Fraker. l982. Meo, T. ‘In: Immunological Methods, pp. 227, (I.Lefkovits and B. Pernis, eds.) Academic Press. N.Y., l979. 91 14. 15. 16. 17. 18. 92 Gill, J. 13; Design and Analysis of Experiments in the Animal and Medical Sciences, pp. 57, The Iowa State University Press, Ames, Iowa, l978. Nakayama, E., W. Dippold, H. Shiku, H. Oettgen, and L. Old. Proc. Natl. Acad. of Sci. 77:2890, l980. Zonzonico, P., G. Fernandes, and R. Good. Cell. Immunol. 60:203, l98l. Flynn, A. and B. Yen. J. Nutr. lll:907, l98l. Nash, L., T. Iwata, G. Fernandes, R. Good, and G. Incefy. Cell. Immunol. 48:238, l979. 93 CHAPTER III EFFECTS OF ZINC DEFICIENCY ON B-LYMPHOCYTE RESPONSES TO TRINITROPHENYL- CONJUGATED LPS AND FICOLL IN THE A/J MOUSE. ABSTRACT Previous data suggested that the responses of immature B-cells might be altered by zinc deficiency. This was further investigated in the present study by examining the responses of zinc deficient mice to TI-l (TNP-LPS) and TI-2 (TNP-Ficoll) antigens which are reported to stimulate B-cells at early and intermediate stages of maturation. .In'vivg immunization with TNP-Ficoll revealed a 30-60% increase in the PFC/l06 lymphocyte response of deficient mice compared to controls. Further both the heterogeneity and affinity of antibody to TNP-Ficoll was altered by the deficiency and reflected a shift towards the type produced by immature B-cells. The in vivo PFC/l06 lymphocyte response to the TNP-LPS was also increased by the deficiency but no change was observed in the affinity or heterogeneity of the antibody produced by the deficient mice compared to controls. In vitro stimulation with TNP-LPS, with and without T-cells, demonstrated that the elevated responses were not a consequence of aberrant T-cell regulation resulting from zinc deficiency. Taken together, the data indicate that zinc deficiency results in increases in either the quanity or responsiveness of B-cells at the stages of maturation required to respond to both TI-l and TI-2 antigens. 94 INTRODUCTION Zinc deficiency is a common nutritional problem in mankind. A prominent feature of the deficiency is the increased vulnerability to disease. Previous studies have defined the striking injury to thymic integrity and subsequent reduction in many T-cell dependent functions resulting from zinc deficiency (l, 2). More recently we have reported on the effects of the deficiency on B-cell function (3). Both antigenic and mitogenic B-cell responses were found to be altered by zinc deficiency. Of special interest was the increased response of the deficient mice to mitogens reported to stimulate immature B-cells. These data suggested that the proportion of immature B-cells or their responsiveness might be modified by the deficiency. To further examine this pr0position, the present study investigated the effects of zinc deficiency on the B-cell subsets responsive to two T-independent (TI) antigens, trinitrophenyl conjugated lipopolysaccaride (TNP-LPS) and trinitrophenyl conjugated Ficoll (TNP-Ficoll). These antigens were chosen since they are thought to provide information regarding the degree of maturity of B-cell populations. Immature B-cells have been reported to respond to the so called "TI-l“ antigens, (TNP-LPS), while the responses to so called "TI-2" antigens (TNP-Ficoll) are acquired later, presumably after additional B-cell maturation has occurred (4). In addition, both the affinity and heterogeneity of the antibody produced in response to these 95 96 antigens are further indices of the maturity of the responding cells (5). For these reasons, the in £119 and in vitrp plaque forming cell (PFC) responses to TNP-LPS and TNP-Ficoll were examined in zinc deficient, restricted and control mice. The affinity and heterogenity of the antibody produced were determined by hapten inhibition. Since T-I responses have been reported to be under T-cell regulation, (6, 7) it was important to be sure any difference in the response to these antigens compared to controls was not a consequence of alterations in T-cell function which can result from zinc deficiency(8). Therefore, the TNP-LPS response was also measured in 11539, in the presence and absence of T-cells. Since normal culture conditions contain substantial quantities of zinc which might modify the responses of lymphocytes from deficient mice, cells were also cultured in medium supplemented with serum from deficient mice. The results will demonstrate that zinc deficiency results in alterations in both the TNP-LPS and TNP-Ficoll responses which do not appear to result from faulty T-cell regulation. The data also suggests that zinc deficiency results in an increase in either the number or activity of B-cells at the early and intermediate stages of maturation. MATERIALS AND METHODS Mice and Diets. 4-6 week old female A/J mice (l7.0t.Zg) obtained from Jackson Laboratory (Bar Harbor, MA) were used in these experiments. Mice were distributed equally into three dietary groups. Two groups of mice were fed ad libitum a biotin fortified egg white diet containing a deficient ((0.7 uan/g diet) or adequate (50 uan/g diet) level of zinc (9). The composition of the diet is listed in previous publications, having been the subject of extensive investigation (9). Since inanition, or reduced dietary intake, accompanies zinc deficiency (l0), a third dietary group was included to distinguish the effects of zinc deficiency from the effects of decreased food consumption. This restricted group was provided the zinc adequate diet in amounts limited to the average daily intake of the deficient mice. Diet consumption was measured daily and the mice were weighed at least once a week. All mice had full access to deionized, distilled water ((0.2 ug Zn/g). To prevent recycling of zinc from body wastes, the mice were housed in stainless steel cages with mesh bottoms. Feed jars and water bottles were washed with 4 N HCl and rinsed with deionized water to remove all residual zinc. Zinc Analysis. Zinc content of the diet (9) and serum (ll) was determined by atomic absorption spectrophotometry (Varian Techron, Springvale, Austrailia) as previously described. Antigens and Immunizations. TNP-LPS and TNP-AECM-Ficoll (TNP-aminoethylcarbamylmethyl80-Ficoll) were obtained from Biosearch 97 98 (San Rafael, CA). The LPS was prepared from E-coli 055:55 by the trichloroacetic acid extraction method. Mice were injected with 2.5, 5, or l0 ug of TNP-LPS or l0, 25 or 50 pg TNP-Ficoll dissolved in sterile phosphate buffered saline. Cell Culture. Single cell suspensions were prepared from spleens of mice from each dietary group using serum free RPMI l640 supplemented as previously described (3). For determination of antigen specific responses of Splenocytes from each dietary group, a modification of the Mishell-Dutton culture system was used as described previouisly. TNP-LPS was dissolved in serum free culture medium and filter sterilized. Appropriate concentrations of this antigen were added to l0 million pooled Splenocytes. Cells were cultivated in tissue culture tubes in l ml volumes of medium supplemented with 0.5% mouse serum for 3, 5 or 7 days in a humidified atmosphere of l0% C02, 7% 02 and 83% N2. Collection of NMS. Mouse serum from unimmunized deficient and control mice was collected as previously described (3). Removal of thy-l Bearing Cells. Splenocytes (2xl07) were suspended in 200 pl of serum free RPMI l640 and treated with 200 pl of a l/l5 dilution of a monoclonal anti-Thyl.2 (New England Nuclear, Boston, MA) for one hour at 4°C. Cells were washed 2 times and then exposed to a 1:4 dilution of rabbit Lowtox-M complement (Cedarlane Laboratories, Hornky, Ontario, Canada) at 37°C for 45 minutes followed by 3 additional washes prior to culture. The efficacy of T-cell removal was validated by the absence of response to Con A stimulation. Haptenation of SRBC. Trinitrophenylated sheep red blood cells (TNP-SRBC) used as indicator cells in the Jerne plaque assay were prepared by the method of Rittenberg (l2) as modifed by Mishell (l3). 99 Preparation of TNP-L-Lysine. TNP-L-lysine was prepared according to the method of Okuyana (l4) for use as an inhibitor of plaque formation. Briefly, L-lysine was reacted with 2, 4, 6-trinitrobenzene sulfonic acid for 2 hours at 25°C in .09 M sodium bicarbonate. The pH was then adjusted to 1.0 with lN HCl and the precipitate was collected and recrystallized from hot methanol. Purity was determined spectrophotometrically (EM=l7,400 at 360nm). Detection of Antibody Forminngells. The total number of anti-TNP direct plaque forming cells was determined using a modification of the Jerne plaque assay described in detail elsewhere (1). All mice were assayed individually in duplicate and corrections were made for the background responses of unimmunized mice. To allow expression of the data as both the average PFC/spleen and per l06 lymphocytes, the number of viable lymphocytes per spleen was determined by the trypan blue exclusion method. The distribution of affinity of the antibody response was determined by the free hapten inhibition of PFC according to the procedure of Anderson (l5). Average affinities are represented by the reciprocal of the inhibitor concentration required for 50% inhibition of plaque formation. The pattern of inhibition was assessed at five hapten concentrations (10'9, 10'8, l0‘7, l0‘6, 10'5M E-TNP-L-lysine). Statistical Methods. All data was examined by analysis of variance. Probability values were determined by the Student's (l6) or Dunnett's t test (l7). RESULTS Weight Gain. As is usually observed, the deficient mice consumed less diet than controls, and at the end of the feeding period weighed only 74% of control body weight (Table 1). Even though the restricted zinc adequate group was limited to the amount of diet consumed by the deficient mice, they weighed 85% of control weight at the end of the experiment. This underate loss in body weight by the restricted mice has been shown to result in minimal effects of inanition on immune function (l0). Determination of the in vivo Response to TNP-LPS and TNP-Ficoll in Mice from the 3 Dietary Groupp. The effect of various doses of antigen on the PFC/spleen response to TNP-Ficoll and TNP-LPS are shown in Figures 1 and 2,respectively. The optimal antigen concentrations were the same for zinc deficient or zinc adequate mice. In subsequent assays, mice of each dietary group were immunized with 25 pg TNP-Ficoll or 10 pg TNP-LPS. The kinetics of the PFC/spleen response to TNP-Ficoll and TNP-LPS are shown in Figures 3 and 4, respectively. The data indicate that zinc deficiency alters both the kinetics and overall magnitude of the response to TNP-Ficoll. On day 4, the deficient PFC spleen response was significantly depressed compared to controls. However, while the control response began to decline at this time, the deficient response continued to rise, and by day 5 was slightly increased compared to the control response. After day 5, the responses of both dietary groups underwent lOO 101 Table 1. Body weight gain and diet consumption of mice maintained on zinc deficient ((0.7 pg Zn/g), restricted (50 ug Zn/g), or zinc adequate (50 ug Zn/g) diet. Dietary Body Weight Food Consumption Group Initial Final g/mouse/28 days (9) (9) Zinc Deficient 17.11.2+ 14.010.23a 61.915.3b Restricted l7.31.3 15.911.0b 61.915.3 Zinc Adequate l7.31.2 l9.010.8 79.015.3 + Mean 1 SEM ap<.00l as compared to zinc-adequate mice bp<.Ol as compared to zinc-adequate mice 102 Figure 1. Dose reSponse curves of the in_vivo anti-TNP Ficoll PFC/spleen reSponse of zinc deficient, and control mice. 103 On 2888 :N ’16 3988 5 01¢ __8_.._..azw e; mm _ 8.88m 8e .329: uaeidS/oacd 104 Figure 2. Dose response curves of the jn_vivo anti-TNP-LPS PFC/spleen response of zinc deficient and control mice. 105 8:88”: 800 mam-7&2:- useldS/Oslci 106 .eewe m we 2mm H sees esp easemesees Lee seem ._weewmiezw m: mm sew: eerseeew mews _eeesee use .eeeeesemww .esewewwee esw~ we emceemes see_em\eme __ee_muezwiweee e>w> sw esp we mewuesw: .m esemwm coze~_ceEE_ sete £60 107 - m n v a _ _ _ / / / // / / / m / / .. // / O . / . / / / // \\ //*\ 850.1 Bgé‘ ......... .. £88 £8 .81.: =8EIQZw. 35 c. l 108 .eews m we 2mm H sees esp easemesees Les seem .memiezw a: O: sew: eersessw mews Fecesee ese .eeeewsemes .usewewwee esz we emceemes see—em\mme memiezwiwuse e>w> mfl.ese we meweeswx .e esemwm 109 :e_6~_ceEE_ Cece 960 h m n v m we _ fl 4 _ 2 ex / A i / \ leeqee / W \ / : / \ / : // \ I 8.88m 9 ......... a / \ 1000.00 2888 5 O.l-o // \ Ewen cN QIIO /& 91.5 mnDIn—IF e>_> 2. uaaidS/oad 110 similar declines. The kinetics of the restricted mice was similar to controls. As with the TNP-Ficoll response, the overall magnitude of the PFC/spleen response to TNP-LPS was also depressed compared to control and restricted mice. With this antigen, the peak control response occurred on day 3 and declined sharply between days 3 and 5. The deficient response however, remained about the same on days 3 and 5, such that by day 5 the response of the deficient mice was similar to the control groups. Between days 5 and 7, the responses of the 3 dietary groups comparably declined. While the extent of the deficient responses to TNP-LPS and TNP-Ficoll were depressed per spleen, this contrasted with the results seen when the responses were expressed per million lymphocytes. When examined in this fashion, at each time point the deficient response to TNP-Ficoll was increased (30-60%) compared to controls (Fig. 5). Similar results were seen for the TNP-LPS response with the exception of the day 3 deficient response which was depressed compared to controls (Figure 6). For both antigens, the responses of the restricted mice were comparable to controls at each time point examined. Thus, contrary to the results of the PFC/spleen response, on a cellular basis the deficient responses to these antigens were usually enhanced compared to controls. Antibody Affinity. The hapten inhibition assay was used to determine if the antibody produced by B-cells from zinc deficient mice differed in affinity. A plot of the relative affinities of the antibody produced, as represented by the concentration of hapten required for 50% inhibition of the plaque forming response, are shown in Figure 7. 111 .eewe m we 2mm H sees esp easemesees Les seem i853: a; me it. 82:35... 8.2. 38:8 eee 683.5%. .esewewwee esz we memeeemes eexeeseex— eo_\eme e>w> s: .m esemwm 112 ce=e~_ceEE_ Etc 360 83858 35 ‘ ......... .‘ =86.on cN 0Il.0 ANA-C =oe_.-Taz._. 95 c. samuidwm 901 xoaa 113 seem .eewe m we 2mm H sees ese mesemesees see .meuiezw m: a: saw: ee~wseeew eewa _esusee ese .eeuewsemec usewewwee esz we memseemes eexeesesxw oo_\mme e>w>.mw .m esemwm 114 se=e~_ceEE_ sewwe £60 - w m v m J. m d e e / // / W / i . // \ // [w\ / l newetwmem 9.... ..d 888 5 9l6 .cesecee :N I L O 0 ID saiflooudwfi‘l QOI/Odd 1009 21.-C mam-Tag. 95 c. 115 .eewe m we memseemes esp we see: esp mesemesees pswee seem .msemwese eeewseeseee sew: seweersessw e>w> mfl.seewe exee : co .m .e .m eeesm useEeeesu seee Eecw mews we memseemes __eewmiezw ese memiezw we sewuwswsew Rom msw>wm meiezw we seweesuseesee .w msemwm 116 8.82558. Cece mg \- s. m m H 1 D9333 ‘ ........... . .- c8828 cN O.l-o 32.8 eN To e we ................. ‘ ........ ................. ““f 88.-+9: _ din N 10 ¢ IO CO [w m 23:4 88:5 98.8 88% (moi) asuodsau io uomqsuuu v.09 6qu uououuaomo SA‘I-dNJ. 117 The same concentration of hapten was required to inhibit 50% of the TNP-LPS response for each dietary group. It would appear then that little difference in affinity of antibody to this antigen occurs as a result of the deficiency. On the other hand, a much higher concentration of inhibitor (8 times) was needed to inhibit the early phase of the TNP-Ficoll response of the deficienct mice compared to controls. This indicates that the deficient mice initially produce antibody of lower affinity to TNP-Ficoll compared to controls. However, by day 7, similar concentrations of hapten were required to inhibit the response of each dietary group which might be expected wince the response had fallen off by this time. Heterogeneity of Antibody Produced. Since the slope of the inhibition curve has been shown to be inversely proportional to the relative heterogeneity of the antibody population (5), the slopes of the inhibition curves for the TNP-LPS and TNP-Ficoll response are plotted in Figure 8. In response to TNP-LPS, the slopes of the curves do not vary with respect to dietary group, indicating little difference in heterogenity as a result of zinc deficiency. In contrast, the slopes of the curves of inhibition of the TNP-Ficoll response are considerably different for the deficient mice compared to controls. Moreover, while the slope of the control curve decreased with time indicating an increase in antibody heterogeneity, the slope of the deficient curve increased slightly which indicates a more restricted antibody population than that produced by control mice. In vitro TNP-LPS Response of Mice From Each Dietary Group. It was important to determine what role, if any, altered T-cell regulation might 118 .eewe m we memseemec esp :e eeswEseuee e>see e we eeewm use we see: esp easemesees eswee seem .__eewmiezw Le memiezw sew; sewuerseeew seewe exec : co .m .e .m ee eeesm useEeeese seee Eesw mews we emceemes e>w> mfl.ese we sewewewssw seeees aom we sewmes esp Lees e>see :ewuwewssw esp we eeewm .w esemwm 119 5.8.2.5. sec 98 \- v n m n _ J4 H _ i o. I )/ S / m 9. .. . / w. l...... / W / N. m. e. e- 3 m m 0638 cN 0.1.0 28:8 cN 01.0 :03ng 84A,:- éeeeeef €8.24 8 8 3410 “ONION“! 1° 90°13 120 play in the response of the deficient mice to these antigens. To this end, the ifl.!i££2 response to TNP-LPS of each of the dietary groups was measured in the presence or absence of T-cells. Because of the possibility that lymphocytes from deficient mice might recover when placed in cultures containing substantial amounts of zinc, cells were cultured in medium supplemented with serum from zinc deficient mice. The zinc level of the serum used for supplementation is shown in Table 2. The zinc content of the deficient serum is at a level usually found in the most severely deficient mice. It was interesting to note that the results obtained in vitro were similar to those observed in vivo when the TNP-LPS response was determined per million lymphocytes. With the exception of the initial time point, when cultured in either zinc deficient (Fig. 9) or zinc adequate (Fig. 10) serum supplemented uedium, the response of lymphocytes from the deficient mice was greater than either the control or restricted responses. Only on day 3 were the responses of each dietary group equivalent. Furthermore, removal of thy-l.2 bearing cells did not alter this pattern of responsiveness. It is clear from this data, that the enhanced response of the deficient mice is not a consequence of altered T-cell function since, even in the absence of T-cells, the deficient response remained elevated compared to controls. 121 Table 2. Zinc levels in culture components Culture Component Zn level (pg/lOO ml) Zn deficient mouse serum 28.2:4+ Zn adequate mouse serum ‘ 101.0:3 RPMI 1640 (supplemented) 8.216 +Mean 1 SEM 122 .pcma_cmaxm Lag azocm xeopm_u comm seem more m we mwpxuo:m_qm do m_ooa m:_m: mpcmewcmaxm N mo 2mm H come on» mucmmmcamg ucwoa comm .mu_e ucmwuvwmu uc_~ Eogy meow cpwz umucmem_aa:m Ea_vme cw m_qu mcwemwn _-»;p mo mucmmnm Lo mocmmmca asp :_ vmczu_:o wows _ocp:ou co .umuuwcpmmc .pcmwuwwmu ocw~ seem mmpxoocm_qm do mablazp op mmcoqmmg on; ocw_> mfl any we mu_um:w¥ .m mc:m_u .l (111 101 . 123 8:2 2938 EN E0: Row K) k [s # In in 1m ‘5 § 31 film/33d U. WWW 3.28m0m ‘323‘ 2883 5 O.l.O 33.2. 5. To .o+N_ E. :2 28 o \ $805. EF 2.5 c. 124 .pcmswceexm Lee eeecm xgeuewe gene cecw mews m we meuxeeeewem we m_eee mcwm: mueeswgeexm N we 2mm “come we» mpcemecemg newee seem .wews eueeeeee ecw~ secw ecmm new: empcwse_ee:m sewees cw wwwme mewceme wuxnp we eeeemee Le eecmmmge esp cw eeeeuwee mews .ecpeee Le .eeuewcpmec .peewewwme eewN meuxeeee_em we meeuezw ep emceemee one eguw> mH.e;p we mewpeewx .eH m2=m_e 125 8:2 eweeeeee eEN 60: Sam m> c. mum/oi ? 3 § § DISCUSSION The results of these experiments indicate that zinc deficiency induces alterations in B-cell responses to the two T-independent antigens TNP-LPS and TNP-Ficoll. There were a number of differences in the response of the deficient mice to these antigens which suggested that there might be an increase in the number or responsiveness of relatively immature B-cells in the spleens of these mice. Since zinc deficiency results in a decrease in lymphocyte numbers it was not surprising that the PFC/spleen response to TNP-Ficoll was reduced in deficient mice. It was surprising, however, that the deficient response was increased 30-60% compared to controls when expressed per million cells. Thus it appears that of the splenocytes remaining in the deficient animals more were responsive to TNP-Ficoll. Since this antigen is reported to stimulate B-cells in an intermediate stage of maturity, an increase in the number of cells at this stage of differentiation could be expected to produce this result. Furthermore, a significant change in affinity, with a shift toward lower affinity antibody and little change in heterogeneity, was observed in the response of deficient mice to TNP-Ficoll. In contrast, the TNP-Ficoll response of the control mice showed little change in affinity and an increase in heterogenity of antibodies produced with time, which is typical of the results reported for TNP responses (l8). Decreases in affinity and restricted heterogeneity are characteristic of responses of immature B-cells (l5). 126 127 Thus the changes in this regard observed in the deficient mice further suggest an increase in responses of cells at the less mature stages of development. The effects of the deficiency on response to a TNP-LPS, an antigen reported to stimulate fairly immature B-cells, produced somewhat different results. No changes were apparent in the affinity or heterogeneity of the antibody produced in response to this antigen as a result of the deficiency. However, as was observed with TNP-Ficoll, at various times after immunization with TNP-LPS, the PFC/l06 lymphocyte response of the different mice was slightly elevated compared to controls. Moreover, when the response was examined in vitro, the increased response of the lymphocytes from deficient mice compared to controls was even more apparent. This increase might be due to an additional component of polyclonal activation in vitro by TNP-LPS. In any case, removal of T-cells had no significant effect on the ig_vitgg_ responses to this antigen. Therefore, it is doubtful that the increased responsiveness to this antigen by the deficient mice is a consequence of some secondary effect of zinc deficiency on T-cell regulatory mechanisms. It is noteworthy that the increased response to TNP-LPS was observed in cultures supplemented with serum from zinc deficient or control mice. Thus the increased response of lymphocytes from deficient mice did not appear to result from the zinc present during the culture. This is an important observation since j__vivo refeeding of zinc has been shown to result in greatly augmented responses to various antigen compared to the control responses (l9). 128 How zinc deficiency results in the increases in the number or responsiveness of B-cells which respond to TI-l and TI-2 antigens is unknown. It is possible that suboptimal levels of zinc interfere with B-cell processing and cause a build-up of B-cells at the earlier stages of differention. The previousy reported increase in responsiveness of lymphocytes from deficient mice to some B-cell mitogens also supports this notion (3). Lymphocytes from deficient mice responded at twice the control level to LPS and dextran $04, mitogens which are exported to stimulate immature B-cells, while their response to the mitogen PPD, which is believed to stimulate a more mature B-cell subset, was equivalent to controls (20-22). Furthermore, Nash's observation of an increase in the number of immature T-cells in the spleens of deficient mice suggests that lymphocyte differentiation might be halted by zinc deficiency (23). More investigation is necessary to detennine if changes in ratios of B-cell populations are indeed induced by zinc deficiency, or if alteration in biochemical parameters such as receptors binding or membrane signaling apparatus might account for these observations. 1. 2. 3. 4. 5. 6. 8. 9. 10. ll. 12. 13. 14. REFERENCES Fraker, R, S. Haas, and R. Luecke. J. Nutr. l07:l889, 1977. Pekarek, R., H. Sandstead, R. Jacob, and D. Barcome. Am. J. Clin. Nutr. 32:l466, 1979. DePasquale-Jardieu, P., and P. Fraker. Cell. Immunol., Chapter 1. McKearn, J. P., and J. Quintans. Cell. Immunol. 44:367, 1979. Goidl, E.A., Siskind, G. M.. J. Exp. Med. l40:l285, 1974. Mongini, P., and W.E. Paul. lng-lymphocytes in the immune response: Functional, developmental and interactive properties. Edited by N. Klinman, D. Mosier, Scher and E. Vitetta. Elsevier North, Holland pg. 369, 1981. Mosier, D.E., and B.M. Johnson. J. Exp. Med. l4l:2l6, 1975. Fernandes, G., M. Nair, K. Onoe, T. Tanaka, R. Floyd, and R. Good. Proc. Natl. Acad. Sci. 76:457, 1979. Luecke, R., and P. Fraker. J. Nutr. l09:l373, 1979. Luecke, R., C. Simonel, and P. Fraker. J. Nutr. l08:88l, 1978. DePasquale-Jardieu, P., and P. Fraker. J. Immunol. l24:2650, 1980. Rittenberg, M., and K. Pratt. Proc. Soc. Exp. Bio. Med. l32:575, 1969. Mishell, B., and S. Shiigi. .1Q:Selected methods in cellular immunology. w. H. Freeman and Company. San Francisco, pg 52, 1980. Okuyama, T., and K. Satake. J. Biochem. 47:454, 1960. 129 15. l6. l7. l8. l9. 20. 21. 22. 23. 130 Anderson, 8.. J. Exp. Med. l32:77, 1970. Downie, N.M“.and R.W. Heath. 1558asic statistical methods. Edited by N. Downie. Harper and Row, N.Y. pg. 128, 1965. Gill, J., Iggoesign and analysis of experiments in the animal and medical sciences. Iowa State University Press, Iowa. pg. 57, 1978. Miller, G.N., and D. Segre. J. Immunol. l09:74, 1972. Zwickl, C., and P. Fraker. Immunol. Comm. 9:6ll, 1980. Gronowicz, F., A. Coutinho, and G. Moller. Scand. J. of Immunol. 3:4l3, 1974. Gronowicz, F., and A. Coutinho. Scand. J. Immunol. 4:429, 1975. Anderson, J., N. Lenhardt, and F. Melchers. J. Exp. Med. 750:]339, 1979. Nash, L., T. Iwata, G. Fennandes, R. Good, and G. Incefy. Cell. Innmnol. 48:238, 1979. 131 CHAPTER IV EFFECTS OF ZINC DEFICIENCY ON PRIMED AND UNPRIMED RESPONSES TO SRBC FOLLOWING ADOPTIVE TRANSFER OR NUTRITIONAL REPLETION ABSTRACT Zinc deficiency has been shown to impair primary and secondary responses to various antigens if the initial antigenic challenge occurs during the deficient state. The consequences of zinc deficiency on an finnune response initiated preceding the deficiency have not been examined. To address this question, A/J female mice were immunized with SRBC prior to being placed on zinc deficient diets. After 4 weeks on the deficient diet, primed mice given a second injection of SRBC exhibited a significant reduction (50%) in responsiveness compared to controls. Furthermore, when equal numbers of splenocytes from primed mice were transferred to syngeneic irradiated hosts to allow comparisons of responses in nutritionally adequate environments, the secondary response of the deficient reconstituted mice was again substantially reduced (50%) compared to control reconstituted mice. Moreover, upon nutritional repletion, at a time when primary responses were fully restored, the zinc repleted primed mice still exhibited a reduced response (30%) to secondary immunization with SRBC compared to primed, zinc adequate fed mice. Taken together, the results suggest that a comparatively short period of zinc deficiency can result in partial, but apparently long-lasting, injury to primed cell populations. 132 INTRODUCTION Zinc deficiency has been shown to result in significant impairment of various hnnune processes (1,2). Severe atrophy of lymphoid organs, lymphocytopenia, and reduction of most T-cell mediated responses have been observed in zinc deficient animals (3). Furthermore, mice immunized with various antigens following short periods of zinc deficiency exhibit decreased primary antibody mediated responses to these antigens (4). Previous studies fran this laboratory (3) and others (5) have shown that the ability to establish a secondary response is also diminished by zinc deficiency. Mice maintained on zinc deficient diets for four weeks and then given two injections of sheep red blood cells (SRBC), spaced l week apart, exhibited a significant reduction in secondary PFC/spleen responses compared to controls. The effects, however, of zinc deficiency on an immune response established before induction of the deficiency have not been examined. It was of interest to determine if a primed cell population might be influenced by zinc deficiency. In addition to the knowledge this could provide about the fundamental role of zinc in lymphocyte function, this information has clinical relevance. It is important to know if periods of zinc deficiency resulting from poor diet or numerous disease states might alter the reSponses to vaccines given prior to initiation of zinc deficiency. For these reasons, mice were immunized with SRBC two weeks prior to being placed on purified diets. After a 28 day feeding period, mice 133 134 maintained on zinc deficient, restricted or control diets were given a second injection of SRBC and the responses of the three groups were compared. At this time, equal numbers of primed cells from each of the dietary groups were also transferred to syngeneic, irradiated hosts to allow examination of the responses in nutritionally equivalent environments. To examine the secondary response following nutritional repletion, another group of the deficient mice were refed diets containing adequate zinc prior to the second injection of SRBC. Similar protocol were followed using unprimed mice to allow direct comparisons of the effects of the deficiency on primed versus unprimed responses. The design of all of these experiments is outlined in Table l. The results reveal that the responses of primed and unprimed mice are impaired during zinc deficiency. Furthermore, following nutritional repletion, at a time when primary SRBC responses were fully restored, the secondary responses of mice primed prior to induction of the deficiency were significantly depressed compared to controls. Thus, it appears that intervals of zinc deficiency can result in permanent damage to primed cell populations. MATERIALS AND METHODS Mice and Diets. Six week old A/J female mice weighing l8.3:.5g were used in this experiment. Mice were divided equally into 3 dietary groups. Two groups of mice were fed ad libitum biotin fortified egg white diet containing a deficient ((0.7 uan/g diet) or adequate (50 pg Zn/g) level of zinc (3). The composition of the diet is listed in previous publications having been the subject of extensive investigation (6). Since inanition, or reduced dietary intake, accompanies zinc deficiency (7), a third dietary group was included to distinguish the effects of zinc deficiency from the effects of decreased food consumption. This restricted fed group was provided the zinc adequate diet in amounts limited to the average daily intake of the deficient mice. Diet consumption was measured daily and the mice were weighed at least once a week. All mice had full access to deionized, distilled water ((0.2 ug Zn/g). To prevent recycling of zinc from body wastes, the mice were housed in stainless steel cages with mesh bottoms. Feed jars and water bottles were washed with 4 N HCl and rinsed with deionized water to remove all residual zinc. Zinc Analysis. Zinc content of the diet (6) was determined by atomic absorption spectrophotometry (Varian Techron, Springvale, Australia) as previously described. Immunizations. To examine the effects of zinc deficiency on a secondary reSponse to SRBC, mice were primed with an intraperitoneal injection of 5 135 136 x 106 SRBC two weeks prior to being fed either zinc deficient, restricted, or zinc adequate diets. After a 28 day feeding period, mice from each dietary group were given a second immunization of l x l08 SRBC and the PFC responses were measured 4 days later. In addition, unimmunized mice, also maintained on the diets for 28 days, were immunized with l X l08 SRBC at this time to determine the relative degree of impairment of the primary response. Detection of Antibody Forming Cells. Subsequent to injection with SRBC, the total number of direct and indirect plaque forming cells (PFC) was determined on day 4 for the secondary response or day 5 for the primary response using a modification of the Jerne plaque assay described in detail elsewhere (3). These time periods were previously determined to be optimal for A/J mice. Corrections were made for the small number of direct plaques which appear on the indirect plates. Background plaques produced by unimmunized A/J mice were negligible. To allow expression of the data as both the average PFC/spleen and per l06 lymphocytes, the number of viable lymphocyte per spleen was determined by the trypan blue exclusion method. Adoptive Transfer. Both the primary and secondary responses of mice from each dietary group were measured following adoptive transfer. To this end, previously irrmunized or unimnunized donor mice from each treatment group were killed after the 28 day feeding period. Washed spleen cells (16 x l06) from these mice were injected intravenously into syngeneic recipient mice which, 6 hours previously, had received 700r of radiation from a 60Co gamma source. Six hours was allowed for the lymphocytes to localize before the hosts were injected intraperitoneally with l x 108 SRBC. The PFC responses of hosts reconstituted with primed 137 cells were measured 4 days after immunization, while the responses of hosts reconstituted with unprimed cells were measured 5 days subsequent to immunization. In addition, a group of mice reconstituted with unprimed cells were immunized with SRBC after a one week, rather than 6 hour period of lymphocyte localization. Unreconstituted, immunized hosts gave less than l0 PFC/spleen at 5 or 12 days after irradiation. Restoration of the Secondary Response to SRBC. After the 28 day feeding period, additional groups of primed and unprimed zinc deficient mice were returned to zinc adequate diet for either 3 or 4 weeks. At the end of these restoration periods, primed or unprimed mice from each dietary group were given an intraperitoneal injection of l x l08 SRBC and a Jerne assay was performed after 4 day or 5 days, respectively. Data Evaluation. Means and standard error of the mean were calculated fran duplicate plates in the case of PFC responses. All data were examined by analysis of variance. Probability values were determined by Dunnett's or Student's t_test. 1353 Table 1. Experimental Design A) Measurement of Immune ReSponses After Dietary Treatment l' Response: Dietary SRBC Jerne Treatment (1X108) Assay Zn (-) Zn (+) l 7 4 weeks 5 days 2° Response: SRBC Dietary SRBC Jerne (5x105) Treatment (lxl03) Assay Zn (-) Rest. Zn (+) v 2 weeks - 4 weeks 4 days 8) Adoptive Transfer Protocol 1‘ Response: Dietary Unprimed Cell SRBC Jerne Treatment Transfer to (IXIDB) Assay Zn (-) Irradiated Zn (+) Hosts 6 hrs or 4 weeks I week y. 5 days 2' Response: SRBC Dietary Primed Cell SRBC Jerne (5x106) Treatment Transfer to (le08) Assay Zn (-) Irradiated Rest. Hosts Zn (+) l 2 weeks 4 weeks , 6 hrs 4 days ____ C) Dietarngepletion Protocol 1° Response: ' Dietary Both SRBC Jerne Treatment Groups (le03) Assay Zn (-) ZlnC (*) Zn (+) Diet 7 4 weeks I 3 weeks 5 days 2° Response: SRBC Dietary All Groups SRBC Jerne (5x105) Treabnent Zinc (+) (1x103) Assay Zn (-) Diets Rest. In (+) 2 weeks 4 weeks 3 or 4 weeks 4 days 139 Table 2. Body Heights of Mice from Each Treatment Group Before and After the Repair Period Body Weight (g) Dietary Group Initial Prior To After 3 wks After 4 wks Repletion 0f Repletion 0f Repletion Zinc Deficient l8.21.3+ l6.3:.23’b 20.9:.4 22.5:.2 (75%) (96%) (96%) Zinc Adequate l8.5:.3 2l.51.3 21.7:.4 23.4i.2 Restricted l8.4:.2 l7.6:.2a 20.7:.5 ND (82%) (96%) +Mean 1 SEM, Dunnett's t test ap<0.01 as compared to zinc adequate mice bp<0.05 as compared to restricted mice Numbers in parenthesis indicate percentage of body weight of zinc adequate mice. 140 .mewe epeeeeee eewN eu emceesee we Leppee Le _o.ve we eeeeewwwcmwm wepeewecww .muws ow we 2mm H ewes we“ mpcemeceee can seem .umxm saw: eewuerceasw cmuwe mace m eeewELepee wee: memceemec one .eewcee mewemew AWN mm m Leuwe mews Pecucee eee peewewwme eewN we emceemec ow e>w> cH ._ ecemwm 141 .9...- n .P. .m f e d n _ Z ‘ _‘L 1° SRBC RESPONSE e .0' a U q 3 d 0 n 2 838853 e98“: O 5 40,000 ._..._. l0,000 60,000 cooimkfii J \n 4» «.Mu .#§ .4. ad fie... t. . J a germs Art RENEW” r “NVA. saw". . . . . .. .9...; v . o . .. J... .. J 3.»... 5...... 30,000 20,000 10,0 - 142 .eePE eueeeeee esz on eeseesee me segues Le mo.ve we eeseewwwsmwm meeeewesww .eewa o_ we 2mm H sees esp musemesees Les seem .seee_ mxee e eeesewsee me; xemme essee e ese ummm we sewpeewsw eseeem e se>wm use: mews mswu mwsu e< .eewsee msweeew see mm e Lepwe mews wesesee Le .eeeewspmes .esewewwee esz we emceemes om e>_>.mm .N essmwm 2" Response to SRBC 143 samooqdwfl'l 90l/0;ld -2 -|00 IgG IgM H ///////// 5 / ” IgG 40,000 - 20,000 - §§ uaaIdS/ 2).-1d 4oo,ooo- RESULTS Effect of Zinc Deficiency on Weight Gain. The deficient mice consumed less diet than controls and after the 28 day feeding period, weighed 5.2 9 less or 75% of control body weight (Table 2). In contrast, the restricted mice, even though limited to the dietary intake of the deficient mice, weighed 82% as much as controls. This minimal degree of weight loss is consistent with previous experiments and has been shown to minimize the effects of inanition on immune function (6). In the zinc repletion experiment, 3 weeks after being refed a zinc adequate diet, the weights of the previously deficient mice and restricted mice were equal to that of the control mice. Effect of Zinc Deficiency on Primed or Unprimed Response to SRBC. The effects of zinc deficiency on the SRBC responses of unprimed or primed mice are shown in Figures 1 and 2, respectively. After the 28 day feeding period, both the direct and indirect PFC/spleen responses to SRBC of previously unimmunized deficient mice were depressed compared to controls (Figure l). This observation has been published previously (3). The response of the primed mice was also impaired by zinc deficiency. Both the direct and indirect PFC/spleen responses of the primed, deficient mice were depressed 50% compared to restricted or zinc adequate mice (Figure 2). On a l06 lymphocyte basis, neither the primed nor the unprimed response of the deficient mice differed statistically from controls. 144 145 Adoptive Transfer Responses of Primed and UnprimedySplenocytes from Zinc Deficient, Restricted and Zinc Adequate Mice. To compensate for the overall decrease in lymphocyte numbers resulting from zinc deficiency and for the possible nutrient limitations of the Zinc deficient mice, splenocytes from primed and unprimed mice from each one of the 3 dietary groups were transferred to syngeneic irradiated mice and the hosts were immunized l2 hours later with SRBC. The results are shown in Figure 3. Even though equal numbers of viable splenocytes from the 3 groups were transferred, the PFC/spleen responses of the deficient reconstituted hosts were depressed compared to zinc adequate and restricted reconstituted mice. When expressed per l06 cells, however, the responses of mice reconstituted with splenocytes from each dietary group were equivalent. The results were very different when unprimed lymphocytes from the deficient mice were transferred to irradiated hosts (Figure 3). In this case, both on a per spleen and per 105 lymphocyte basis, mice reconstituted with splenocytes from deficient mice gave significantly higher responses to a primary injection of SRBC than did control reconstituted mice. Identical results were obtained whether a 6 hour or l week period of localization was allowed prior to the initial immunization (Figure 4). The one week period of localization and maturation was included to increase plaquing efficiency, thus there are more plaque forming cells present in mice from each group after a one week period compared to six hour period of localization. However, the relative difference between the two groups was unchanged. Primary and Secondary Responses of SRBC Following Nutritional Repletion. The primed deficient mice were refed diets containing adequate zinc for 146 .eewE eeeeeeee eswe eu eeseesee me seuees so mo.ve we eeseewwwsmwm meeeewesww .eewe up we sees esp mesemesees Les seem .seee_ Aemseemes o_v exee m Le Aemseemes omv mxee e eeEsewsee me; xemme essee e eee seweaewemseees Leewe msees e ummm sew; eeewseesw mew; mew: .eewe epeeeeeeueew~ Le .eeeewsemes .esewewweeueswe sesw meexeesewem Ao_v eeswses: Le Aomv eeswse sew; eeeeewemseees memes eeeeweessw ewesemsxw we memseemes ems .m eczmwm 147 salflooudwfi‘l QOI/Odd 0000 . 0000 000.0. ///./. ...“..mw. / j sewmce: //// L* / 000. *1; 000m 000m 000¢ I "//////////// E... .n.“ L I L 523988 839.2 emmm 2 8838 8. .N ezaeee chesew ommm ew emceemem we. a. / I D 3"." . "CHI OI... n. ..wu...... ...». l I I". IHII “means. I I I D .”.u.u.u.”.n. 333" I C I". t I". I C I "3” we. ........n.n u “Mun”. .... I I 000.0? 000.0m 000.0N. 1 000.00. USGIdS/Odd 148 .eewe _esesee ee eeseesee me segues Le mo.ve we eeseewwwsmwm meueeweswe .seee_ exec m eessewsee me: xemme essee e ese seweaewemseees seewe see: _ ummm sew: eeewsessw use: eewz .eewa meeeeeee esz Le usewewwee eswe sesw meexeesewem eeswces: sew; eeeeewemseees memes empeweessw ewesemsxm we memseemes ems .e eszmwm PFC/Spleen 149 ADOPTIVE TRANSFER & Zn deficieni SRBC RESPONSE Zn odequoie , IgG : J r “F : , .. 1400 l ' a; 3 ° -. 1300 d: 1} g -2 $200 3 5.0 _ if 5 :2, _ -5 E IOO E : :r 3% 3 .. . -I m .lgM -E i 8 y. -: I 0 :_ d: :400 & 10,000:- -5 ’ i300 -: 3200 spool -3- 2 _ -3 - IOO 150 three weeks or 4 weeks prior to the second injection of SRBC. A refeeding period of 3 weeks had been shown to be more than adequate to completely restore a primary response to SRBC (8). The secondary response of the formerly deficient primed mice is shown in Figure 5. On a per spleen basis, the secondary IgG response of the zinc repleted mice was depressed 30% compared to controls, while on a per l06 cell basis, the responses of the dietary groups were again equivalent. As expected, 3 weeks of refeeding was, however, adequate to completely restore both the direct and indirect primary responses of the deficient mice (Figure 6). Refeeding even resulted in an increase in both the IgM and IgG responses to SRBC compared to controls. 151 .eews wesesee ee emceesee me seeees Le mo.ve we eeseewwwsmwm meeeewesww .eews NF we 2mm w sees esp mesemesees Les seem .cmee_ mxee e eeELewsee we: xemme essee e use ommm sew: eerszzs;es esez mews __e eewsee msweeewes esp Leew< .meeesm __e eu “ewe eeeeeeee esz msweeew we mseez m xs ee3e__ew meewe e>wpeeemes se exee mm Leewe mews _esesee Le .eeeewsemes .esewewwee eswe we emceemes om e>w> mm .m esemwm 2° Response to SRBC offer refeedmg semaoudwm 90I/Ozld O O (D l 4400 4200 O (D l ... ...-.....- é UGGIdS/Qdd BOQOOOL 400. 153 .eepe _esesee ee eeseegee me segues se mo.ve we eeseewwwsmwm meueewesw4 .eewe e we 2mm w sees esp mesemesees ses seem .seeew mxee m eeEsewsee we; xemme essee e ese emmm sew: seweeewssasw _ewews_ se se>wm esez eews wwe eewe mwse u< .meeesm sees ee uewe eeeeeeee eswe msweeew we mseez m xs eeze__ew meewe e>weeeemes se maee mm seuwe eews _esesee se .esewewwee esz we emceemes ow e>w>.qw .o eszmwm 154 00m 0 0 <1’ § § 29 .29 , l 6 q. S, e SGWOHGUJA'I 90I/0sld § § 8 § e9 ea. 8822 5% 8%. 2 seems ._ DISCUSSION Zinc deficiency has been shown to impair both l° and 2° response to SRBC in which the initial challenge occurred during the deficient state. These studies revealed that the deficiency can also interfere with secondary responses to SRBC even when the primary challenge is given 2 weeks prior to initation of the deficiency. Since 2 weeks has been shown to be adequate to establish both T-helper cell and B-cell memory to heterologous red cell antigens (9,l0,ll), these results suggest that zinc deficiency reduces the responses of memory as well as virgin lymphocytes. These studies further suggest that fundamental changes in the primed cell population result from zinc deficiency. When equal numbers of viable splenocytes from each dietary group were transferred to irradiated normal hosts, upon secondary immunization with SRBC, the PFC/spleen response of deficient reconstituted mice was depressed compared to control reconstituted mice. This was unexpected since, prior to transfer, the secondary response of the deficient and control mice were equivalent on a per cell basis. Therefore, transfer of equal numbers of splenocytes from deficient and control mice to nutritionally adequate environments might have been expected to produce equivalent secondary responses. Whether zinc deficiency results in aberrant migration, localization or responsiveness of these cells which might explain the decreased response remains to be examined. 155 156 The results obtained following nutritional repletion further support the idea that zinc deficiency induces alterations in the primed cell population. After 3 weeks on the zinc adequate diets with completely restored body weights, the PFC/spleen response of the formerly deficient mice was still depressed (30%) compared to controls. Three weeks has been shown previously to be adequate to completely restore spleen and thymus weights and, as was apparent in this study, primary responses to SRBC (l). Furthermore, refeeding the diet for an additional week did not improve the responses of the formerly deficient mice, since their response remained at 30% of control values. However, the response of the formerly deficient primed mice, while depressed, was still better than the response seen prior to refeeding. This implies that some of the inhibition observed during the deficient state is in part due to nutrient limitations of the zinc depleted environment. Since zinc has been reported to be important for enzymes involved in cell proliferation (12) and also in factors involved in immune responses such as factor serum thymes and T-cell replacing factor (13), alterations in any or all of these might be responsible for the added reduction in the secondary response of these mice. Nevertheless, taken together, the data following adaptive transfer and nutritional repletion suggest that primed cell populations are modified by zinc deficiency. The observation of a lasting effect of zinc deficiency on a primed immune response is important in light of its clinical significance. In addition to dietary induced zinc deficiency, there are a number of disease states such as cirrhosis, intestinal obstruction, and renal disease which can result in zinc deficiency (2). Since zinc deficiency, as previously mentioned, is known to interfere with primary 157 antibody responses, the findings of a reduced secondary response during zinc deficiency suggest that the immune response of these patients is more severely compromised than had previously been proposed. The further observation of a 30% decrease in the response even after nutritional repletion suggests that intervals of zinc deficiency resulting from poor diet or any of the outlined diseases, could result in some decrease in memory cell responsiveness. The rather substantial secondary response observed upon refeeding, however, suggests that while less than normal, this response might still be protective in the repleted host. As might be expected, the results of zinc deficiency on the unprimed population were very different than those observed on primed cells. Both per spleen and per l06 lymphocytes, the PFC response of hosts reconstituted with Splenocytes from unprimed deficient mice was considerably elevated compared to hosts reconstituted with equal numbers of spleen cells from control mice. These results were somewhat predictable based on the results of other studies. Previous experiments have suggested that zinc deficiency leads to an accumulation of lymphocytes at immature stages of differentiation. The responses to certain B-cell mitogens and T-I antigens, which are reported to stimulate immature B-cells, are increased significantly by zinc deficiency (l4,l5). In addition, increases in the number of immature T-cells in the spleens of deficient mice have been reported (l6). The presence of a higher proportion of immature cells in the donor population which could be processed rapidly into mature cells by a normal host could explain the enhanced responses to SRBC by hosts reconstituted with splenocytes from deficient donors. Layton, E£.£l°s have shown that the primary adoptive transfer response is mediated by a so called "pre-progenitor" cell. The 158 19 surface markers of these cells are IgMTIgD‘ which are typical of immature cells. Howard, gt al., (18) have shown that priming can inhibit the responsiveness of these cells which might explain why the ad0ptive transfer response of the primed deficient mice was not elevated. We cannot, however, rule out the possibility that decreases in T-suppressor function or increases in T-helper function might contribute to this result in light of the known effects of the deficiency on T-cell function (l9,20). The fact that no such enhancement occurred in the memory cell response would seem to argue against altered T-cell effector function. The effects on the primary response were similar when zinc was resupplied by refeeding of the zinc adequate diets. After immunization with SRBC's both the direct and indirect PFC/spleen responses of the restored mice were enhanced compared to controls. This "overshoot" phenomena has been observed in adult and weaning mice upon nutritional repletion (4,8). It seems reasonable to assume that the enhanced responses are a result of differences in the cellular composition of repaired spleens compared to controls. This is further evidence of the alteration in ratios or functionality of splenocyte populations resulting from zinc deficiency. Taken together, the results suggest that dietary zinc deficiency results in modifications of both virgin and primed lymphocyte papulations. But unlike virgin lymphocytes which can be renewed by nutritional repletion, primed cells appear to be a stationary rather than a dynamic population, which when damaged by zinc deficiency cannot completely recover. 10. ll. 12. 13. 14. 15. 16. REFERENCES Pekarek, R., H. Sandstead, R. Jacob and D. Barcome. Am. J. Chm. Nutr. 32:l466, l979. Prasad, A., H. Sandstead, A. Schubert, A. Cale and Z. Farid. Am. J. Clin. Nutr. l2:437, l963. Fraker, P., S. Haas and R. Luecke. J. Nutr. l07:l889, l977. Zwickl, C. and P. Fraker, Immunol. Comm. 9:6ll, l980. Fernandes, G., M. Nair, K. Onoe, T. Tanaka, R. Floyd and R. Good. Proc. Natl. Acad. Sci. 76:457, l979. Luecke, R. and P. Fraker. J. Nutr. l09:l373, l979. Luecke, R., C. Simmonel and P. Fraker. J. Nutr. 108:88l, l978. Fraker, P., P. DePasquale-Jardieu, C. Zwickl and R. Luecke. Proc. Natl. Acad. Sci. 75:5660, l978. St. C. Sinclair, hnnunol. l2:559, l967. Cunningham, A.J. and E. Sercarz. Eur. J. Immunol. l:4l3, l97l. Cunningham, A.J., Immunol. l6:62l, l969. Prasad, A. and D. Oberleas. J. Lab. Clin. Med. 83:634, 1974. Dardenne, M., J. Pleau, P. Lefrancier, and J. Bach. C.R. Acad. Sci. Paris 292:793, 1981. DePasquale-Jardieu, P., Chp. l, Thesis. DePasquale-Jardieu, P., Chp. 3, Thesis. Nash, L., T. Iwata, G. Fernandes, R. Good and G. Incefy. Cell Immunol. 48:238, l979. 159 17. 18. I9. 20. 160 Layton, J. J. Immunol. 126:1227, 1981. Howard, M.J. Baker, J. Teale, and K. Shortman. Scand. J. Immunol. 11:327, 1981. Fraker, P., C. Zwickl and R. Luecke, J. Nutr, ll2:309, l982. Chandra, R., Am. J. Clin. Nutr. 33:736, l980. SUMMARY AND CONCLUSIONS The worldwide incidence of zinc deficiency coupled with its detrimental effects on immune processes warranted investigation into the specific effects of suboptimal zinc levels on lymphocyte subsets. Since lymphopenia accompanies zinc deficiency, it was important to determine if subpopulations of lymphocytes were uniformily affected by zinc deficiency. Focusing initially on B cells, these studies revealed that zinc deficiency did, indeed, alter B cell responses and, furthermore, these alterations appeared to be selective among 8 cell subsets. B cells from deficient mice responded at twice the level of controls to mitogens reported to stimulate immature B cells, LPS and Dextran 504, while the response of the PPD stimulated population, believed to be a more mature B cell subset, was unchanged by the deficiency. These data implied that the number or responsiveness of immature B cells were influenced by zinc deficiency. This possibility was further investigated using TNP-LPS and TNP-Ficoll,antigens which have been demonstrated to activate B cells at early and intermediate stages of maturation. Responses to these substances were also increased as a result of the deficiency. I vitro examination revealed this elevation was not a consequence of faulty T cell regulation. The characteristic of the antibody produced to TNP-Ficoll was also altered by the deficiency. Both the affinity and 161 162 heterogeneity of antibody produced by the deficient mice were characteristic of that made by immature B cells. The results of adoptive transfer experiments also suggested that B cell migration and/or maturation might be influenced by supoptimal zinc levels. The primary adoptive transfer response has been demonstrated to be mediated by a cell with surface characteristics of immature B cells (sIgMTSIgD‘). The elevated PFC responses observed in syngeneic irradiated hosts reconstituted with splenocytes from deficient mice compared to controls may be indicative of an increase in the proportion of these sIgM+ B cells in the spleens of deficient mice. Preliminary fluorescent activated cell sorter profiles (Appendix 1) of splenocytes labeled with anti u chain antibody also revealed an increase in both the number of sIgM+ cells and in the amount of sIgM found on splenocytes from deficient mice compared to controls. Taken together, the results of surface marker analysis, adoptive transfer data, mitogenic and antigenic B cell probes provide rather convincing evidence that B cells at early or intermediate stages of maturation are accumulating in the spleens of the deficient mice. The reasons(s) for this is not known, but suggests an undetermined role for zinc in B cell maturation. Additional studies revealed that the effect of the deficiency on B cells was not limited to immature and virgin B cells since mature, antigen primed cells were also impaired by zinc deficiency. Moreover, while cells responsible for primary immune responses are completely renewable following periods of zinc deficiency, the detrimental effects on antigen primed cells appear to be longlasting. T cell populations also demonstrated a differential sensitivity to zinc deficiency. After a 96 hour culture period, splenocytes from 163 deficient mice gave elevated responses to allogeneic cells in the mixed lymphocyte response; equivalent proliferative response to PHA and reduced proliferative response to Con A compared to control mice. Careful examination of the kinetics of these responses revealed that the differences in these responses were, however, influenced by the length of the culture period. Although the Optimal Con A and PHA responses of lymphocytes frmn each treatment group were actually equivalent, very significant differences in the kinetics and maturation of the responses were observed. Failure to account for these differences nay explain some of the discrepancies in earlier reports 0f.ifl.!i££2 investigations of T cell responses of deficient mice. Further, the collective results of the experiments presented herein also indicate that the effects of dietary induced zinc deficiency may differ from those produced by in yitrg zinc depletion. Literature reports show that unlike T cells, B cells responses were unaffected when cultured in zinc chelated serum, which the authors suggested might also be true for in vivo induced zinc deficiency. Indeed, we also found T cell, but not B cell responses to be impaired when cells were cultured in serum from deficient mice. In contrast, the results 0f.ifl.!l!2 induced zinc deficiency reported here clearly reveal that B cell as well as T cell responses are impaired by zinc deficiency. Thus it appears that suboptimal zinc levels-13.11339 may have adverse affected on some lymphocyte populations, but these effects are different than those observed 10.11129 It would seem that the affects produced in yjyg_are a product of alterations on the immune system as a whole leading to changes in ratios on populations of cells rather than the immediate effects on cellular functions which appear to result fran in yitrg zinc depletion. 164 In conclusion, the results of the experiments presented demonstrate significant alterations in T and B cell functionality as a result of dietary zinc deficiency. The data strongly suggest that B cell maturation processes may be altered by zinc deficiency resulting in an accumulation of less mature B cells in the spleens of the deficient mice compared to controls. Further experimentation such as examination of additional surface markers for B cell maturation states or ease of tolerance induction are necessary to confinn this hypothesis. 165 Appendix I Preliminary Fluorescence Activated Cell Sorter Analysis of IgM and 190 bearing Splenocytes from Zinc Deficient Restricted and Zinc Adequate Mice Introduction Examination of the alterations in B-cell responses resulting from zinc deficiency suggested that B-cell maturation process might be impaired. If so, this would account for the observed increase in responses to antigens and mitogens believed to stimulate immature B-cells which result from zinc deficiency. Since at early or intermediate stages of maturity B-cells express high levels of IgM and nondetectable or low levels of 190 (l), examination of surface Ig characteristics of splenocytes from deficient mice offered an additional method to determine whether or not immature B-cells were altered by zinc deficiency. In this preliminary set of experiments, splenocytes from zinc deficient, restricted and control mice were stained with FITC conjugated anti IgM or 190 and independently analyzed by means of a fluorescently activated cell sorter (FACS). This technique allowed quantitation of the total number of IgM and 190 bearing B-cells as well as the ratio of cells expressing high or low levels of IgM or 190 on their surface. Currently the only commercially available anti IgD antibodies are two monoclonal allotype specific antibodies, neither of which are specific for 6 chains of A/J mice. One of the monoclonals is specific for a Balb/c a allotype. Thus, while A/J mice have been used in all previous experiments, it was necessary to include both A/J and Balb/c mice in these experiments. Fluorescent profiles of splenocytes from zinc deficient Balb/c and A/J mice revealed an increase in the number of 519 M+ cells and in the number of cells expressing high levels of sIgM compared to control and restricted mice. Furthermore, the profiles of sIgM+ cells from 166 167 deficient mice, resembled those of CBA/N splenocytes, a mouse strain reported to have a block in B-cell maturation (2). In contrast, no differences were seen in the $190 profiles among the three dietary groups. Thus, these data suggest that the deficiency leads to an accumulation of splenocytes with large amounts of IgM on their surfaces which corroborate the results of functional studies of B-cells during zinc deficiency. Collectively these results support the probability of an alteration in B-cell processing as a result of zinc deficiency. Materials and Methods Mice and Diets: Inbred Balb/c (Igh-Sa) and A/J (Igh-Se) mice used in this experiment were obtained from Jackson Laboratories, Ban Harbor, ME. Protocal for establishing the dietary deficiency were exactly as outlined in Chapter l-4 of this thesis. Antiserum: Anti-mouse IgM was purchase from Litton Bionetics, (Kensington, MD). This is an affinity purified antibody made specific for u chain by absorption with light chain and heavy chain classes. It is shown by Ouchterlony to be free from any cross reactive antibodies to other mouse immunoglobulin components. Fluorescein isothiocyanate (FITC) conjugated goat anti rabbit 19 was ordered from Miles Yeda (Rehovot, Israel) and when used alone it stained <1% of spleen cells from A/J or Balb/c mice. Monoclonal anti Igh-5a (IgD) (IgGZa subclass) specific for the Igh-Sa allotype of murine 190 was obtained from Dickinson (Sunnyvale, CA). FITC - conjugated goat-anti-mouse IgGZa was purchased from Nordic Immunologicals, (Tilberg, The Netherlands). The FITC-anti 192a does not cross-react significantly with IgM or IgD since spleen cells stained only with this antibody contain (5% positive cells. 168 Cell Surface Labeling: Single cell suspensions of splenocytes from each dietary group were obtained by pressing spleens through a fine wire mesh. Splenocytes were washed three times in Hank's balanced salt solution, which contained .l% bovine serum albumin and .l% NaN3, to remove cytophilic immunoglobulin bound to Fc receptors. Red blood cells were lysed with Tris buffered .84% NH4Cl and dead cells were removed on a fetal calf serum gradient. Splenocytes were adjusted to l x 107 cells/ml, following two additional washes. Two-hundred ul aliquots of the cell suspension were incubated with 50 pl of l:2 dilution of rabbit—anti u sera or 4 pg of rabbit- antimouse Igh-Sa antibody for 30 minutes at 4°C. Following three washes with cold medium, cells were incubated with 50 ul of a l:l0 dilution of FITC conjugated goat-anti-rabbit Ig or lDOul of a l:8 dilution of FITC-goat anti-mouse IgG 2a for 30 minutes at 4°C. Cells were washed three times and the degree of fluorescence was analyzed by means of a fluorescence activated cell sorter. The principles underlying this analysis are outlined in the next section. FACS Qperating Principles The fluorescence profiles were obtained using the Ortho cytofluorograf 50 (Ortho Diagnostic Systems, Raritan, N.J.). The basic Operating principles described in this section are similar to those applied by Bonner, gt 31., (3) in his prototype of a fluorescence activated cell sorted. For cell sorter analysis, a pressurized single cell suspension is driven into a quartz. flow cell, where it joins a saline sheath of circular cross section. The laminarly flowing sheath serves to protect the sample flow from mixing and boundary effects imposed by the interior surface of the flow path. Concentric flow 169 streams of sample within the sheath, a condition known as hydrodynamic focusing, prevent mixing of the sample and the sheath. A low pressure is used to maintain a slow flow rate of the cells through the quartz cell which result in a small sample stream diameter and improved accuracy of measurement. Next, this stream is illuminated by a laser beam. Cells bearing fluorescein conjugated sIgM or $190 emit a signal as a result of excitation of the bound fluor. The amount of light blocked or scattered by encounter with the sample stream is also monitored. The blocked light is measured directly by a photoelectric diode and fed to a processor. The fluorescence emission and scattered light are optically focussed onto fiber optics which terminate in photomultiplier tubes. Amplified photomultiplier outputs are fed into a signal processor. Processor outputs were selected to provide a graphical oscilliscope display of fluorescence intensity versus size or a trace of the number of fluorescent events as a function of fluorescent intensity. Data Presentation: The data is presented graphically as either the fluorescence or natural log of fluorescence intensity vs. cell number. Figure l represents a plot of fluorescence intensity of cells stained with anti-IgM along the x axis versus cell counts along the y axis. In this, and all plots, the analysis was derived from examination of l00,000 cells. The brighter the intensity of fluorerscence staining, the higher the channel nunber, the more $19 an individual cell is assumed to express. Channels 25-400 contain nonfluorescent cells while channels 400-1000 contain the fluoresence cells. It is possible to expand the fluorescence portion of this curve using a log scale to more clearly depict the populations of cells with an intermediate or high density of 170 surface Ig. Thus, Figure 2, represents a plot of the log of the fluorescence intensity vs. cell count. When the data is expressed in this manner two populations of fluorescent cells are apparent, in addition to the large peak of nonfluorescent cells (channel 25-400). Peak l, channels 400-725, represents low to intermediate density sIgM bearing cells and peak 2, channels 725-925, represents cells with a high density of sIgM. Figure 3 represents a plot of the log of fluorescence intensity of cells stained with anti 190. In this case channels 75-500 contain nonfluorescent cells, while channels 500-725 contain cells bearing low to intermediate 5190, and channels 725-925 contain cells with a high density of surface IgD. Results and Discussion Representative profiles of FACS analysis of sIgM and splenocytes from deficient, restricted and control A/J mice are shown in Figures l and 2. (Similar profiles have been obtained using Balb/c mice). Two major differences are noted when the profiles of sIgM+ cells from zinc deficient and zinc adequate or restricted mice are compared. There appear to be more cells from deficient mice (68%) staining with FITC anti-IgM compared to controls (48%). Furthermore, a higher proportion of cells from the deficient mice had a high density of sIgM (5l%) compared to controls (28%). This can best be seen in Figure 2 where a log scale has been used to expand the fluorescent portion of the curve. Figure 2 reveals a clear shift in the ratio of cells from deficient mice bearing a high density of IgM (peak II) versus cells bearing a low to intermediate density of IgM (peak I) compared to controls. 171 .xeeewese : wpse eeeemensee luwmm sew: eesweem eews _esesee use esewewwee esw~ Eesw mepxeese_em we me_wwese eeseemeseewm .H esemwm Counts x 1 00 172 Anti p. Zn odequoie __ unlabeled 2.0 - Fltcantip 12 .‘3 C 3 o O 1 .0 .- Channel number Zn deficienl — — unlabeled Fltc anti [1. 2.0 - I I l I I I | 1.0 - l | \ ‘ ~ - _ - — - _ - l _ - — l 250 500 750 Channel number 173 .xeoasece : wese eeeemehseeluwmm sew: eesweem eews _esesee ese .eeeewspmes .esewewwee esw~ sesw mepxeesewem we maesmeexe eeseemeseewm .m esamwm Counts x 1 00 Counts x 100 Counts x 1 00 174 Anti p. Zn (+) _. - - unlabeled — Fltc anti p. - ‘~~-—~ -~— l l 500 750 Channel number - - .. unlabeled ~~-- I 1“— 500 750 Channel number --.. unlabeled -— Fltc anti p. ‘N- ‘ ‘--~--- Q ‘---‘ 500 750 Channel number 96 Fluoreacent 48 96 peak l 71 (low intensity) 96 peak ll 28 (high intensity) 96 Fluoreacent 68 96 peak I 48 (low Intensity) 96 peak ll 51 (high intenalty) 96 Fluorescent 49 96 peak i 68 (low intensity) 96 peak ll 31 (high intensity) 175 Since cells bearing high amounts of sIgM are reported to be the less mature B-cells, (4) the findings of an increase in both the number of IgM+ cells and expression of sIgM on splenocytes from deficient mice corroborate the results of the functional studies previously presented. For example, the stimulation of splenocytes from deficient mice with mitogens specific for immature B-cell resulted in twice the control response; FACS analysis revealed twice as many high density sIgM bearing splenocytes in deficient mice compared to controls. In addition, FACS prolifes of IgM bearing splenocytes from neonatal mice, which have high number of immature B-cells (5) and from CBA/N mice (6), which are proposed to have a defect in B-cell maturation which results in large numbers of immature B-cells, are strikingly similar to the profiles obtained for splenocytes from deficient mice. It should also be noted that the profiles obtained for zinc adequate mice revealed that the percentage of IgM+ cells, and the percentage with high versus low intensity of IgM were comparable to those reported for adult mice from various strains (6). In contrast to the sIgM profiles, analysis of Balb/c splenocytes stained with FITC-anti-IgD failed to detect differences in either the number of sIgD+ cells or the degree of expression of 5190 among the three dietary groups (Figure 3). Since collectively the results of these studies indicated an increase in the number of immature splenic B-cells as a result of zinc deficiency, and these cells are reported to bear low 5190, it is not clear why no change in intensity of 190 bearing cells was observed in splenocytes from deficient mice. Since the amount of $190 is much lower than the amount of sIgM orlsplenocytes, it is possible the changes were more subtle. Furthermore, an increase in the number of 176 .erQJJCe m wese eeeemehseeluwmm sew; eesweem eewa _esesee ese .eeeewsumes .esewewwee esz sesw mepxeese_em we maesmepxe eeseemeseewm .m esemwm Counts x 100 Count: I: 100 Counts x 100 177 Awuia Zn (+) .. .. unlabeled Fitc anti 6 ~~~’~’--‘ Channel number In H -— - — unlabeled Fltc anti 6 ‘-~‘- “ -- - Channel number Rest. " - "' unlabeled Fitc anti 6 96 Fluorescent 56 96 Peak l 59 96 Peak ll 41 96 Fluorescent 56 96 Peak i 52 96 Peak ll 48 96 Fluorescent 58 96 Peaki 60 96 Peak ll 40 Channel number 178 cells expressing low or undetectable amounts of 190 may not be easy to detect since the majority of splenic B-cells already express low amounts of 190. Thus, it is possible that the deficiency leads to a block in B-cell maturation at an intermediate stage where cells express high amounts of sIgM and low 5190. The plausibility of this alternative is suggested by experiments described in Chapter 3 of this dissertation. Those data demonstrated that zinc deficiency resulted in an increase in the response to TNP Ficoll, an antigen reported to stimulate B-cells at an intermediate stage of maturation. More experimentation is required using dual labeling to allow simultaneous examination of sIgM and 5190 to properly answer this question. Taken together, the results support the idea of an alteration in B-cell subsets subsequent to zinc deficiency. These alterations appear to result in an increase in the number of cells with surface 19 phenotypes and functional properties characteristic of less mature B-cells. 2. 3. 4. References Goding, J. In: Lymphocyte Surface Immunoglobulins (Vitelia, gt 31., eds.) pp. 203. RavenPress, N.Y. l982. Fidler, J. E. Morgan and W. Weigle. J. Immunol. l24:13, 1980. Bonner, W., H. Hulett, R. Sweet and L. Herzenberg. Rev. Sci. Instruc. 43:404, 1972. Kearney, J., M. Cooper, J. Klein, E. Abney, R. Parkhouse and J. Lawton. J. Exp. Med. 146:297, l977. Mosier, D., I. Zitron, J. Mond, A. Ahmed, I. Scher and W. Paul. Immunol. Rev. 37:89, l977. Scher, I., S. Sharrow and W. Paul. J. Exp. Med. 144:507, 1976. 179