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Effects of psychosocial stressors on splenic lymphocyte proliferation and natural killer cell activity… Rowan, Rosemary Eva 1992

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EFFECTS OF PSYCHOSOCIAL STRESSORS ON SPLENIC LYMPHOCYTE PROLIFERATION AND NATURAL KILLER CELL ACTIVITY IN THE PRESENCE OR ABSENCE OF A MOUSE MAMMARY TUMOUR. By ROSEMARY EVA ROWAN B.Sc, Trinity Western University, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Anatomy) We accept this thesis as conforming to the reqired standard THE UNIVERSITY OF BRITISH COLUMBIA August 1992 © Rosemary E. Rowan, 1992 In presenting this thesis in partial fulfi lment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my wri t ten permission. (Signature) Department of Anatomy The University of British Columbia Vancouver, Canada Date 8 October 1992 DE-6 (2/88) ii ABSTRACT Weinberg and Emerman (1989) demonstrated that differential housing conditions alter the growth of the transplantable androgen-responsive Shionogi mouse mammary carcinoma (SC115). In addition it was shown that changes in the immune system are influenced both by housing and the presence or absence of tumour. The objective of this thesis was to determine if the immune system and, more specifically, if natural killer (NK) cells may be involved in mediating the effects of differential housing on SC115 tumour growth. In a preliminary study in our laboratory, T lymphocyte proliferation in the spleens of mice in different housing conditions was measured. The initial study of this thesis was designed to replicate these experiments in order to obtain sufficient data for statistical analysis. At the time of tumour-cell injection, mice raised individually from the time of weaning either remained individually housed (II) or were placed in groups of 5 (IG); mice raised in sibling groups of 3 from the time of weaning either remained in their rearing groups (GG) or were separated and housed individually (GI). Three wk after tumour-cell injection, spleens were removed and cells were cultured with or without the mitogen Concanavalin A (Con A) to stimulate proliferation of T lymphocytes. This proliferation was measured by [3H] - thymidine incorporation. In the absence of Con A, there were no differences in spleen cell growth among animals from the 4 housing iii groups, whether or not they were injected with tumour cells. With Con A stimulation, lymphocyte proliferation was decreased in IG mice. However, changes in response were eliminated in animals injected with tumour cells. The major studies of this thesis focused on the investigation of the role of NK cell activity in modulating tumour growth rate in our model. NK cell lytic activity is modulated by both the presence of a tumour and psychosocial stressors. Further, it has been shown that the immune system responds within days to stressors. In this thesis, NK cell activity at early time points following formation of experimental groups and tumour-cell or vehicle injection was examined. First, a protocol to measure splenic NK cell cytolytic activity in our mice was established. Using this protocol, NK cell activity was examined 3 d and 1 wk following tumour-cell or vehicle injection and rehousing. Following vehicle injection, groups did not differ significantly in NK cell activity. All vehicle injected groups were significantly lower in NK cell activity than tumour-cell injected groups and there was no significant change in NK cell activity from 3 d to 1 wk. Significant stimulation of splenic NK cell activity occurred 3 d post-injection of SC115 cells. However, no correlation was observed between the level of splenic NK cell activity and tumour growth rate induced by the housing condition. It was concluded that either splenic NK cell activity does not accurately reflect NK cell activity at the tumour site or that NK cells are not a significant regulator of the differential tumour growth rates seen in this model. iv TABLE OF C Page THESIS ABSTRACT ii LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix ACKNOWLEDGMENTS x CHAPTER 1. INTRODUCTION 1 A. Stress and Cancer 1 1. Human Studies 1 2. Animal Studies 4 B. The Shionogi Mouse Mammary Carcinoma 7 C. Stress and Immune Function 16 D. Natural Killer Cells 19 1. Cytotoxic Effector Cells 19 2. NK cells: Characteristics and Functions 21 3. Regulation of NK cells 23 E. Stress and NK Cell Activity 27 1. Human Studies 27 2. Animal Studies 28 F. Thesis Objective 29 V CHAPTER 2. MATERIALS AND METHODS 31 A. Tumour Model 31 1. Dissociation 31 2. Freezing 33 3. Transplantation of Tumour Cells 33 4. Monitoring Tumour Growth 34 B. Animal Model 35 C. Spleen Cell Proliferation Assay 37 1. Effector Cell Preparation and Stimulation 37 2. Radioactive Labelling 41 3. Harvesting 41 D. Natural Killer Cell Cytolytic Activity Assay 43 1. Target Cell Preparation 44 a) Freezing of target cells 44 b) Thawing of target cells before assay 45 c) Radioactive labelling 46 2. Effector Cell Preparation 46 a) Preparation of spleen cell suspension 46 b) Preparation of nylon wool columns 47 c) Cytotoxicity assay 48 d) In vivo boosting of effector cell activity 51 vi 3. Calculation of Results 50 4. Statistical Analysis 52 CHAPTER 3. RESULTS 53 A. Completion of Spleen Cell Proliferation Data 53 B. The [5,Cr]-Release Assay 58 1. Propagation of YAC Cells 58 2. NK Cell Assay and Target Cell Labelling 61 3. Test Assay Using a Mouse with Tumour 65 4. Four Housing Groups and Novelty Stress 67 5. Test Assay Using Known Responder Strains 69 6. In Vivo Boosting 71 7. In Vitro Boosting 73 8. Removal of Red Blood Cells 75 9. Processing of 6 Spleens at One Time Using the Established Protocol 78 C. NK Cell Assay Timecourse Study 80 vii CHAPTER 4. DISCUSSION 87 A. Introduction 87 B. T-Lymphocyte Proliferation in Response to Con A 90 C. Natural Killer Cell Cytolytic Activity 92 1. Modification of the [51Cr] - Release Assay 92 2. Timecourse Study of NK Cell Activity 94 BIBLIOGRAPHY 101 viii Table No. Page Table I: Secondary antibody response in groups GG, GI, IG and II 14 Table II: Comparison of 2 different target cell labelling procedures 63 Table III: Comparison of 2 different target cell labelling procedures with modifications 64 Table IV: Test NK cell assay using tumour-cell injected mice 66 Table V: NK cell assay using tumour-cell injected animals and psychosocial stress conditions 68 Table VI: NK cell assay using known responder mouse strains in addition to the DD/S mouse strain 70 Table VII: NK cell assays after in vivo boosting 72 Table VIII: NK cell assay using in vitro boosting 74 Table IX: Test NK cell assay with removal of red blood cells before nylon wool treatment 76 Table X: Confirmation of protocol with nylon wool treatment and red blood cell lysis 77 Table XI: Confirmation of the ability to process a greater number of animals at one time using the established assay protocol 79 Table XII: NK cell lytic activity at 3 d in mice from the 4 experimental groups . . . 85 Table XIII: NK cell lytic activity at 1 wk in mice from the 4 experimental groups . . 86 ix LIST OF FIGURES Figure No. Page Figure 1: Tumour growth with and without acute daily novelty stress 10 Figure 2: Tumour growth in second study 13 Figure 3: Use of wire mesh screen in dissociation of spleens 39 Figure 4: Plating protocol for spleen cell proliferation assay 40 Figure 5: Apparatus for harvesting in spleen cell proliferation assay 42 Figure 6: Plating protocol for cytotoxicity assays 50 Figure 7: Splenic T-lymphocyte proliferation in the presence (stimulated) or absence (unstimulated) of Con A. a) Vehicle injected mice 56 b) Tumour-cell injected mice 57 Figure 8: Growth of YAC cells 60 Figure 9: NK cell activity at 3 d in mice from the 4 experimental housing groups 83 Figure 10: NK cell activity at 1 wk in mice from the 4 experimental housing groups 84 X LIST OF ABBREVIATIONS Con A Concanavalin A [51Cr] Radioactive S1 chromium GG Set of animals raised in sibling groups of 3 and remaining group housed GI Set of animals raised in sibling groups of 3 and rehoused individually [3H]TdR Radioactive tritiated thymidine IFN Interferon IG Set of animals raised individually and rehoused in groups of 5 II Set of animals raised individually and remaining individually housed IL-2 Interleukin-2 LGL Large granular lymphocyte LN2 Liquid nitrogen NK Natural killer cell Poly I:C Polyinosinic-polycytidylic acid SCI 15 Shionogi mouse mammary carcinoma 115 xi ACKNOWLEDGEMENTS First and foremost, I wish to thank my supervisor, Dr. Joanne T. Emerman. Thanks, Joanne, for taking that first chance with me; for all that you have taught me and the way you have guided me through this thesis with the patience, endurance and encouragement of a mother, and especially for not giving up on me throughout my various personal trials! As for my 'partner in crime', Gerry J. Rowse, my 'other half in the lab, what can I say but thanks for being there? Our mouse gala events will stay with me forever. To Dr. Joanne Weinberg, thank you for making yourself available to me as a second mentor and supervisor, and for all your friendly advice and encouragement. To Darcy and Shannon, Kevin and Greta, thank you all for your help with experiments and for making our labs a place where good research is done and laughter abounds. I also wish to thank the Dept of Anatomy for this opportunity and in particular Dr. W. K. Ovalle for spurring my interest in this great department. Thanks also go to Dr. Waterfield for advice in protocol matters as well as the valued use of the gamma counter. The Biomedical Research Centre also provided valuable assistance with the use of their equipment in the initial stages of this work. To the most important person in my life, my husband Thomas, my loving family, especially my parents and my grandmother, and my special friends, I can never fully express my appreciation for all the encourgement and support, the babysitting and the technical help, all offered unstintingly and steadily throughout this time. Thank you so much! Finally I wish to thank my God and Saviour for His unfailing love, for giving me the strength to carry on and see this through to the end. To Him be the Glory. 1 CHAPTER 1 INTRODUCTION A. STRESS AND CANCER The possibility that disease processes may be affected by stress is not new. In 1884, a leading article in the British Medical Journal suggested that at funerals "the depression of spirits under which chief mourners labour at these melancholy occasions peculiarly predisposes them to some of the worst effects of chill" (Leader, 1884). A classic example of the influence of stress on infection is acute necrotizing ulcerative gingivitis which got the name of 'trench mouth' in the 1914-1918 war because of its occurrence in men exposed to the appalling stress of trench warfare. It is an invasion of the gums by common mouth bacteria probably due to failure of the normal immunological defences (Baker, 1987). However it is not just infections that stress and the ability to cope with stress may play a role in, but something that is less easily curable and that is responsible for many more deaths, and that is cancer. 1. Human Studies The clinical observation that psychosocial stress may influence the development and course of cancer is one that physicians of the 18th and 19th centuries made when 2 having to rely on their own understanding, rather than on more objective, statistically analyzed laboratory results and medical technology. Reports from these physicians point to an association between severe emotional trauma and the onset of cancer (LeShan, 1959). In the past few decades, many epidemiological studies suggest a link between psychological factors and cancer. At Kings College Hospital, London, women coming for breast biopsy had psychological assessments carried out before the results of the biopsy were known. It was found that women whose biopsies are later found to be malignant are likely to be women who more frequently suppressed anger (Greer & Morris, 1975). All the cancer patients had an interview 3 months after diagnosis and at the 5 year follow-up it was found that a greater percentage of those who had expressed feelings of hopelessness in the interview had died, in contrast to those who expressed either denial or a fighting spirit. This suggested that a woman's psychological reaction to the diagnosis may be strongly predictive of the 5 year outcome (Greer, et al., 1979). Other studies have shown that stressful life-events, such as separations or losses from an important emotional relationship and disruptions in family, school or work life occur statistically more often in children and adults who develop cancer than in matched control groups (Greene & Miller, 1958; Greene, 1966; Home & Picard, 1979; Jacobs & Charles, 1980; LeShan, 1966; see also reviews by Baker, 1987; Dorian & Garfinkel, 1987; Fox, 1981; Sklar & Anisman, 1981). Males with primary depressive illness, or measured as depressed by the Minnesota Multiphasic Personality Inventory, experience an increased death rate from malignancies (Shekelle, et al., 1981; Whitlock & Siskind, 3 1979). Relapse of malignant melanoma is found to be more frequent among individuals who express difficulty in adjusting to the disease and surgery than among individuals who report relatively less difficulty in adjustment (Rogentine, et al., 1979). A recent study has confirmed these and other studies (Cheang & Cooper, 1985; Cooper et al., 1989; Lehrer, 1980; LeShan, 1966) that breast cancer patients have significantly more important losses and difficult life situations than controls prior to the discovery of the breast tumour (Forsen, 1991). It was suggested that the more remote stressful life events affect the development of breast cancer but not the progression of the disease. Only the scores of the 12 month SRRS test (Social Readjustment Rating Scale) remain significantly associated with recurrence-free and overall survival. The field of psychosocial cancer has had many contradictory results, however, often as a result of methodologic inadequacies. For example, some researchers have found that coping strategies of patients are related to the progression of their malignant disease while others have not (Buddeberg, et al., 1991). Amidst the confusion of these epidemiological studies are emerging studies showing the benefit of psychotherapy on survival of patients with metastatic breast cancer. One such study found that cancer patients who have undergone psychotherapy not only have a longer survival time than patients not so treated, but that psychotherapy and chemotherapy have a synergistic effect on survival time (Grossarth-Maticek & Eysenck, 1989). Results of another study were consistent with this, but greater in magnitude (Spiegel, et al., 1989). 4 Even though a relationship appears to be established between higher cancer incidence and psychological factors in humans, including inability to cope with stress, some understanding of the underlying physiological mechanisms involved needs to be elucidated so that this relationship may have more clinical relevance. 2. Animal Studies A relation between stress and cancer has been demonstrated in more controlled animal studies. A number of these studies have shown, in fact, that stressors do change tumour growth and still others are looking at the underlying mechanisms involved. Unfortunately, there is a great deal of inconsistency between these studies (see reviews of Justice, 1985; Sklar & Anisman, 1981). Researchers have found that stress may cause an increase, a decrease, or no change at all in tumour growth rate. This is due in part to the particular animal model used and in part to the fact that stress is a widely and somewhat loosely used term for describing a wide range of biological responses to a novel or difficult situation (Riley, et al., 1981). The magnitude and type of response has been shown to change according to a number of variables. These include the type of stressor, the intensity and duration of the stressor, when the stressor is applied, that is, either before or after tumour formation, whether the stressor is chronic or acute, whether the stressor is physical or psychological and whether or not a coping response is available, for example, being able to turn off an electric shock. In addition, in the investigation of the relationship between stress and tumour growth, a large variation in response exists 5 simply because of the difference in species and type of tumour, whether it be spontaneous or virally induced, benign or metastatic, and the measurements of end point, whether it be tumour size or survival of the animal. Many studies use a variety of physical stressors such as restraint, footshock, loud noise, rough handling or cold and have shown that these affect both tumour incidence and growth. Again, results are varied according to the stressor. The length of exposure time produces different results; chronic exposure to these stressors generally inhibits tumour incidence and growth (Baker & Jahn, 1976; Burchfield, et al., 1978; Newberry, et al., 1976; Newberry & Sengbusch, 1976; Pradhan & Ray, 1974; Riley, 1981; Sklar & Anisman, 1979), while acute exposure enhances tumour incidence and growth (Greenberg, et al., 1984; Lewis, et al., 1983; Niebergs, et al., 1979; Peters & Kelley, 1977; Solomon & Amkraut, 1979; Steplewski, et al., 1985). Exposure to the stressor before injection of tumour cells of an immunoresponsive tumour inhibits tumour growth, while the same stress administered after tumour-cell injection increases tumour growth (Amkraut & Solomon, 1972; Greenberg, et al., 1984; Riley, 1981; Burchfield, et al., 1978). Stress-induced immunosuppression could be responsible for increased growth and rebound immunoenhancement could cause the decreased growth. However, for nonimmunoresponsive tumours, growth is frequently inhibited during stress and may increase when stress is terminated (Justice, 1985). 6 As in human studies, the coping response available is another variable at work in this complex relationship between stress and cancer. Escape from shock, for example, is one form of a coping response. Exposure to inescapable shock has been shown to markedly increase tumour growth and decrease tumour rejection or survival time (Lewis, et al., 1983; Sklar & Anisman, 1979; Visintainer, et al., 1982) compared to the same amount of escapable shock. Another form of a coping response may be fighting (Conner,et al.,1971; Sklar & Anisman, 1980; Weinberg, et al., 1980; Weis, et al., 1976); this has been shown to reduce tumour growth rate (Amkraut & Solomon, 1972; Sklar & Anisman, 1980). More relevant to psychological factors in the human situation is the use of psychosocial stressors, such as housing conditions, which have also been shown to influence tumour induction and growth rate. These studies date back to 1944, when Andervont (1944) investigated the influence of population density on spontaneous mammary tumour latent periods. It was found that isolation-reared mice develop mammary tumours earlier than did mice raised in social groups. Other studies utilizing transplanted tumours and looking at the influence of population density on the survival time of mice found that an abrupt change in social environment, either from isolation to a group of 10 mice per cage or vice versa, results in an increased growth of the tumour (Dechambre & Grosse, 1973; Sklar & Anisman, 1980). 7 However, as mentioned before, the behavioural response of animals to such psychosocial stressors can change this effect. For example, Sklar and Anisman found that in group housed animals who fight after social change, tumour growth does not increase as much as it does in groups where fighting does not occur (Sklar & Anisman, 1980). B. THE SHIONOGI MOUSE MAMMARY CARCINOMA The model that we have been working with utilizes the Shionogi mouse mammary carcinoma (SCI 15). This is a transplantable, androgen-responsive tumour that originated spontaneously in a female mouse of the DD/S strain. Following 19 generations of transplantation, an androgen-responsive subline arose, which grew faster in males than in females (Bruchovsky & Rennie, 1978; Emerman & Siemiatkowski, 1984; King & Yates, 1980; Minesita & Yamaguchi, 1965). When 3 x 106 tumour-cells are injected subcutaneously (s.c.) into the interscapular region of a male mouse 2 - 4 months old and housed in the standard housing condition used in our laboratory, 3 sibling mice/cage, a palpable tumour typically arises in 7 - 10 days and grows to a mass of 2 - 3 grams in 18-21 days. This mouse mammary tumour subline is similar to some human breast cancers in its sensitivity to different classes of steroid hormones, including androgens (Bruchosvsky & Rennie, 1978; King & Yates, 1980), estrogens (Noguchi, et al., 1987; Nohno, et al., 1982) and glucocorticoids (Watanabe, et al., 1982). These hormones have 8 also been shown to be modulated by psychosocial stressors (Andrews, 1977; Axelrod & Reisine, 1984; Riley, 1981). Another factor that has been suggested to be involved in regulating SCI 15 tumour growth is the immunological status of the host. This has been observed in studies performed using nude athymic mice, where SCI 15 tumours develop equally in both male and female mice. Immunopotentiation caused by bacterial infection results in decreased growth of SCI 15 tumours and increased growth is seen with glucocorticoid administration, possibly due to suppression of the immune system (Watanabe, et al., 1982; Kitamura, et al., 1980; Nohno, et al., 1986). Using this animal-tumour system, our laboratory has have been examining the effects of psychosocial stressors on mammary tumour growth. The model was originally adapted from those previous studies which found that being housed individually accelerated tumour growth and that a change in housing conditions had an even more dramatic effect. Based on this data, experiments were designed to investigate if 3 conditions of change in social housing groups would differentially alter the growth rate of the SCI 15 tumour (Weinberg & Emerman, 1989). For the study, male mice, at the time of weaning, were housed either individually or in sibling groups of 2 - 3. Following tumour cell injection, the mice were subjected to 1 of 3 changes in housing condition: mice raised individually were either placed in non-sibling groups of 4 - 5 (IG) or paired with a female (IP); mice raised in sibling groups were separated and housed individually (GI) or remained as they were (GG). The GG 9 group was the group against which tumour growth in the other 3 housing groups were compared because, as described above, sibling groups of 3 mice/cage are the standard housing condition in the laboratory. Interactive effects of a change in housing environment and an acute psychological stressor were also examined by subjecting half the groups in each condition to a daily acute stressor in the form of 15 min/d, 5 d/wk, in one of five different novel environments. This daily exposure to a novel environment was designed to raise the levels of glucocorticoids consistently (Friedman & Ader, 1967). It has been shown previously that exposure of mice to daily novelty stress results in consistently elevated levels of glucocorticoids which are slow to habituate (Hennessy & Levine, 1977; Pfister & King, 1976). Therefore, this protocol acts effectively as an acute psychological stressor. The other half received no acute stressor and served as a control. Experiments were terminated after 3 weeks. It was found that in the absence of acute daily novelty stress, tumour growth in GI animals is similar to that of GG controls, while growth rate is reduced in both IG and IP animals, p < 0.05 (Fig.l). With acute daily novelty stress, tumour growth rate is increased in GI animals and decreased in IG animals compared to all other groups, p < 0.05. 10 Figure 1. Tumour growth with and without acute daily novelty stress. No Acute Novelty Stress Acute Novelty Stress 18 23 0 7 11 Days following tumor cell injection Tumour growth in male mice in the four housing groups: GG, raised and maintained in sibling groups of 2 - 3; GI, raised in sibling groups of 2 - 3, then separated and housed singly; IG, raised singly housed, then rehoused in nonsibling groups of 4 - 5; IP, raised singly housed, then rehoused with a female. ( ) = n per group. On day 23 following tumour-cell injection, tumour growth, collapsed across group, was greater in the presence of acute daily novelty stress than in its absence, p < 0.05. In addition, collapsed across novelty stress, all groups differed significantly from each other in tumour size by day 23, GI > GG > IP > IG, p's < 0.05 (Weinberg & Emerman, 1989). Since it is known that stressors activate the hypothalamo-pituitary-adrenal axis (Friedman & Ader, 1967; Selye, 1973) and that SCI 15 tumour growth is stimulated by glucocorticoids either by direct action on these tumour-cells (Watanabe et al, 1982) or indirectly by suppression of the immune system (Nohno et al, 1986), some adrenocorticoid parameters were measured at the end of the three week experimental 11 period. These included adrenal weights and plasma and adrenal corticosteroid levels. No significant differences were found among groups in adrenal weights or in adrenal weight/body weight ratios. In addition, housing condition does not differentially affect basal corticosterone levels or the corticoid increase that occurred following exposure to the novel environments. The data in these experiments demonstrate that the growth rate of the SCI 15 tumour can be significantly affected both by housing condition and by acute daily novelty stress. However, it reveals that a change in housing condition does not necessarily alter the growth rate of this tumour. Our lab undertook a second study to investigate further if group versus individual housing as well as being moved from one housing condition to another affects SCI 15 growth (Weinberg & Emerman, 1989). This study used a model similar to the previous one with some modifications. Since the acute novelty stress maximized the differential tumour growth in the first study, all animals in all the groups received the daily acute stress. In addition, immune parameters as well as endocrine parameters were going to be measured. Therefore, for every housing condition, animals were injected either with tumour-cells in vehicle or with vehicle alone as the effects of stressors on the immune system have been shown to differ in tumour-bearing and non-tumour bearing animals (Steplewski et al, 1985). Finally, a third change involved the replacement of the individual to pair condition (IP) with the individual to individual condition (II). In this condition, mice that were housed as 12 individuals from time of weaning were left as individuals for the study. This controlled for the effects of isolation without a change in housing condition from time of weaning. A number of immune and endocrine parameters were examined. Measures of endocrine function included testis and seminal vesicle weights and plasma levels of testosterone and dihydrotestosterone (DHT). These were all included because the SCI 15 tumour is androgen-responsive (Bruchovsky & Rennie, 1978; Emerman & Semiatkowski, 1984; Emerman & Worth, 1984) and testosterone levels are known to be altered by stress (Andrews, 1977; Axelrod & Reisine, 1984). Because stress can affect the immune system (Stein.et al, 1976) and host immunological status has been suggested to be important in regulating SCI 15 tumour growth (Nohno et al, 1986), immune functioning was also examined. As data suggest that stressors can modify both humoral (antibody-mediated, B cell) and cell-mediated immunity (T lymphocyte) (Coe et al., 1988; Esterling & Rabin, 1987), both those aspects were examined by looking at antibody production and mitogen-induced T-lymphocyte proliferation. (Completion of the lymphocyte proliferation data formed a component of this thesis.) In addition spleens were weighed as a general measure of immune responsiveness to stress (Peters & Mason, 1979) as typical stress syndromes seem to manifest as a decrease in spleen weight along with changes in other organs, such as adrenal hypertropy (Riley et al, 1981). The data on the growth of tumours in this study replicate that seen in the previous study (Fig. 2). On the day of termination, i.e. the end of the 3 week experimental 13 period, group to individual mice (GI) have the largest tumours and the individual to group mice (IG) have the smallest tumours. Figure 2. Tumour growth from second study. 3 o E 0 7 11 14 18 Days following tumor cell injection Tumour growth in male mice in the four housing groups: GG, raised and maintained in sibling groups of 2 - 3; GI, raised in sibling groups of 2 - 3, then separated and housed singly; IG, raised singly housed, then rehoused in nonsibling groups of 4 - 5; II, raised and maintained singly housed. ( ) = n per group. All groups were subjected to acute daily novelty stress. On day 18 following tumour-cell injection, GI = II > GG > IG, p < 0.05 (Weinberg & Emerman, 1989). Interestingly, the individual mice who did not experience a change (Q) have a tumour growth rate which closely approximated that of the group to individual condition (GI). In all of the endocrine measures, ie. plasma levels of testosterone and DHT as well as testis and seminal vesicle weights, no significant differences among groups were found. 14 The enzyme linked immunosorbent assay (ELISA) was used to evaluate humoral (B cell) functioning by measuring the secondary antibody response to a repeated innoculation of antigen. Analysis revealed that overall, the ability of tumour-bearing animals to synthesize antibody is suppressed compared to non-tumour bearing animals. In addition, there is a main effect of group: antibody response of IG animals (the group with the smallest tumours) is increased compared to that of GI animals (the group with the largest tumours), p < 0.05 (Table 1.) (Weinberg & Emerman, 1989). Table 1. Analysis of secondary antibody response Secondary Antibody Response Group* No Tumour Tumour2 GG 1.11 ± 0.021 0.91 ± 0.08 GI3 0.92 ± 0.12 0.95 ± 0.07 IG 1.18 ± 0.03 1.02 ± 0.05 n 1.02 ± 0.05 0.98 + 0.05 * n = 4-9 1 Absorption at 400 nm, mean ± S.E.M. 2 Tumour < No Tumour, p < .05. 3 GI < IG, p < .05. Analysis of T cell proliferation in response to the mitogen Concanavalin A (Con A) in a spleen cell proliferation assay is part of this thesis and is therefore discussed later. 15 Both the presence of a tumour and housing condition affect spleen weights. Animals injected with tumour-cells have markedly enlarged spleens compared to animals injected with vehicle alone. However, there was no differential effect of housing group in tumour-bearing animals. In contrast, vehicle-injected IG animals had somewhat enlarged spleens compared to vehicle-injected animals in the other groups. The data from these experiments clearly indicate dramatic and replicable effects of social housing condition and exposure to novel environments on tumour growth. However, despite the fact that this tumour is androgen- and glucocorticoid-responsive, the endocrine measures taken at the end of the 3 week experimental period provide no direct evidence for the involvement of the pituitary-adrenal or pituitary-gonadal systems in mediating the effects of stressors on SCI 15 tumour growth. The immune measures do suggest that the immune system is involved, although the results, taken at 3 weeks after tumour-cell injection, are difficult to interpret. However, housing as well as the presence of a tumour affects the immune system. Previous observations suggest that the growth of SCI 15 tumours can be modulated by the host immune system. First, it has been demonstrated that the original androgen-dependent SCI 15 tumour, which does not grow in females or castrated males, can be induced to grow in these mice by the administration of pharmacological doses of glucocorticoids (Watanabe, et al., 1982). The authors conclude that the growth promoting effect of glucocorticoids is due in part to suppression of host immunity and in 16 part to stimulation of a glucocorticoid receptor in the SCI 15 cells. Second, it was found the development of the SCI 15 tumour is retarded by the injection of Staphylococcus aureus on the day of tumour-cell injection (Nohno, et al., 1986). As a result of these and numerous other demonstrations that host immunity is important in the regulation of tumour growth (Cerottini & Brunner, 1974; Herberman & Ortaldo, 1981), this research project focused on the immune system and its possible role in the differential growth rates observed. C. STRESS AND IMMUNE FUNCTION Data suggest that psychiatric patients with diagnoses of major depressive disorders have poorer immune function, manifested by poorer blastogenic responsiveness (Schleifer, et al., 1984) and lower percentages of helper T lymphocytes than nondepressed comparison subjects (Krueger, et al., 1984). Others also suggest that depressed patients may have a lower percentage of peripheral T lymphocytes (Schleifer, et al., 1985) than non-depressed patients. One of the most stressful life experiences appears to be marital disruption, either through divorce or death (Bloom, et al., 1978; Jacobs & Ostfeld, 1977; Rees & Lutkins, 1967). An association has been shown between bereavement and impaired lymphocyte proliferation response to mitogen (Bartrop et al., 1977; Schleifer, et al., 1983). Marital 17 disruption through divorce or separation, in both the short and the long term, is associated with poorer immune function and greater depression (Kiecolt-Glaser, et al., 1987). In addition, poorer marital quality is associated with a poorer immune response. Another study showed that family caregivers of patients with Alzheimer's disease, at high risk for depression, have poorer immune functioning in most of the cellular immunological assays than comparison subjects (Fiore, et al.,1983). Thus, these data provide good evidence for chronic distress-related alterations in the immune system of humans. In animal studies, the chronicity of the stressor seems to have various effects on immunological responses. The acute or short-term consequences of a stressor appear to be immunosuppressive, with mitogen responsiveness falling below base-line levels. However, this seems to be followed by a rebound immunoenhancement (Monjan & Collector, 1977). In addition, the effects of stressors on the immune system have been shown to differ in tumour-bearing and non tumour-bearing animals (Steplewski, et al., 1985). In tumour-bearing animals, leukocytes are decreased by restraint stress and increased after the recovery period. Lymphocytes are increased and neutrophils and large granular lymphocytes (LGLs) are decreased after the recovery period. Total T cells and suppressor T cells are decreased during stress and increased during recovery. The percentage of suppressor T cells increased during the recovery period. In healthy animals bearing no tumour, leukocyte numbers are decreased after stress, but returned to normal after the recovery period (tumour-bearing animals had higher than normal counts after 18 recovery). The percentage of lymphocytes decreases in healthy animals, but increases after recovery. In addition, neutrophils and LGLs increase during stress, whereas monocytes and total T cells and helper/suppressor T cells are unaffected. After recovery, neutrophils decrease, monocytes are unaffected and LGLs, total and helper T cells increase. A further variable altering the immune-stress relationship is the intensity of the stressor. For example, a relationship has been reported between the intensity of an acute stressor and the degree of suppression of both the number of T lymphocytes and their responses to the mitogen phytohaemagglutinin (PHA) (Keller, et al., 1981). Social interactions among animals have also been shown to influence immunological function. Infant monkeys show depressed lymphocyte proliferation responses to both PHA and Con A when separated from their mothers, returning to normal at reunion (Laudenslager, et al., 1985). Early weaned rats demonstrate a highly significant decrease in lymphocyte response to PHA compared to normally weaned rats (Keller, et al., 1983). It is likely that the mediation of stress effects on immune function is a complex phenomenon, involving direct (central nervous system) effects of stress, stress-induced changes in neuroendocrine and neurochemical activity, and interactions between neuroendocrine and immune networks. This has been suggested from several findings. All of the major lymphoid organs are known to be innervated directly, suggesting possible control by neurotransmitters (Felton, et al., 1985; Dion & Blalock, 1988). It 19 has also become apparent that various components of the immune system are responsive to neuroendocrine peptide hormones (Blalock, 1984). In addition, data demonstrate the presence of high-affinity receptors for neurotransmitters and neuropeptides on cells of the immune system (Johnson, et al., 1982; Mann, et al., 1985; Weber & Pert, 1984; Wybran, et al., 1979). The enkephalins have been shown to enhance lymphocyte responsiveness to mitogens and increase lymphoid organ size (Gilman, et al., 1982; Plotnikoff, et al., 1985). In contrast, adrenocorticotropin (ACTH) has been shown to suppress the in vitro antibody response as well as the y interferon response of murine lymphocytes (Johnson, et al., 1982; Johnson, et al., 1984). It seems that components of the immune system can in turn directly affect the neuroendocrine system, with the production of such peptides as ACTH, endorphins and thyroid stimulating hormone (TSH) (Smith & Blalock, 1981; Smith, et al., 1983). D. NATURAL KILLER CELLS 1. Cytotoxic Effector Cells Several cytotoxic effector cells have been described and are considered to play an important role in immune surveillance against cancer, infection and graft rejection. These effector cells are: 1) Cytotoxic T lymphocytes (CTL), which are major histocompatibility complex (MHC)- restricted and can lyse antigen-specific target cells, 20 2) Natural killer (NK) cells, which can lyse a variety of NK-sensitive tumour-cells in an non-MHC restricted manner, 3) Killer cells for antibody-dependent cell-mediated cytotoxicity (ADCC), which can lyse antibody-coated cells and 4) Macrophages and neutrophils, which, when activated, can also lyse tumour-cells or inhibit tumour growth. While all these effector cells have the ability to destroy undesired target cells, the mechanisms involved in target cell lysis are not clearly understood. It appears, however, that the various cytotoxic systems have certain features in common and lysis can be divided into at least three stages (Bonavida & Wright, 1986): 1) the effector cells bind to the target cells through specific receptors, 2) the effector cell is activated or triggered in a stage called programming for lysis or the lethal hit with the extracellular release of cytotoxic granules and 3) target cell is damaged in a killer-cell independent stage. The immune cells which are involved in the modulation of the growth rate of the SCI 15 tumour have not yet been determined. However, NK cells are reported to play a significant role in the in vivo destruction of transplanted syngeneic tumour-cells (Hanna, 1986) and to be involved in the first line of the defense mechanism (Bonavida & 21 Wright, 1986). Thus the investigation of these effector cells seemed a logical step in elucidating immunologic factors affecting the differential growth of the SCI 15 tumour. 2. NK Cells: Characteristics and Functions The development of the in vitro 51Chromium [5,Cr] release assay by Brunner et al. (1968) provided a simple technique for measuring target cell lysis by cytotoxic cells. The assay rapidly gained widespread acceptance as the optimal technique for quantifying cytotoxic T lymphocyte-mediated lysis in terms of [^CrJ-release by labelled target cells (Cerottini & Brunner, 1974). In vitro assays of nucleated cells obtained from experimental animals or human patients became major research tools for many laboratories. It soon became clear, however, that a significant proportion of the in vitro cytotoxicity observed in many of these systems could not be accounted for by antigen-specific T cell mediated lysis. This occured when leukocytes, regardless of where they were obtained, i.e. from normal or tumour-bearing experimental animals or humans, were assayed against [51Cr]-labeled tumour target cells (Takasugi, et al., 1973; Herberman, et al., 1974). By 1975, it was recognized that the in vitro "background" lysis of [51Cr]-labeled tumour target cells observed in these systems was a phenomenon in its own right: "natural killing". Herberman et al. (1975) and Kiessling et al. (1975) originally described NK cells. 22 Natural killer cells are functionally defined as white blood cells with the ability to recognize and lyse certain malignant and undifferentiated or virus-infected normal cell types in the absence of any prior immunization (Roder & Pross, 1982). NK cells appear to be a distinct population of cells with lymphoid morphology, the major phenotype referred to as large granular lymphocyte (LGL). Since the mid-1970s when NK cells were first recognized to be different from classical cytotoxic cells, intensive efforts have been made to characterize the effector cell type or types. Despite these efforts, no single definition of NK cells has emerged, i.e., NK cells are phenotypically and functionally heterogeneous (Pollack, 1986). A number of laboratories have investigated the cellular characteristics and functions of natural killer cells. Although initially recognized for their anticancer activity, NK cells appear to be involved in a multitude of biological processes. These include mediating resistance to microbial and viral infection (Lopez, et al., 1982; Santoli, et al., 1978), resistance to parasites (Eugui & Allison, 1982; Hatcher & Kuhn, 1982), resistance to haemopoietic transplants (Lotzova- & Savary, 1977; Kiessling, et al., 1977; Lotzova\ et al., 1983), regulation of the growth and/or differentiation of erythroid, myeloid and lymphoid cell types (Mangan, et al., 1983; Hansson, et al., 1982; Arai, et al., 1983) as well as immunosurveillance to primary tumours and their metastases (Haller, et al., 1977; Warner & Dennert, 1982). Thus NK cells may represent a population whose complex functional activities arise from a basic reponsibility of maintenance of homeostasis within the biological system (Savary & Lotzovd, 1986). 23 3. Regulation of NK Cells Animal studies have examined the in vivo activation of NK cell activity by a variety of bacterial agents which include Bacillus Calmette Guerin (BCG) and Corynebacterium parvum (C. parvum), as well as synthetic agents such as polyinosinic-polycytidylic acid (poly I:C) and pyran copolymer (Welsh, 1984; Brunda, et al., 1981; Brunda, et al., 1982; Henney, et al., 1978; Herberman & Holden, 1978; Oehler, et al., 1979; Dyck, 1986; Savary, 1987). A number of these studies (Herberman, et al., 1981; Herberman, et al., 1982; Ortaldo, et al., 1983; Timonen, et al., 1982) led to the direct observation that activity of NK cells could be augmented by interferon (IFN). These early results demonstrated that highly purified IFNs result in the dose-dependent activation of NK cells. Only a short period of exposure is required for maximal IFN-mediated NK activation (Trinchieri & Santoli, 1978; Welsh, 1984; Djeu, et al., 1979a) and b); Ortaldo, et al., 1980) and IFNs (a, B, amd 7) have all been shown to be very potent modulators of NK cell activity (Ortaldo & Herberman, 1986). Although IFN can augment NK cell activity, this cytokine is not necessary for normal NK lytic function in vitro (Minato, 1980; Copeland, 1981). There is, however, evidence which suggests that IFN may play an important role in vivo by enhancing pre-existing NK cell activity or by recruiting active NK effector cells to the site of infection (Minato, 1980; Saksela, 1978; Ullberg, 1981). 24 One agent which recently has received a great deal of attention regarding its ability to augment mouse, rat and human NK cells has been interleukin-2 (IL-2) (Kurabayashi, 1981; Domzig & Stadler, 1982; Weigent, et al 1983; Handa, et al., 1983; Lotzova\ et al., 1987). This lymphokine, which was first reported as a growth-promoting factor for T cells, has recently been shown also to affect growth of NK cells. In addition to its ability to promote the proliferation of NK cells, it has also been shown to augment NK cell activity. The IL-2-mediated mechanism of activation (previously thought to be independent of IFN), involves the production of IFN-7 (Ortaldo & Herberman, 1986). However, IFN produced by IL-2 treatment of human peripheral blood lymphocytes does not play an essential role in increasing NK cell activity in most donors and it has been shown that IL-2 induced augmentation of NK cell activity is due to the direct action of IL-2 on LGLs (Ortaldo & Herberman, 1986). It has also been recently reported that in addition to their responsiveness to IFN, NK cells are a major producer of IFNs in response to various stimuli. Under a variety of conditions, highly purified LGLs have been shown to be able to produce substantial amounts of IFN (Djeu, et al., 1982) and also IL-1, IL-2, B Cell Growth Factor (BCGF) and Colony Stimulating Factor (CSF) (Scala, et al., 1986). Stimuli have included tumour-cell lines, viruses, BCG, C. parvum and various mitogens and bacterial products. These observations of production of IFNs and other cytokines by a highly enriched NK cell population, have several important and interesting implications: 25 1) these results demonstrate that NK cells appear to be a major source of IFN production, which would allow them to function as immunoregulatory cells; 2) the ablilty of NK cells to produce IFN suggests that they have an alternative mechanism for host defense in addition to their direct cytotoxic activity; 3) they raise the interesting possibility that the effector cell population may be self-regulated in its ablilty to recognize and activate other populations of LGLs or themselves by the production of cytokines (Ortaldo & Herberman, 1986). The negative regulation of NK cells is also an important concern. Experimental evidence has demonstrated that NK cell antitumour activity can be regulated by suppressor cells of various histological types. These include macrophages (Bordignon, et al., 1982; Bash & Vogel, 1984; Seaman, et al., 1982; Yang & Zucker, 1984), T cells (Zoller & Wigzell, 1982; Tarkkanen, et al., 1983), granulocytes (Seaman, et al., 1982; Kay & Smith, 1893) and other cell types not belonging to any of the known lymphoid or myeloid cell classes (Blair, et al., 1983). Regulation of NK cell activity by suppressor cells was observed in both young and old animals, in individuals treated with putative immunopotentiating agents, in tumour-bearing animals and cancer patients, and in several strains of mice exhibiting low natural immunity (Savary & Lotzov£, 1978; Koren, et al., 1981; Riccardi, et al., 1981; Allavena, et al., 1981; Uchida & Micksche, 1981; 26 Bordignon, et al., 1982; Tarkkanen, et al., 1983; Lotzova" & McCredie, 1978; Blair, et al., 1983). Concordant with the role of NK cells in resistance against tumours is the high incidence of neoplasia in strains of mice with low NK cell responder status (Lotzova" & McCredie, 1978). Evidence is accumulating which indicates that NK cell function can be affected by tumour-cells and their products and visa versa. For example, NK cell activity is depressed in patients with large tumour burdens, including cancer patients whose ability to produce interferon is normal (Takasugi, et al., 1977). Recent animal studies indicate that NK cells can inhibit metastasis formation (Hanna & Burton, 1981; Hanna & Fidler, 1980; Talmadge, et al., 1980a; Talmadge, et al., 1980b). In addition, in several other animal studies elevated levels of NK cell activity have been correlated with decreased growth rates of tumour-cells introduced into host animals (Herberman & Holden, 1979; Hanna, 1982; Haller, et al., 1977; Petranyi, et al., 1976; Riccardi, et al., 1979). However, the role of NK cells in modulating the growth of established solid tumours remains unclear as it is still unresolved whether elevated NK cell activity has any therapeutic potential against primary tumours (Moy & Golub, 1986). 27 E. STRESS AND NK CELL ACTIVITY There have been many studies examining the effects of stress on NK functioning. It is generally found that NK cell activity decreases in response to stressors. This has been found in human as well as animal studies. 1. Human Studies In human studies, NK cells have been isolated from peripheral blood of patients and activity measured by the [51Cr]-release assay. In healthy donors, an association has been demonstrated between various stressors and significantly reduced NK cell activity. For example, medical students subjected to an examination stress have suppression of NK cell activity in response to the examination, with those scoring high in self-reported loneliness showing the most depressed NK cell activity (Glaser, et al., 1986). Locke, et al.(1984) found a significant correlation between high life change stress over one year combined with poor coping and a decline in NK cell activity. Others have found that there is depressed NK cell activity in patients with schizophrenia or depression compared to normal or healthy control individuals (Urch, 1988). On the other hand, studies of accountants during income tax season (April) and in the post-tax period (November), found an increase in NK cell activity at the time of peak stress followed by suppression during the post-stress period (Dorian, et al., 1985). In studies of patients with breast cancer, stress factors such as patient "adjustment", lack of social support and 28 fatigue/depression symptoms have been correlated with sustained depression of NK cell activity (Levy, et al., 1987). It has been suggested that depression of NK cell activity can be used as a predictor of prognosis for patients with breast cancer (Levy, et al., 1987). Studies in breast carcinoma patients have found that their NK and CTL cells are defective in their ability to respond to IL-2 (Hakim, 1987). 2. Animal Studies In animal studies, NK cells have been isolated from many organs besides peripheral blood, including spleen, lymph nodes and liver. They have also been found infiltrating solid tumours. Several studies specifically examining the effects of stress on NK cells have found stress depresses NK cell activity (Greenberg, et al., 1984; Kandil & Borysenko, 1987). These studies have included stressors such as surgical stress (Pollock, 1987), tail electric shock (Kraut, 1986) and rotation-induced stress (Kandil & Borysenko, 1987). A recent study using forced swimming stress found that stress decreases NK cell activity against the syngeneic MADB106 tumour and increases lung metastases of the same tumour after intravenous inoculation. The timing of the stress was found to be important: lung metastases increased when stress was applied 1 h but not 24 h before tumour-cell injection (Ben-Eliyahu, et al., 1991). On the other hand, exposure to stress is also known to release opiod peptides from both central and peripheral sites. Morphine and B-endorphin have both been shown to 29 enhance NK cell activity (Kraut & Greenberg, 1986; Mandler, et al., 1986). Another stress-responsive hormone, growth hormone, has also been shown to be involved in NK enhancement (Saxena, et al., 1982). Since most evidence suggests that stress depresses NK cell activity and that a depression in NK cell activity can be correlated with increased tumour incidence and growth, NK cells seemed a logical effector cell to examine for their role in the course of the development of the SCI 15 tumour. Of particular interest was the possible role of NK cells in the increased and decreased tumour growth rates of the mice in the various housing conditions and subjected to the daily acute novelty stress. Data suggests that NK cells are active very early in the antitumour immune response when the tumour burden is low and later are subject to immunosuppression as the tumour grows in size (Gerson, et al., 1981; Wei & Heppner, 1987). For this reason, measurement of NK cytolytic activity at early times following tumour-cell injection was deemed important. Time points of 3 d and 1 wk post-tumour-cell injection were used in order to elucidate a time course of NK cell activity in our model. F. THESIS OBJECTIVE The objective of this thesis is to investigate the possible involvement of the immune system in the modulation of tumour growth observed in response to psychosocial 30 stressors. In our model, the psychosocial stressors consisted of differential housing conditions (individual vs group housing, change vs no change) and exposure to daily acute novelty stress. Initial studies are focused on a general measure of cell-mediated immune activity, ie. the mitogen-induced (Con A) proliferation of splenic lymphocytes. Preliminary data had already been obtained by others in the laboratory, and these experiments are replicated in order to obtain sufficient data for statistical analysis. The major focus of this thesis is the investigation of the possible role of NK cells in mediating the effects of differential housing on SCI 15 tumour growth. First, the protocol to measure NK cell activity in the spleen is adapted to obtain specific lytic activity in our strain of DD/S mice. The resulting protocol is then used to measure splenic NK cell activity at 3 d and 1 wk post injection and rehousing in order to elucidate a timecourse of activity. 31 CHAPTER 2 MATERIALS AND METHODS A. TUMOUR MODEL The androgen-responsive SCI 15 mouse mammary carcinoma, subline class I (Bruchovsky & Rennie, 1978), was maintained by serial transplantation in male mice of the DD/S mouse strain. Dissociation of cells for serial transplantation was the same as that described below for experiments and has been described previously (Emerman & Siemiatkowski, 1984). Male mice were 2 - 4 months old and housed in the standard colony condition when injected. The standard colony condition consisted of male mice raised and housed in sibling groups of 3 mice/cage. 1. Dissociation Tumours weighing approximately 2 g were dissected free of extraneous tissue with sterile technique and finely minced with opposing scalpel blades. The pieces were transferred to a dissociation flask and Saline-Trypsin-Versine (STV) added. STV consisted of 0.05% trypsin (1:250) and 0.025% EDTA (Sigma Chemical Co., St. Louis, MO) in Ca2+- and Mg 2+-free Saline A, pH.7.3. The flask was then gently hand swirled 32 for 2 min, the contents transferred to a 50 ml conical centrifuge tube and spun at 80 x g for 1 min in a bench top clinical centrifuge (ICU). The supernatant was then transferred to another 50 ml centrifuge tube, an equal volume of Dulbecco's Modified Eagle's Medium (DMEM; Terry Fox Laboratory, Vancouver, B.C) + 5 % Calf Serum (CS) added (CS to inactivate the trypsin) and the tube spun at 200 x g for 4 min to enrich the epithelial cell population. The pellet was then resuspended in 5 ml DMEM and placed in a 37 °C waterbath. The tissue left in the original centrifuge tube was left shaking at 100 rpm on a gyrator shaker (Junior Orbit Shaker, Lab-Line Instruments, Inc., Ill) in a 37 °C incubator for 7 min. The contents of this flask were then centrifuged at 80 x g for 1 min, the supernatant collected and an equal volume of DMEM 4- 5% CS added to the supernatant. This was spun for 5 min at 400 x g, resuspended in 5 ml DMEM and placed in a 37 °C waterbath. The remaining tissue was put back in the flask for a third and final dissociation with STV for 7 min. The supernatant was collected as described above, to which an equal volume of DMEM + 5% CS was added. The cell suspension was then centrifuged and resuspended in 5 ml DMEM. All 3 cell suspensions were pooled and passed through a 150 urn Nitex filter (Tetko, Inc., Elmsford, NY) to collect cell aggregates and single cells. The resulting cell suspension was centrifuged at 340 x g for 5 min and the pellet resuspended in 20 ml DMEM. An aliquot of this cell suspension was diluted 1:10 and counted on a haemocytometer using trypan blue exclusion to determine the number of viable cells. The plasma membranes of dead cells are not able 33 to prevent trypan blue (pH 7.2) from entering the cytoplasm, and therefore the dead cells stain blue. The suspension was then adjusted to the concentration desired for either freezing (see below) or injecting into mice (see below). 2. Freezing Although the SCI 15 carcinoma was maintained in vivo, SCI 15 cells were also stored at -70 °C in a liquid nitrogen (LN^ tank. The cell suspension from the dissociation was adjusted to 1 x 107-1.5 x 107 cells/ml in freezing media [50% DMEM + 44% CS + 6% dimethylsulfoxide (DMSO)]. Freezing vials were labelled with cell type and date of dissociation and 1 ml of cell suspension in freezing media was aliquoted to each vial. Vials were then slowly frozen according to a schedule provided by the manufacturer of the freezing tank (Handi-Freeze freezing tray, Union Carbide), before being transferred to a liquid nitrogen storage tank (MVE Cryogenics, TA 60). 3. Transplantation of Tumour Cells In order that each mouse received 3 x 106 cells in 0.1 ml of media, the cell suspension was adjusted to 3 x 107 cells/ml in DMEM. 0.1 ml extra of cell suspension was necessary because of partial loss due to retention in the head of the syringe. The mice were lightly anesthetized with ether and then injected s.c. into the interscapular region. Care had to be taken to lift the skin high up so as not to penetrate underlying 34 tissue and to plunge the needle in deep enough to minimize the amount of cell suspension that leaked back out. Male mice were used in all experiments and were housed 3 siblings/cage, which was the standard housing condition in our colony. 4. Monitoring Tumour Growth For tumour propagation, mice were palpated twice weekly after tumour injection, beginning on days 5 - 8 when a palpable tumour generally first appears and terminating at 18-21 days, when tumour size was between 2 and 3 g. When tumours were measurable, caliper measurements were taken and tumour weights calculated according to the formula (Simpson -Herron & Lloyd, 1970) length (cm) x fwidthfcm)]2 = g 2 For experiments, mice were palpated twice a week, starting on day 6 or 7. Tumour weights were calculated when caliper measurements were able to be taken and the last measurement was taken on the day of termination. 35 B. ANIMAL MODEL Mice were housed in polycarbonate cages (18x29x 13 cm) with stainless steel lids and had food (Purina mouse chow pellets) and water ad libitum. Cages were placed on stainless steel racks in a room with a 12 h dark/light cycle (0700h - 1900h). The room was relatively free from extraneous building noise and remained at a constant temperature of 22 °C. Following weaning, male mice were housed individually or in sibling groups of 3, and at 2 - 4 mo of age were used in experiments. They were injected s.c. in the interscapular region with a single cell suspension of 3 x 106 cells in 0.1 ml of DMEM (tumour(T) groups) or with DMEM alone (no tumour(NoT) groups). Four groups were formed immediately following injection: 1) IG - males raised individually housed were placed in non-sibling groups of 5 animals, 2) II - males raised individually housed remained individually housed, 3) GI - males raised in sibling groups of 3 were separated and housed individually and 4) GG - males raised in sibling groups of 3 remained in their sibling groups. 36 All the animals in all of the groups were subjected to an acute daily stressor consisting of a 15 min exposure, prior to 1200h, to 1 of 5 different novel environments, which were rotated in the following order: 1. a round clear plastic jar 9 cm in diameter and 7 cm high with a white plastic screw top lid, 2. a polypropylene box 12 x 10 x 4 cm with a lid, 3. a covered cardboard box with cardboard divisions forming compartments 7 x 7 x 14 cm high, 4. a plastic cup (220 ml - 10 cm in height and top diameter of 6.5 cm, base . diameter of 4.75 cm) with lid, 5. a clean cage (empty of bedding, food or waterbottle) with a standard cage top. All lids had holes punched in them for adequate ventilation. During experiments, the room was closed off each night at 1800 h and no one entered the room prior to the start of the experiment. Previous data demonstrate that basal hormone levels can be obtained under these conditions (Weinberg & Bezio, 1987). On the day of termination, the cage was carried from the colony room to an adjacent lab, the animals removed from their home cages, weighed and decapitated immediately. Trunk blood was collected and spleens were removed, weighed and dissociated as described below. 37 These experiments were conducted at 3 d, 7 d and 3 wk following tumour-cell injection. For the latter 2 time points, mice were exposed to 5 acute novelty stress periods a week, Monday through Friday. If the termination day fell on a Monday the mice were exposed to the acute novelty stress the day before (Sunday) and not the two days previous. For the earlier time point of 3 d, mice were stressed the days following tumour-cell injection, regardless of the day of the week. C. SPLEEN CELL PROLIFERATION ASSAY The purpose of this assay was to test the general immune responsiveness of splenic T lymphocytes by measuring their proliferative response to the mitogen Con A. This method was based on previously described procedures of [3H]thymidine ('HTdR) incorporation into DNA as a measure of proliferation (Anderson, Et al, 1972; Bradley, 1980; Nowell, 1960). 1. Effector Cell Preparation and Stimulation Spleens were removed aseptically from mice terminated by decapitation, weighed and placed in a 35 mm petri dish containing RPMI (Terry Fox Laboratory, Vancouver, B.C.). After being washed in RPMI, they were transferred to a wire screen (grid size approximately 0.5 mm) sitting in another petri dish, containing RPMI + 10 mM HEPES 38 (Fig 3). Each spleen was cut into small pieces and then gently pressed through the mesh with the plastic end of a 10 ml syringe plunger; the cells were collected in the RPMI + 10 mM HEPES. The screen was washed once with RPMI and the pooled suspensions were transferred to a 50 ml centrifuge tube and brought to 40 ml with additional medium if necessary. This was centrifuged at 600 x g for 2 min and the pellet resuspended in 3 ml Tris-NHjCl and left to sit for 3 min for maximum red blood cell lysis. It was then centrifuged at 600 x g for 2 min and the resulting pellet washed twice with RPMI before being resuspended in exactly 10 ml of RPMI + 10 mM HEPES from which an aliquot was removed to count viable cells identified by trypan blue exclusion on a haemocytometer. Spleen cell suspensions were adjusted to 1.25 x 10s cells/ml in RPMI medium supplemented with 10% fetal calf serum (FCS) (Grand Island Biological Co., Burlington, ON) + 5 x 10"s M 2-mercaptoethanol (2 ME; Sigma). A suspension of 80 /*1 was plated into each well of a round-bottomed 96-well plate for a total of 1 x 105 cells. For each spleen, 2 rows of 8 wells were first plated with spleen cells (Fig. 4.). Following this, 20 jd RPMI + 10% FCS + 2ME was added to the first row of spleen cells. To the second row of spleen cells, 20 /tl of a Con A solution (20 ^g/ml in RPMI +10% FCS + 2ME) was added. The plate was then lightly tapped to mix the contents and placed in a 37 °C, 95% air, 5% C02 incubator for 48 h. 39 Figure 3. Use of wire mesh screen in dissociation of spleens. Petri dish containing RPMi • HEPES. Wire mesh screen is placed into the dish and the spleen into the wire screen for dissociation. 40 Figure 4. Plating protocol for spleen cell proliferation assay A C J C J C unstimulated row B (~ ) (~) {stimulated'umbo* o o o o o o c O O O O O O O O D f J C J C unstimulated row o o o E Q Q QimuMed^itPtonP^ Q Q O O O O O O O O unstimulated row receives 20 /xl media stimulated with Con A receives 20 /il Con A Rows A and B are both plated with 80 /xl of spleen cell suspension A Rows D and E are both plated with 80 /il of spleen cell suspension B 41 2. Radioactive Labelling Plates were removed from the incubator after 48 h and examined for evidence of proliferation using a phase contrast microscope (Leitz Weztlar, Diavert). Then, 0.5 jtCi H3TdR was added to each well. The plate was gently mixed by tapping the sides and placed in the incubator for an additional 18 h. 3. Harvesting To remove all traces of residual radioactive materials of previous experiments, the harvester (Nunc Cell Harvester 8; Fig. 5) was first rinsed with 1% Count-off solution and then rinsed 12 times with distilled water. Filter paper (Whatman glass microfibre 934-AH, 1.0" x 28.0") was added to the harvester and rinsed with 3 washes of 70% methanol. These filter paper discs were then placed in vials as a measure of background radiation. Filter paper was again placed in the harvester and the cell suspensions in the first row of the multiwell plate were aspirated into the harvester and onto the filter paper, followed by 12 washes with distilled water to obtain all cells. A drop of methanol was placed on top to kill all the cells before each filter paper disc was removed from the harvester and placed in a vial. The harvester was then washed with one rinse of methanol, followed by 6 rinses with distilled water, one Count-off rinse, 12 distilled water rinses and finally 3 methanol rinses. This washing procedure was performed 42 Figure 5. Apparatus for harvesting in spleen cell proliferation assay. • vacuum hose / OOOOOOOO Nunc Cell Harvester 8 water hose clamp ^— tube to let out water ^— tube for aspiration Cwj ^ — press to release ^ - ^ water 43 between each row of spleen cells and background filter paper discs were included between each spleen sample, if cells from more than one spleen were plated. Each row of cells from each spleen was harvested in the same manner as described for the first row. When all the wells had been harvested, the vials containing the filter paper discs were placed in a 65 °C oven for 20 min to remove all traces of moisture. Scintillation Cocktail, 3.8 ml, (Aquasol; New England Nuclear) was then dispensed into each vial, the vial vortexed and left to stand in the dark for 1 hour before counting in a Liquid Scintillation Counter (Beckman, LS 5801). D. NATURAL KILLER CELL ACTIVITY ASSAY The natural killer cell activity present in the spleens of the experimental animals was determined by measuring NK cell activity against target cells susceptible to NK cell lysis, using the [5,Cr]-release assay. Various ratios of splenic lymphocytes to ^Cf]-labelled target cells (150:1, 75:1, 37.5:1, 18:1, 9:1 and 4.5:1) were placed in the wells of a 96-well plate and the lysis of target cells measured by the amount of radioactive chromium released into the supernatant over a period of 6 h. 44 1. Target Cell Preparation The target cells used, called YAC, were originally derived from a cell line of a Moloney virus-induced lymphoma (Cikes, et al., 1973) that had been shown to be susceptible to lysis by NK cells. The YAC cells, obtained as a gift from Dr. D. Chow (Manitoba Institute for Cell Biology), were maintained by serial passage in vitro in a cell suspension of RPMI +10% FCS with cell concentrations of a minimum of 4 x 10* cells/ml and a maximum of 7 x 10s cells/ml. After 2 or 3 passages, YAC cells were frozen (see below) and stored for future use. a) Freezing of cells The cell suspension was adjusted to 1 x 107 - 1.5 x 107 cells/ml in freezing media [50% DMEM + 44% CS + 6% DMSO] and 1 ml of this suspension aliquoted into each freezing vial. These vials were slowly frozen in LN2 and then placed in a liquid nitrogen storage tank. 45 b) Thawing of cells before assay The YAC cells were thawed and cultured the day before an experiment so that they were in the exponential phase of growth at the time of radioactive labelling. Since each vial contained approximately 1 x 107 cells and viability after freezing was about 60%, the appropriate number of vials were removed from liquid nitrogen storage for each experiment. Thawing was achieved by rubbing briskly between the hands for a few minutes, then placing in a 37 °C waterbath with frequent shaking. A Kim-wipe wetted with 70 % ethanol was held over the lid to avoid aerosol release upon the opening of the tube. The contents of all the vials were pooled in a 15 ml centrifuge tube and an equal volume of warmed RPMI added. The suspension was spun at 600 x g for 2 min and the pellet washed twice with RPMI. After resuspending in 10 ml of RPMI + 10 mM HEPES, an aliquot of cells was diluted 1:10 and viable cells determined by trypan blue exclusion, were counted on a haemocytometer. The cell suspension was then adjusted to the final concentration in RPMI +10% FCS + 10 mM HEPES and placed in large tissue culture flasks (75 mm2), which were then laid on their sides with loosened lids, in a 37 °C, 95 % air: 5% C02 incubator. For assay 18 - 24 h later, the concentration was 6 - 7 x 104 cells/ml in a total of 50 - 75 ml/flask. 46 c) Radioactive labelling After counting viable cells on a haemocytometer, 107 cells in RPMI +10% FCS + 10 mM HEPES were placed in a 15 ml round-bottomed centrifuge tube and centrifuged at 600 x g for 2 min. The supernatant was removed and the pellet resuspended by shaking in the small amount of medium remaining (80 - 100 /xl). To this was added 100 /U (500 fid) of f'Cr] (Na[5,Cr]04 - 5 mCi/ml; specific activity of 350 - 600 mCi/mg chromium) and the suspension mixed well. This was then placed in a 37 °C waterbath for 80 min, with periodic shaking to ensure constant exposure to [^Cr]. Following this incubation, the centrifuge tube was spun at 600 x g for 2 min and the pellet washed twice with RPMI. It was then resuspended in 10 ml RPMI + 10 mM HEPES to count and the cell suspension adjusted to 1 x 107 cells/ml in RPMI +10% FCS + 10 mM HEPES for the assay. 2. Effector Cell Preparation a) Preparation of spleen cell suspension Spleens were removed aseptically from mice terminated by decapitation, weighed and placed on a 35 mm petri dish containing RPMI. After washing in RPMI, they were 47 transferred to a wire screen sitting in RPMI + 10 mM HEPES in another petri dish (Fig. 3). The procedure that follows is also described on p. 37. Each spleen was cut into small pieces and then gently pressed through the mesh with the plastic end of a 10 ml syringe plunger and the cells collected in the medium. The screen was washed with medium and the pooled suspensions transferred to a 50 ml centrifuge tube and brought to 40 ml with additional medium if necessary. This was centrifuged at 600 x g for 2 min and the pellet resuspended in 3 ml Tris-NHtCl and left to sit for 3 min. This was necessary in order to lyse all red blood cells. The suspension was then spun at 600 x g for 2 min and the resulting pellet washed twice with RPMI and resuspended in 1 ml of RPMI +10% FCS + 10 mM HEPES; the cell suspension was now ready for passage through nylon wool columns. b) Preparation of nylon wool columns Nylon wool (Fenwal Laboratories, 111.) was boiled 6 times in distilled water and dried in 150 °C oven for rapid and complete drying of the wool. It was then carded by hand (using gloves) and 0.7 g was packed into 10 ml plastic syringes up to the 7 ml mark. They were then covered in foil and sterilized. When needed to separate cells, columns were removed aseptically from their foil covering and placed in a clamp stand. The wool was wet with 10 ml RPMI so that no air bubbles formed. Once the column was wet, 20 ml of warm RPMI + 10% FCS + lOmM HEPES medium were poured through the column and it was placed in the incubator for at least 30 min to equilibrate. 48 When the effector cell suspension was ready, it was placed drop-wise onto equilibrated nylon wool columns to separate out T lymphocytes and NK cells from B lymphocytes and macrophages. The cell suspension was followed by 0.5 - 0.8 ml RPMI + 10% FCS + 10 mM HEPES. The columns were then incubated for 35 - 60 min in a 37 °C, 95% air: 5% C02 incubator. Following incubation, cells were eluted from the column by the slow drop-wise addition of 10 ml RPMI -I- 10% FCS + 10 mM HEPES and collected in a 15 ml centrifuge tube. The tube was then centrifuged at 600 x g for 2 min, the pellet washed once in RPMI and then resuspended in RPMI + 10 mM HEPES to count. The cell suspension was centrifuged for 2 min at 600 x g and then resuspended to a concentration of 1.5 x 107 cells/ml in RPMI + 10%FCS + 10 mM HEPES, ready for the cytotoxicity assay. c) Cytotoxicity assay Spleen cell suspension (100 itl) was placed into the 150:1 and 75:1 wells of a 96 well V-bottomed plate (fig. 6). Media (100 /xl) was added to the 75:1, 37.5:1, 18:1, 9:1 and 4.5:1 wells and then the suspensions serially diluted. This was accomplished by pipetting up and down the 75:1 ratio suspension to mix the suspension, removing 100 /xl and placing it in the next ratio well (37.5:1). Then, that ratio well was mixed to resuspend, 100 yX removed and added to the 18:1 well and so forth until the final ratio well is reached (4.5:1). After resuspending the final ratio well, 100 it 1 is removed and just discarded. Thus, each ratio well has 100 pi of cell suspension in it and each of these 49 is done in triplicate. To these wells, 100 /*1 p'CrJ-labelled YAC cells were added, bringing the final volume in each well to 200 /il. Additional wells containing YAC cells only were plated in order to determine spontaneous and total release of radioactivity (Fig. 6). Spontaneous release is a measure of the radioactivity released by the YAC cells themselves during the course of the assay, with no outside influences. Only additional media was added to the wells measuring spontaneous release to bring the volume to 200 fd. Total release is a measure of the total [^Cr] that was incorporated into the cells (D. 1. c), p. 46) and therefore 100 jtl of IN HC1 was added to bring about total lysis of all cells, resulting in total release of all ["CrJ. Plates were centrifuged gently at 25 x g for 2 min to settle the cells together and placed in an incubator (37 °C, 95 % air: 5% CO2) for 6 h. After incubation, plates were centrifuged at 150 x g for 10 min, an aliquot of supernatant (100 /*1) was removed and placed in small glass tubes (6 x 50 mm). These were placed in larger 12 x 75 mm glass tubes for counting in a gamma counter (Packard Minaxi 7 Auto-Gamma 5000 series). 50 Figure 6, Plating protocol for cytotoxicity assays. 10 A© © © G50O © © © O O 0 0 0 0 0 O O G»0 O O O O O 0 0 o o o o o c«o o o o o o C«C O O O O O o o o o o o o o o o H 0 © © 0 © © © © © © (?) = experimental wells: 100 p\ spleen cell suspension places into these wells (Al through B3) @> = experimental wells: 100 p\ media was added to these wells (Bl through F3) (s) = spontaneous wells: containing 100 pi labelled YAC cell suspension + 100 fil media © = total wells: containing 100 p\ labelled YAC cell suspension + 100 fil 1 N HCl columns 1,2,3 contain spleen cells from spleen A columns 6,7,8 contain spleen cells from spleen B 51 d) In vivo boosting of effector cell activity Polyinosinic-polycytidylic acid (Poly I:C) is a synthetic helical complex of the homoribopolynucleotides polyinosinic and polycytidylic acids. It has been shown to augment NK cell activity both in vitro and in vivo (Talmadge, et al., 1985). In vivo boosting of NK cells was accomplished by the intraperitoneal (i.p.) injection of 100 fig Poly I:C in 0.2 ml PBS, 18 h prior to the removal of the spleen for the cytotoxic assay. 3. Calculation of Results Specific lysis was determined according to the following formula (Kraut & Greenberg, 1986): % specific = experimental release - spontaneous release lysis total release - spontaneous release where experimental release is the counts in experimental cultures of target cells and effector cells. Spontaneous release is the measure of the counts in cultures containing only target cells. Total release is the counts obtained by adding 100 fd of IN HC1 to target cells to lyse all cells. 52 4. Statistical Analysis Statistical analyses were performed with appropriate analyses of variance for the factors of Group, Tumour and Ratio, with repeated measures on the last factor. Significant main effects were analysed by Tukey's post-hoc tests. 53 CHAPTER 3 RESULTS A. COMPLETION OF SPLEEN CELL PROLIFERATION DATA The spleen cell proliferation assay is an immunological assay that measures the proliferation of T lymphocytes isolated from spleens in response to the mitogen Con A. Proliferation is determined by [3H]TdR incorporation into DNA, based on previously described procedures (Andersson, et al., 1980, Bradley, et al., 1980 and Nowell, 1960). The assay was used to provide a general measure of immune responsiveness of T cells in the spleens of experimental mice. Briefly, male mice were reared either singly housed or in sibling groups of 3 from weaning to adulthood. At 2 - 4 mo of age, animals in each rearing condition were injected with either tumour-cells in vehicle or vehicle alone. The four experimental housing groups were formed immediately following injection, i.e. individually housed animals either stayed singly housed (II) or were placed in non-sibling groups of 5 (IG), and mice raised in sibling groups were either separated and housed individually (GI) or remained in their same sibling rearing group (GG). They then were exposed to the acute daily stressor for 15 min/d, 5 d/wk, for a total of 18 - 19 d. At this time, mice were 54 terminated and spleens removed. Spleens were dissociated and cell suspensions were incubated in the presence or absence of Con A and proliferation measured by [3H]TdR incorporation. The results of the experiments are shown in Fig. 7 a) and b). These results are a combined effort of several people in the laboratory. Data were analyzed by 2 (vehicle/tumour) X 4 (group) X 2 (Con A/no Con A) analyses of variance (ANOVA). Significant main effects or interactions were further analyzed by Newman-Keuls post-hoc tests. A Tumour X Group X Stimulation interaction F(3,42) = 3.23, p < 0.05, was revealed when proliferation was analyzed in the presence or absence of Con A. The Newman-Keuls tests indicated that in the absence of Con A, there were no differences in proliferative activity among animals from the 4 housing groups, whether or not they had been injected with tumour-cells. However, differential effects of housing group were observed following exposure to Con A in vehicle-injected animals; IG animals showed significantly less lymphocyte proliferation than GG controls, p < 0.05. A differential effect of housing condition was not observed in animals injected with tumour-cells although there was reduced proliferative activity in all tumour-bearing animals compared to vehicle-injected controls, F(l,42) = 5.24, p < 0.05. 55 figure 7i Splenic T-lvmphocvte proliferation in the presence (stimulated) or absence (unstimulated) of Con A. Spleen cell proliferation was assessed by the incorporation of fH]TdR into spleen cells either stimulated with the mitogen Con A or unstimulated (absence of Con A). Spleen cells were obtained from mice rehoused in the 4 experimental housing groups 3 wk prior to termination. II - Individually housed animals stayed singly housed IG - Individually housed animals were placed in non-sibling groups of 5 GG - Mice raised in sibling groups of 3 remained in their same group GI - Mice raised in sibling groups of 3 were separated and housed individually. Before formation of experimental groups, half the animals in each condition were injected either with vehicle alone (No Tumour - Fig. 7 a) or with tumour cells in vehicle (Tumour - Fig. 7 b). All groups were exposed to the acute daily stressor. Number of mice in each group: • vehicle (Con A) - IG (6), II (6), GI (6), GG (5) (no Con A) - IG (6), H (6), GI (6), GG (5) tumour (Con A) - IG (7), II (6), GI (7), GG (7) (no Con A) - IG (7), II (6), GI (7), GG (7) 56 Figure 7a). Vehicle injected mice. 400 In the presence of Con A, IG < GG, p < 0.05. 57 Figure 7b). Tumour-cell injected mice. 4 0 0 3 2 0 2 4 0 160 -8 0 O Gl GG IG There is reduced proliferative activity in all tumour-bearing animals compared to vehicle injected controls, p < 0.05. 58 B. THE [5ICr]-RELEASE ASSAY A series of experiments were performed to adapt the [51Cr]-release assay to measure NK cell activity in the DD/S strain of mice, tumour and target cells. The protocol was adapted from Kiessling, et al, (1970) and Kraut & Greenberg, (1986). Specifically, the assay was used to measure the activity of NK cells present in the spleens of the experimental mice against the NK-sensitive target cells, YAC. It was to determine if NK cells were involved in the differential growth of the tumours in mice housed under the various conditions and exposed to acute daily novelty stress. For these experiments, male mice housed in the standard condition of 3 mice per cage were used unless otherwise mentioned. 1. Propagation of YAC Cells. The target cells used for the assay, YAC, were derived from the cell-line of a Moloney virus-induced lymphoma (Cikes et al, 1973) and have been demonstrated to be susceptible to NK cell lysis. Any cell that will lyse these cells in a short term assay (3 - 6 h) without prior exposure, is described as having natural cytotoxicity. The growth characteristics of YAC cells were investigated by culturing these cells for a total of 10 days. This was done in the following manner. 59 A vial of frozen cells was thawed as described in Materials and Methods. Cultures were set up at approximately 6 x 10* cells/ml in a total of 10 ml and placed in a 25 mm2 tissue culture flask that was left standing upright with the cap loosened in a 37 °C, 95% air: 5% C02 incubator. Approximately 24 h later, the cells were resuspended and a small aliquot was removed and counted. Cell numbers were calculated and the cell concentration adjusted to 2 different concentrations with the addition of fresh medium (RPMI +10% FCS); the original concentration of 6 x 104 cells/ml and a higher concentration of 1.6 x 105 cells/ml. Again, the cell suspensions were counted the following day and then subcultured. However, only the lower concentration was used at this point and. subsequently subcultured for a total of 10 days. This was because the higher concentration resulted the next day in a concentration of cells that approached the upper limit too closely. A growth chart of the YAC cells is shown in Fig. 8. Cell number generally increased approximately 4 fold over 24 hours, although increases of 5 - 6 fold were also observed. The culture set up at the higher concentration of 1.6 x 105 cells/ml on day 1 approached the upper limit of 7 x 105 cells/ml the next day. On day 2, cell cultures were set up lower than usual (2 x 104 cells/ml) so that a longer time period (2 d) could pass before the need for subculturing. After 2 d, the resulting concentration fell into the range that had been previously achieved by setting up at 6 x 104 cell/ml and leaving for 1 d. 60 Figure 8. Growth of YAC cells. 2 3 4 5 6 7 8 9 10 DAYS SUBCULTURED Cultures of YAC cells were set up at approximately 6 x 104 cells/ml (A) Approximately 24 h later, cell concentration was determined after counting cell numbers (A), and new cultures were set up at that time ($. On day 1, the cell culture was set up at an initial higher concentration of 1.6 x 10s cells/ml (o-«). 61 2. NK Cell Assay and Target Cell Labelling. Protocols for the NK cell cytotoxicity assay from 2 sources (Kiessling et al, 1970; Kraut & Greenberg, 1986) were tested. Two experiments were performed to answer the following questions. 1) Did the NK cell assay produce specific lysis of the target cells by the spleen cells from DD/S mice? 2) Which target cell labelling protocol would give the maximum incorporation of [51Cr] while keeping the spontaneous release of the [^Cr] incorporated to a minimum? In the first experiment, the assay utilized effector to target cell ratios of 80:1, 40:1, 20:1, 10:1, 5:1 and 2.5:1, using the cells from 1 spleen. The length of the incubation period of the cytotoxicity assay was 4 h. The target cell labelling was accomplished in the following ways. 2 pellets of approximately 107 YAC cells each (growing in an exponential phase of growth) were obtained. To each pellet was added 210 fid ["Cr] (1 mCi/ml). The pellet was gently resuspended and placed in a 37 °C waterbath. One sample was left in the waterbath for a total of 90 min, removed, washed 3x and counted. The other sample was removed after only 60 min in the waterbath and washed twice. It was then resuspended in RPMI +10% FCS + lOmM HEPES, returned to the waterbath for an additional 20 min, resuspended and counted. Both target cell preparations were then used in the assay. 62 Both a difference in the total incorporation of radioactive label and a difference in spontaneous release was observed between the 2 methods in the first experiments (Table II). Although the 1.5 h incubation gave a higher incorporation, the spontaneous release was higher. However, the variability in the results of the specific lysis obtained was substantial. Cells from the same spleen cell suspension were used for both labelling procedures, but it appeared that only the 1.5 h incubation resulted in specific lysis. This experiment was repeated with a few modifications. Spleen cells from two different animals were used (A and B) for both labelling procedures. The amount of radioactive label added to the cells was increased from 210 /*Ci [^Cr] to 500 jtCi ["Cr] and the ratio of effector to target cells was increased to 150:1. (Only the lower ratio was used earlier because higher lysis had been expected.) Maintaining the use of a total of 6 ratios meant that the resulting ratios were 150:1, 75:1, 37.5:1, 18:1, 9:1 and 4.5:1. Neither the spontaneous release nor the specific experimental lysis obtained seemed to be significantly affected by the increased labelling procedure of the second experiments (Table III). When both incubation periods for labelling were used, the results for procedures were very similar, 18.9% and 19.8% for spleen A, 11.1% and 12.9% for spleen B at 150:1. However, the total amount of radioactive label incorporated was higher in the 1.5 h incubation. 63 Table II. Comparison of 2 different target cell labelling procedures Effector:Target cell (E:T) ratio [5lCr] Incubation time Specific Release(%) 1 h 1.5 h 80 40 20 10 5 1 1 1 1 1 2.5 : 1 2.5 1.1 4.4 0.8 0.5 0.4 31.6 17.1 10.1 10.9 2.9 19.5 Release Total (cpm) Spontaneous (%) 5053.0 15.2 7924.3 23.8 Incubation was in 210 juCi [51Cr] for 1 h or 1.5 hr. Total incubation time for the cytotoxicity assay was 4 h. Each ratio was performed in triplicate (S.E. < 5%). 64 TABLE III. Comparison of 2 different target cell labelling procedures, with modifications E:T ratio Specific lysis (%) A (1 h) B (1 h) A (1.5 h) B (1.5 h) 150:1 75:1 37.5:1 18:1 9:1 4.5:1 Release: Total Spontaneous 18.9 15.5 15.3 9.8 5.5 4.3 4629, 9, .4 .2 cpm % 11.1 9.1 9.9 7.6 2.8 1.5 19.8 16.7 11.7 8.7 4.1 3.2 7005. 10. .6 ,7% 12.9 10.3 9.2 5.5 3.7 2.0 cpm Incubation was in 500 ^ Ci [51Cr] for 1 h or 1.5 h. Total incubation time for the assay was 4 h. Each ratio was performed in triplicate (S.E. < 5%). 65 3. Test Assay Using a Mouse with Tumour This experiment included the spleen from a mouse that had been injected with SCI 15 tumour-cells 3 wk prior (Spleen A) as well as a spleen from a mouse injected with vehicle alone (Spleen B). This experiment would determine if the presence of a tumour would enhance or suppress NK cell activity. The presence of a tumour after 3 wk appeared to suppress NK cell activity (Table IV). At a ratio of 150:1 spleen cells of mice injected with tumour cells (T) had a specific release of 15% while spleens of mice injected with vehicle alone (NoT) had a specific release of 29.1%. 66 TABLE IV. Test NK cell assay using tumour-cell injected mice. Specific Lysis (%) E:T Ratio Spleen A (T) Spleen B (NoT) 150:1 15.0 29.1 75:1 8.9 23.0 37.5:1 2.3 36.5 18:1 2.9 5.3 9:1 0.08 2.1 4.5:1 0.0 1.6 Spleen cells from an animal injected with tumour-cells 3 wk previously (Spleen A) and spleen cells from an animal not injected with tumour-cells (Spleen B) were used. NK cell assay using the 1.5 h labelling procedure for the target cells. All other conditions remained the same. 67 4. Four Housing Groups and Novelty Stress. In the previous experiments, male mice between 2 and 4 mo old were housed in the standard condition of sibling groups of 3. These experiments now focus on NK cell responses in animals from the 4 different experimental housing groups (i.e. II, IG, GG and GI) and exposed to acute daily stress. Animals (total n = 14) were injected with tumour cells and placed in the four different housing groups as described in Materials and Methods. They were then subjected to 15 min/d, 5 d/wk of novelty stress, i.e. placement into 1 of 5 different containers described in Materials and Methods. This was continued for a total of 19 d while tumour growth was monitored. At the end of this time, animals were terminated and spleens removed for the NK cell assay. These results are outlined in Table V. A clear difference among animals in the different conditions was not present, 150:1 ratios averaged 16%, 11%, 10% and 13%, and the variability among the animals in the groups was very high. 68 TABLE V. NK cell assay using tumour-cell injected animals and psychosocial stress conditions Mice in Effector:Target (E:T) cell ratios Housing groups 150:1 75:1 37.5:1 18:1 9:1 4.5:1 16 II GG GI # 1 # 2 # 3 # 4 # 5 mean ±S.E.M # 1 # 2 # 3 mean ±S.E.M # 1 # 2 # 3 mean ±S.E.M # 1 # 2 # 3 mean ±S.E.M 23.86 15.66 10.32 14.02 17.11 16.2 ±2.2 7.4 8.7 17.62 11.2 ±3.3 15.68 4.81 8.36 9.6 ±3.2 9.14 14.33 15.9 13.1 ±2.0 19.42 12.10 7.45 12.58 9.37 12.2 ±2.0 5.09 7.68 10.56 7.8 ±1.6 9.3 4.7 8.24 7.4 ±1.4 5.55 12.87 13.4 10.6 ±2.5 11.83 8.41 5.26 11.14 7.5 8.8 ±1.2 2.35 5.0 8.59 5.3 ±1.8 4.58 3.27 1.69 3.2 ±0.8 3.95 8.59 9.17 7.2 ±1.6 8.88 6.36 2.74 7.04 4.38 5.9 ±1.1 1.69 2.5 4.41 2.9 ±2.9 2.5 1.65 1.88 2.0 ±0.3 2.35 2.65 4.63 3.2 ±0.7 3.25 3.76 1.85 2.19 1.87 4.3 ±0.4 * 4.02 1.09 2.6 ±1.5 1.46 0.7 -1.1 ±0.4 3.84 2.99 1.16 2.7 ±0.8 3.42 2.1 0.27 0.27 0.54 1.3 ±0.6 -1.69 0.71 1.2 ±0.5 2.02 5.95 -4.0 ±2.0 0.51 0.81 0.03 0.5 ±0.2 After receiving tumour-cell injection, the mice were housed in the different conditions and received acute daily novelty stress for 3 wk. -* counts obtained for these wells were not above counts for spontaneous release. 69 5. Test Assay Using Known Responder Strains To test the protocol established thus far, the assay was used to measure the NK cell activity of 3 known responder mouse strains. These strains were C57B1-6, CBA and Balb/c (Kiessling, et al., 1975). Spleens from 4 mice were tested from each strain in addition to spleens from 2 DD/S mice. The results showed that the assay protocol was measuring specific release of NK cell activity in the DD/S strain (Table VI), although the assay produced variable results. 70 Table VI. NK cell assays on known responder strains of mice in addition to the DD/S mouse strain. Effector:Target Cell Ratios Mouse 150:1 75:1 37.5:1 18:1 9:1 4.5:1 C57B16 CBA Balb/C DD/S #1 #2 #3 #4 mean ±S•E.M• #1 #2 #3 #4 mean IS•E.M• #1 #2 #3 #4 mean ±S.E.M. #1 #2 mean iS•E•H. 11.2 15.2 19.4 18.5 16.1 ±.9 15.3 20.5 9.8 10.9 14.1 ±1.2 6.9 5.1 22.1 27.8 15.6 ±2.8 13.7 13.8 13.8 ±.04 6.1 7.4 11.4 9.5 8.6 ±.58 10.1 14.6 6.3 7.7 9.7 ±.91 8.9 3.2 16.9 14.9 11.0 ±1.6 10.9 8.7 9.8 ±.78 8.1 11.8 7.3 6.0 8.3 ±.62 4.6 7.0 2.1 4.2 4.5 ±.5 2.9 1.5 13.1 8.1 6.4 ±1.3 5.1 5.2 5.2 ±.04 1.9 1.9 3.0 1.7 2.1 ±.15 1.6 3.2 0.4 5.6 2.7 ±.56 1.2 0.1 7.3 4.7 3.3 ±.8 3.4 1.2 2.3 ±.78 0.04 -1.6 --0.4 0.9 0.9 --0.3 -5.1 2.2 -0.6 1.7 1.15 ±.4 ------0.4 -1.0 ---2.0 0.2 -0.1 ---* counts obtained in these wells did not differ from spontaneous release 71 6. In Vivo Boosting Polyinosinic-polycytidylic acid (Poly I:C) is a synthetic helical complex of the homoribopolynucleotides polyinosinic and polycytidylic acids. It has been shown to augment NK cell activity both in vitro and in vivo (Talmadge, et al., 1985). In vivo boosting of NK cells was accomplished by the i.p. injection of 100 /*g Poly I:C in 0.2 ml PBS, 18 h prior to termination of mice. Significant stimulation of NK cell activity was achieved (Table VII). However, in a tumour-bearing animal this boosting was not as evident as in a mouse free of tumour. 72 Table VII. NK cell assays after in vivo boosting Specific lysis (%) E:T ratio Spleen A Spleen B Spleen C Spleen 0 P-I:C, NoT P-I:C, NoT P-I:C, NoT P-I:C, T 1 5 0 : 1 7 5 : 1 3 7 . 5 : 1 1 8 : 1 9 : 1 4 . 5 : 1 6 1 . 1 5 0 . 1 3 0 . 5 18 .2 1 1 . 7 7 . 5 5 2 . 3 50 .2 38 .9 2 2 . 4 15 .9 1 2 . 3 7 6 . 8 6 4 . 7 4 0 . 8 2 4 . 2 14 .4 7 . 2 2 7 . 4 1 6 . 2 1 0 . 6 2 5 . 5 1 . 8 3 . 9 All mice received Poly I:C (P-I:C) 18 h prior to termination. Mice A, B and C had no tumour (NoT); mouse D was injected with SC115 tumour cells 3 wk prior to termination (T). 73 7. In Vitro Boosting In vitro boosting was performed by increasing the ratio of effector to target cells to 300:1, increasing the length of the assay incubation time to 6 h and using nylon wool columns. This assay was performed on spleens from 4 different animals (A, B, C and D). Although the standard length of an NK cell assay is 4 h there are reports of assays that are longer in length (Krant and Greenberg, 1986). Cells from a spleen (A) separated on a nylon wool column (Materials and Methods) was used as another method of boosting in vitro cytotoxicity, since this enriches the T cell and NK cell populations from the spleen by removing B cells and most macrophages (Trizio & Cudkowicz, 1974). 300:1 ratios did not substantially increase cell lysis, but seemed to reduce the variabilty among the animals. The change from a 4 h to a 6 h incubation period did increase the lysis by almost 10% (Table VIII). Since these results confirmed that the increase in incubation length to 6 h increased lysis, this incubation time was used in future protocols. Results using the nylon wool column showed no substantial increase in lysis. 74 Table VIII. NK cell assay using in vitro boosting E:T ratio 300:1 150:1 75:1 37.5:1 18:1 9:1 Spleen 34.2 22.8 17.8 13.3 10.6 7.2 A* Speci Spleen 33.0 29.6 23.2 17.3 12.4 7.7 B .fic lysis Spleen 26.2 26.1 18.7 ** 13.2 5.2 (%) C Spleen D 35.7 28.4 19.7 ** 12.2 5.8 Ratio of effector to target cells was increased to 300:1 and the incubation length of the cytotoxic assay was increased to 6h. * - Spleen cells from A were passaged through nylon wool. ** - counts were not obtained from these wells 75 8. Removal of Red Blood Cells Experiments were performed to establish if lysing the red blood cells with Tris NH4CI before passage through nylon wool columns would improve NK cell activity. An additional experiment using 4 mice was conducted to compare the performance of spleen cells in the NK cell assay after lysing red blood cells and passing through nylon wool (Spleens A, B and C), with those not so treated (Spleen D). The cytotoxic assay length was only 4 h as the results these would be compared with utilized this time. It had already been established that a longer incubation length increased lysis. Spleen cells with (Spleen A) and without (Spleen B) Tris lysis and passed through nylon wool columns did show dramatic differences in their NK cell activity (Table IX). Although not enough cells could be obtained to perform a 300:1 ratio with the spleen that had the red blood cells lysed, the lysis obtained from 100:1 was still higher than the 200:1 ratio of the spleen not exposed to Tris lysis. In the repeated experiments the results were again clear (Table X). Lysis was clearly consistently raised compared with spleen cells that were not passed through nylon wool. Thus red blood cell lysis using Tris NH4CI and passage through nylon wool columns were used in all subsequent effector cell preparations. 76 Table IX. Test NK cell assay with removal of red blood cells before nylon wool treatment Specific release (%) E:T ratio Spleen A (Tris) Spleen B (No Tris) 2 0 0 : 1 1 0 0 : 1 5 0 : 1 2 5 : 1 1 2 . 5 : 1 6 . 2 5 : 1 3 : 1 ** 31 .4 2 2 . 3 16 .2 1 0 . 5 6 . 4 4 . 8 2 5 . 6 1 7 . 5 11 .7 10 .8 7 . 5 9 . 3 * Spleen A had red blood cells lysed with Tris NH4C1 prior to the use of the columns. Spleen B had not. Cytotoxic assay incubation length was 4 h. ** - not enough cells were obtained to perform this ratio -* counts obtained from these wells did not differ from spontaneous release 77 Table X. Confirmation of protocol with nylon wool treatment and red blood cell lysis Specific Release (%) E:T Ratio Spleen A Spleen B Spleen C Spleen D 200:1 100:1 5 0 : 1 2 5 : 1 1 2 . 5 : 1 6 . 2 5 : 1 3 : 1 ** 2 4 . 3 1 6 . 2 1 6 . 2 6 . 5 7 . 3 3 . 6 ** 16 .6 11 .8 7 . 7 5 . 4 3 . 4 1 . 5 ** 18 .3 1 5 . 1 8 . 0 8 . 0 4 . 8 2 . 3 8 . 3 8 . 1 5 . 0 2 . 5 2 . 4 1 . 0 • NK cell assay using spleen cells from 4 mice. Animals A, B and C were passaged through nylon wool, while D was not treated. All spleens had red blood cells removed prior to this. Cytotxicity assay incubation length was 4 h. ** - not enough cells were obtained to perform this ratio -" counts obtained from these wells did not differ from spontaneous release 78 9. Processing of 6 Spleens at One Time The processing of spleen cells from a total of 6 animals in one day was attempted to determine the feasibility of processing this number of spleens and to confirm the success of the assay protocol established thus far. Animals were either injected with tumour cells and vehicle (A, B and C) or vehicle alone (D, E and F). This experiment was exactly as described in the Materials and Methods, including the use of Tris lysis and nylon wool columns, ratios of 150:1, 75:1, 37.5:1, 18:1, 9:1 and 4.5:1, and an incubation time of 6 h. The results of the NK cell assay are detailed in Table XI. Specific lysis ranged form 37.4% to 52.2% in spleens from animals terminated 3 wk following tumour-cell injection and 35.1% to 41.3% in spleens from animals injected with vehicle only. Total f'Cr] uptake was high, about 0.8 counts/cell and spontaneous release was very low, about 6%. 79 Table XI. Confirmation of the ability to process a greater number of animals at one time using the assay protocol established Specific lysis (%) B:T r a t i o 1 5 0 : 1 7 5 : 1 3 7 . 5 : 1 1 8 : 1 9 : 1 4 . 5 : 1 A (T) 3 8 . 2 3 2 . 6 2 2 . 9 1 4 . 9 9 . 7 3 . 8 B (T) 5 2 . 2 4 3 . 2 3 5 . 8 2 2 . 2 ** ** C (T) 3 7 . 4 3 3 . 8 2 4 . 9 1 5 . 7 ** 5 . 5 D(NOT) 4 0 . 1 3 7 . 3 2 9 . 6 2 2 . 1 1 5 . 0 9 . 3 E(NoT) 3 5 . 1 2 8 . 7 1 7 . 9 1 1 . 7 8 . 1 4 . 9 P(NOT) 4 1 . 3 3 4 . 5 2 6 . 4 1 8 . 6 1 1 . 6 6 . 8 NK cell assays were performed on 6 animals. A, B and C were injected with tumour-cells 3 wk previously (T); D, E and F were injected with vehicle (NoT). All spleen cell suspensions were subject to Tris lysis and passaged through nylon wool columns. Incubation length of cytotoxicity assay was 6 h. ** - counts were not obtained for these wells 80 C. NK CELL ASSAY TIMECOURSE STUDY A series of experiments was undertaken to investigate the possible role of NK cells in mediating the differential tumour growth observed in the animals in the 4 housing conditions and exposed to daily acute novelty stress. As NK cell activity has been implicated to be important in the early stages of tumourogenesis (Keller, 1981; Lotzova* & Herberman, 1986), a time course of 3 d and 1 wk was chosen to investigate enhancement or depression of NK cell activity and the possible implication this might have on SCI 15 growth. Male mice were raised and housed as described in Methods and Materials. As it was only physically feasible to dissociate and assay 8 mice in one experimental day, 2 different groups, e.g. IG (5 mice) + GI (3 mice) could be tested on one day, or the same group with or without tumour, e.g. GG-tumour (3 mice) + GG-no tumour (3 mice). An effort was maintained to ensure that all possible combinations were tested on 1 day, for each time point (except for IG tumour and no tumour which would include 10 mice). Mice in all groups were balanced for age and degree of wounding due to fighting and each condition was replicated to produce the following n's: IG - 3 different groups for a total of 15 mice, II - a total of 6 individual mice assayed 3 at one time, GG - 3 different groups for a total of 9 mice, GI - 2 different groups producing a total of 6 separated individuals. Whenever groups were injected with tumour-cells and terminated at early 81 time points, at least 3 - 5 males were injected at the same time and kept for 3 wk to ensure the tumours grew. Mice terminated at 3 d received 2 acute stress sessions, while those mice terminated after 1 wk received 5 acute stress sessions (the last being the day before termination in all cases). All protocols for housing and assays are found in Materials and Methods. Statistical analyses were performed with appropriate analyses of variance for the factors of Group, Tumour and Ratio, with repeated measures on the last factor. Significant main effects were analyzed by Tukey's post-hoc tests. Maximal stimulation of splenic NK cell activity occured at 3 d following the injection of SCI 15 tumour cells (Fig. 9 and Table XII) rather than at 1 wk (Fig. 10 and Table XIII). At 3 d, NK cell activity was significantly greater in tumour-cell injected mice than in vehicle injected mice. The ANOVAs revealed significant main effects of Group, F(3,66) = 5.92, P = 0.001, Tumour, F(l,66) = 757.18, P < 0.001 and Ratio, F(5,330) = 911.48, P = 0.018 and a Group X Tumour X Ratio interaction, F(15,330) = 4.59, P < 0.01. The post-hoc analysis indicated that for vehicle injected animals, groups did not differ significantly in NK cell activity. However, following tumour-cell injection, GI animals had greater NK cell stimulation overall than II and GG animals 82 (P < 0.05). Further, at the 4 lower effector to target cell ratios, GI animals also had significantly greater NK cell activity than IG animals. NK cell activity in tumour-cell injected animals was significantly lower at 1 wk than at 3 d, F(l,72) = 204.68, P < 0.001. In addition, NK cell activity was again significantly greater in tumour-cell injected than in vehicle injected animals at 1 wk, F(l,61) = 29.9, P < 0.001 (Fig. 10). However, the groups were not significantly different from one another at this time. 83 Figure 9. NK cell lytic activity at 3 d in mice from the 4 experimental groups 1 0 0 i 1 4 .5 :1 9:1 18:1 37 .5 :1 7 5 : 1 150:1 EFFECTOR : TARGET RATIOS Lytic activity of splenic NK cells 3 d post-injection. Mice from the 4 experimental groups were either injected with SCI 15 cells ( ) or vehicle alone ( ). Three days later, the lytic activity of splenic NK cells was measured. NK cell activity was significantly greater in tumour-cell injected than in vehicle injected mice, P < 0.05. Within the tumour-cell injected condition, GI > GG and II, P < 0.05. Groups are as described in Fig. 7. Number of mice in each group: vehicle II (6), IG (15), GG (8), GI (5) tumour II (6), IG (11), GG (9), GI (6) 84 Figure 10. NK cell lytic activity at 1 wk in mice from the 4 experimental groups 1 0 0 GO if) > o LL u LU a CO 75 50 25 . A IG D GG • Gl 4.5:1 9:1 18:1 37.5:1 75:1 150:1 EFFECTOR : TARGET RATIOS Lytic activity of splenic NK cells 1 wk post-injection. Mice from the 4 experimental groups were either injected with SCI 15 cells ( ) or vehicle alone ( ). One week later, the lytic activity of splenic NK cells was measured. Number of mice in each group: vehicle II (6), IG (14), GG (8), GI (6) tumour II (6), IG (15), GG (9), GI (5) 85 Table XII. NK cell lytic activity at 3 d Specific lysis (%) Tumour Vehicle E:T IG II GG GI IG II GG GI ratio n=15" n=6 n=9 n=5 n=l5 n=6 n=8 n=5 1 5 0 : 1 7 5 : 1 3 7 . 5 : 1 1 8 : 1 9 : 1 4 . 5 : 1 9 1 . 9b ± 3 . 3 7 9 . 0 ± 4 . 0 6 9 . 4 ± 2 . 7 5 2 . 8 ± 2 . 3 4 0 . 1 ± 2 . 0 2 6 . 5 ± 2 . 2 7 6 . 6 ± 1 . 0 7 1 . 1 ± 0 . 9 6 3 . 8 ± 1 . 9 5 4 . 9 ± 2 . 1 4 2 . 9 ± 1 . 9 2 9 . 9 ± 1 . 9 7 9 . 4 ± 3 . 2 7 1 . 9 ± 4 . 4 6 0 . 3 ± 3 . 4 4 7 . 9 ± 3 . 3 3 4 . 9 ± 2 . 8 2 4 . 2 ± 2 . 5 8 3 . 4 ± 2 . 2 8 2 . 3 ± 1 . 6 7 8 . 1 ± 1 . 0 6 6 . 1 ± 1 . 4 5 3 . 2 ± 0 . 8 3 9 . 3 ± 1 . 3 3 2 . 0 ± 2 . 8 2 6 . 6 ± 2 . 1 1 9 . 1 ± 1 . 5 1 2 . 5 ± 0 . 9 7 . 9 ± 0 . 6 4 . 3 ± 0 . 4 3 7 . 9 ± 1 . 8 3 1 . 6 ± 1 . 4 2 4 . 0 ± 0 . 9 1 5 . 5 ± 0 . 7 8 . 7 ± 0 . 8 5 . 0 ± 0 . 9 3 5 . 1 ± 3 . 1 2 8 . 6 ± 3 . 2 2 3 . 6 ± 2 . 8 1 6 . 5 ± 2 . 0 1 1 . 4 ± 1 . 6 7 . 1 ± 1 . 1 4 0 . 4 ± 2 . 2 3 6 . 3 ± 2 . 3 2 6 . 8 ± 1 . 7 1 9 . 6 ± 1 . 3 1 4 . 5 ± 2 . 1 8 . 6 ± 1 . 0 NK cell activity of spleens from mice from the 4 experi-mental groups terminated 3 d following tumour-cell or vehicle injection and group formation. NK cell activity was determined by measuring the [5,Cr]-release from YAC target cells lysed by NK cells using the established protocol. a: n = number of animals terminated in each condition b: values are expressed as the mean ± S.E.M. 86 Table XIII. NK cell lytic activity at 1 wk Specific lysis (%) Tumour Vehicle E:T ratio 150:1 75:1 37.5:1 18:1 9:1 4.5:1 IG n=15" 51. 8b ±3.2 46.3 ±3.1 35.5 ±2.6 24.6 ±1.9 16.0 ±1.3 9.2 ±1.0 II n=6 59.6 ±5.8 49.0 ±4.4 37.8 ±4.5 26.2 ±3.8 17.2 ±3.2 10.0 ±2.4 GG n=9 49.8 ±3.4 44.6 ±3.2 36.1 ±3.2 24.7 ±2.4 16.4 ±1.5 8.4 ±0.7 GI n=5 60.9 ±8.5 54.8 ±7.1 42.5 ±6.7 32.9 ±6.6 21.6 ±4.6 13.2 ±3.2 IG n=l4 40.4 ±3.1 35.7 ±3.2 28.5 ±2.8 19.9 ±2.1 13.3 ±1.3 7.5 ±0.9 II n=6 33.6 ±1.6 29.2 ±1.5 24.1 ±1.8 14.9 ±2.2 10.0 ±1.3 6.6 ±1.1 GG n=8 35.0 ±4.6 32.2 ±4.3 25.3 ±3.6 18.2 ±2.7 12.6 ±2.0 7.2 ±0.8 GI n=6 33.4 ±6.2 28.2 ±5.1 21.7 ±4.0 15.4 ±3.0 10.6 ±2.3 5.5 ±1.2 NK cell activity of spleens from mice from the 4 experi-mental groups terminated 1 wk following tumour-cell or vehicle injection and group formation. NK cell activity was determined by measuring the [5ICr]-release from YAC target cells lysed by NK cells using the established protocol. a: n = number of animals terminated in each condition b: values are expressed as the mean ± S.E.M. 87 DISCUSSION A. INTRODUCTION This research project focused on the immune system and its role in differential tumour growth observed in our animal-tumour model used. This model is one which clearly and dramatically shows effects of social housing condition and exposure to novel environments on mouse mammary tumour growth. Both increases and decreases in tumour growth rates are evident. Being reared in the standard social housing group of 3 mice/cage and then singly housed (GI) or reared individually housed and remaining individually housed (II) following tumour-cell injection markedly increases tumour growth compared to that in mice reared and remaining in their same sibling social housing group (GG), provided animals are also exposed to acute daily novelty stress. In contrast, being reared individually and then transfered to a large social housing group of 5 non-sibling mice/cage (IG) following tumour-cell injection markedly reduces tumour growth compared to that in GG mice. Interestingly, this occurs both in the presence and absence of acute daily novelty stress (Weinberg & Emerman, 1989). In part, this model supports data from previous studies that suggest that tumour growth may be increased in individually housed animals compared to group housed animals (Sklar & Anisman, 1980). However, other studies show that an even greater 88 influence on tumour growth is the change in housing condition rather than just being individually housed (Dechambre & Grosse, 1973; Steplewski, 1987). They demonstrate an increase in tumour growth in animals that experience a change in housing condition compared to that in animals that do not, whether they are group housed or individually housed. In our model, however, the change in social housing condition does not appear to be as important in the modulation of tumour growth as the condition of being individually housed. Singly housed animals, either with (GI) or without (II) a change in housing condition, have an increased tumour growth, whereas the change from individual housing to social group housing reduces tumour growth compared to that in mice in the control group, which consists of social housing with no change (GG). At this time, however, it is difficult to pinpoint the critical parameters present in our animal-tumour model accounting for the differential growth observed. This is because of the built in asymmetry in the model, that is, the use of different group sizes, group compositions or sibling relationships, change versus no change as well as the familiarity of the group housed animals to each other. These different factors, any or all of which could have an influence on tumour growth, arose as a result of the initial experimental design - an effort to create conditions involving change (IG, GI) that were as different as possible from the groups that did not experience a change (II, GG). Despite the asymmetry, these conditions have contributed to the creation of a model that demonstrates both an increased and a decreased tumour growth rate in response to psychosocial factors. The initial goal, therefore, has been accomplished; now, the 89 significance of each of these variables in influencing tumour growth rate are being investigated by others in the laboratory. The psychosocial stress of housing has been reported to have different effects with differences in gender (Rabin et al., 1987; Steplewski, 1987), strain (Lyte, et al., 1990), housing density (Brain, 1975; Gamello, et al., 1986; Sklar & Anisman, 1980) and specific components of the immune system studied (Rabin, et al., 1987). Our model has a unique factor that seems to be critical in determining the extent of modulation of tumour growth. This is the acute daily novelty stress superimposed on the various housing conditions. Although there is differential tumour growth in mice in the different housing conditions, these differences are exaggerated if the mice are also exposed to an acute daily novelty stress. The exaggerated results from novelty stress may be related to the coping response to stress. A mediating factor in reducing tumour growth may be the frequent fighting observed in IG animals in our model. It has been suggested that fighting may provide a behavioural response that reduces arousal and enables animals to cope with stress (Weinber, et al., 1980). Sklar & Anisman (1980) found that tumour growth rate increases in animals changed from individual to group conditions only if fighting among group members does not occur. In addition to psychological mediation, fighting may directly affect physiological responses. Increased wounding that occurs in fighters may stimulate the immune and/or endocrine systems, which could play a role in reducing tumour growth in the IG group. 90 Conversely, the lack of opportunity for individually housed animals to fight, and therefore possibly less able to cope with stress, may be a mediating factor in increasing tumour growth in our model. Individual housing may have increased the impact of acute daily novelty stress in both the II and GI groups, resulting in an increase in tumour growth rate compared to that in GG controls. The complex relationship between stress and cancer likely involves several physiological mechanisms. Immune functioning was initially examined because host immunological status has been shown to be important in regulating SCI 15 growth (Nohno, et aL, 1986) as well as the fact that the immune system in general plays a role in regulating tumour growth and that stress can affect the immune system. General measures of immune response have been taken to examine the role of the immune system in mediating the effects of stress in our animal-tumour model. Spleens were weighed, antibody production determined and the mitogen-induced proliferation of spleen cells was determined. The latter formed part of this thesis. B. T-LYMPHOCYTE PROLIFERATION IN RESPONSE TO CON A. A general measure of cell-mediated immune responses is the proliferative response of spleen cells to the mitogen Con A. The method used in this thesis was based on previously described procedures of [3H]TdR incorporation into DNA as a measure of 91 proliferation (Bradley, 1980). These results did indicate that housing condition has some differential effects on spleen cell proliferation in vehicle-injected animals, but differences in response are eliminated in animals injected with tumour cells, at least 3 wk following tumour-cell injection. It is likely that the presence of a tumour may have suppressed immune functioning beyond the point that would permit modulation by psychosocial factors. In our model, humoral responses have been shown to be suppressed by tumour at 3 wk (Weinberg & Emerman, 1989), and this suppression phenomenon has been previously noted in measures of humoral response as well as cell-mediated immunity (Coe, et al., 1988, Esterling & Rabin, 1987). The immune response of mice in the different housing groups may well be suppressed by tumour to varying degrees at earlier time points. Other studies indicate that tumour growth is influenced by changes in the endocrine and/or immune systems initially, but that these changes are no longer evident at 3 wk (Coe, et al., 1988; Greenberg, et al., 1984; Riley, 1981; Steplewski & Vogel, 1986; Steplewski et al., 1985). The IG animals appeared to have the best humoral response, although this was in contrast with measures of cell-mediated responses, where the IG vehicle-injected animals appeared to be the most immunosuppressed. This could perhaps be a result of an earlier exposure to higher levels of corticosteroids. Corticosteroid has long been known as a general suppressor of immune function (Riley, 1981). On the other hand, immune parameters may not have any bearing on the mechanisms mediating the differential growth of this tumour in mice in the different housing conditions. 92 Although the spleen cell assay is a good measure of general immune reponsiveness, it involves several subsets of cells responsible for the cell-mediated immune reponse. If the psychosocial stress is altering one subset or ratios of subsets, that would in turn affect tumour growth, this alteration would not be detected in the spleen cell assay (Sei, et al., 1991). Therefore, more specific immune responses must be looked at, at timepoints that may be more relevant. Natural killer cells are currently thought to be one of the first lines of defense against tumour cells and are active early in the antitumour response (Haller, et al., 1977; Herberman & Ortaldo, 1981; Wei & Heppner, 1987). The in vitro assay used to measure the activity of these cells was adapted from Kiessling, et al. (1970) and Kraut & Greenberg (1986). C. NATURAL KILLER CELL ACTIVITY 1. Modification of the fCrJ-Reiease Assay The [5,Cr]-release assay to measure NK cell activity (Kiessling, et al.,1970; Kraut & Greenberg, 1986) was optimized for our DD/S strain of mice. The growth characteristics of the target cells (YAC) were investigated, as it was necessary to obtain cultures growing in the exponential phase for sufficient uptake of radioactive chromium 93 and low spontaneous release. YAC cells were cultured and subcultured for a total of 10 days. It was established that to have the cells in a state of exponential growth, cultures should be initiated at a concentration of 6 - 7 x 104 cells/ml approximately 24 h prior to an assay. This reliably gave enough cells in the exponential state of growth for both good incorporation of radioactive label (minimum of 0.1 counts/cell, although the more it approached 1 count/cell the better) and low spontaneous release of that label (not to exceed 20%). Lysis of YAC target cells with spleen cells from our DD/S mice was achieved. The target cell labelling procedure utilized a 1.5 h labelling period. An appropriate amount of [^Cr] and appropriate effector to target cell ratio were determined. Experiments indicated that specific lysis of YAC cells obtained from spleen cells from DD/S mice was low and the variability among the animals was high. This led to the concern that it may not be possible to detect any significant differences among the groups. To determine if this low responsiveness was a characteristic of the DD/S mouse strain or if the assay protocol was not sensitive enough, the NK cell activity of 3 known responder mouse strains was tested. These included the strains C57B1-6, CBA and Balb/c (Kiessling, et al., 1975), in addition to DD/S mice. The results show that the DD/S strain did appear to have specific NK cell activity, and this not substantially less than other strains, although the assay produced variable results. Lysis had to be increased to be significant and perhaps this would reduce variability. Moreover, increasing the lysis 94 might aid in identifying any differences in the NK cell activities of mice raised in the various housing groups. Increasing NK cell activity was achieved by boosting the activity in vitro in the following manner. The ratio of effector to target cells was increased to 300:1, the length of the assay incubation time was increased to 6 h and the red blood cells were lysed with Tris NI^Cl prior to passaging the cell suspension through nylon wool columns. Preliminary data using this modified assay suggested lysis would be high enough to demonstrate differences in NK cell activity among animals in the 4 housing conditions, if differences existed. The specific release obtained was in the range of many other studies in the literature (Brunner, et al., 1968; Cikes, et al., 1973; Kiessling, et al., 1975; Kraut & Greenberg, 1986). 2. Timecourse Study of NK Cell Activity NK cell activity was assayed in spleens of mice from the 4 housing groups, with and without tumour, at 3 d and 1 wk post-injection and rehousing. NK cell activity is stimulated by the injection of SCI 15 tumour cells. This is the first demonstration that SCI 15 tumours are capable of stimulating NK cells. This stimulation follows a definite time course. The activity of the NK cells appear to be maximal at 3 d and begins to decline at 1 wk. Preliminary data from others in the lab of the 24 h timepoint suggested that NK cell activity is lower than at 3 d or 1 wk and that tumour-cell injected and 95 vehicle injected groups do not differ at 24 h. In addition, data obtained from modifying the NK assay revealed that at 3 wk, there is no difference in NK cell activity between tumour-cell and vehicle injected groups (Table XI, p. 79). Thus, the stimulation appears to have fully declined by 3 wk. Inducers of NK cell activity such as IFN maximally stimulate NK cell activity 18 - 24 h following administration of exogenous IFN (Ortaldo et al., 1989; Talmadge et al., 1985). However, the maximal stimulation that is observed in this thesis at 3 d is not unexpected, since the immune system must first recognize the tumour and then respond with the production of lymphokines. This phenomenon has been demonstrated in other forms of immune stimulation. Lymphokine-activated killer (LAK) cells are produced by co-culturing human peripheral blood lymphocytes with IL-2 (Ben-Aribia, 1987). After 3 or more days of proliferation in vitro, LAK cells are able to lyse fresh or cultured solid tumour cells usually insensitive to classical NK cell killing. In addition, augmentation of NK cell activity is maximal at 3 d following an injection of virus or bacterial cells (Klein & Kearns, 1989; Savary & Lotzova\ 1987). Other studies have shown tumour-induced stimulation of splenic NK cell activity in the same range of time and even up to 21 days post-injection (Hoffman-Goetz, et al., 1992; Lala, et al., 1985; Maccubin, et al., 1989; Pollack, et al., 1984; Wei & Heppner, 1987). The decline in NK cell activity seen in tumour-cell injected animals at 1 wk may be due to tumour-mediated suppression of NK cell activity. The tendency of NK cell stimulation to decline with time or the actual 96 suppression of NK cells by tumours is also a well documented phenomenon (Hartmann, et al., 1986; Fulton, 1987; Savary & Lotzova\ 1987; Wei & Heppner, 1987). SCI 15 tumour cells injected into male mice raised under our standard laboratory conditions (groups of 3 siblings, GG) produce a palpable tumour within 6 - 8 days, which grows to mass of 2 - 3 g in approximately 3 weeks. Growth rate of the SCI 15 tumour may be increased or decreased by housing mice under different conditions (Weinberg & Emerman, 1989). Tumour growth rate is increased in GI and II mice and decreased in IG mice compared to that in the standard GG condition. We had hypothesized that the psychosocial stressor of differential housing may have affected the immune systems of the mice in the various groups to produce the variable tumour growth rates. In this study an effect of differential housing on NK cell activity in the 4 experimental groups has been demonstrated. At 3 d, NK cell activity is markedly stimulated in all tumour-cell injected mice. GI animals injected with tumour cells have the greatest NK cell activity, whereas in mice that are injected with vehicle alone, the groups do not differ from each other. At 1 wk, although NK cell activity is still stimulated to some extent in tumour-cell injected mice, mice in the 4 experimental housing groups do not differ from one another. As it has been shown that stress depresses NK cell activity (Greenberg, et al., 1984; Kandil & Borysenko, 1987), we hypothesized that GI and II animals, which have 97 the largest tumours, would have decreased NK cell activity and that IG animals, which have the smallest tumours, would have increased NK cell activity. Surprisingly, it was found that at 3 d post-injection, when NK cell stimulation is the greatest, GI animals have greater NK cell activity than IG animals. Further GI and II animals do not have similar NK cell activity at this time. One explanation for these results is that NK cell activity in the spleen does not accurately reflect the activity of NK cells at the tumour site. A number of experimental findings support this possibility. Antigen-stimulated lymphocytes and macrophages have been shown to release substances that are capable of recruiting other lymphocytes, including NK cells, from the circulation (Greenberg, et al.,1986; Migliorati, et al., 1987; Zangermeister-Wittke,et al., 1989). Studies of tumour-infiltrating lymphocytes in preneoplastic mouse mammary lesions, show that NK cell activity at the tumour site is higher than in the spleen (Wei & Heppner, 1987). In addition, it has been suggested that in solid tumour models the ability of NK cells to infiltrate their target is as important as the lytic ability of the immune cells themselves (Jaaskelainen et al.,1989). Thus, although splenic NK cell activity 3 d post injection is greater in GI than in IG mice, it is possible that because of differences in recruitment or infiltrative abilities, NK cell activity at the tumour site may be greater in IG than in GI mice. Alternately, NK cell activity may not be a significant regulator of SCI 15 tumour growth, although it is difficult to believe that the stimulation observed is merely a 98 byproduct of the activation of a different pathway. The difference in NK cell activity between the II and GI groups, which both develop large tumours, may be due to the factors discussed above or could indicate that different mechanisms may be regulating tumour growth in these two groups of mice. It is interesting to note another difference between NK cells in the spleen of IG and GI groups. Close examination of the data reveals that when both are maximally stimulated at 3 d, although the activity in GI mice is overall greater than IG mice, this is not true for the highest ratio. The activity in IG mice is greatest at 150:1 and then the curve drops sharply (almost a straight line on log paper) to well below the GI level. However, examination of the GI curve reveals a tapering off at the higher ratios, almost as if a "saturation point" had been reached. One possible explanation for this, is that there is a qualitative as well as quantatative difference between the NK cells in the spleens of the various housing groups. It could be that in the IG group, there is a small proportion of NK cells present in the spleen, but these cells are more highly efficient killers. In contrast, the NK cells present in the spleens of the GI groups could be less activated, although a higher proportion could be present. Differences in quantity could be tested by immunofluorescence staining, using an NK marker such as asialo GM, (Kasai,et al., 1980). Qualitative differences are harder to test, although it is possible to perform single-cell kinetic assays which may provide some answers. On the other hand, this phenomenon may have no significance. In vivo studies may provide more direct answers as to the role of NK cells in the differential tumour growth observed. Such 99 studies would involve either stimulation of NK cell activity with an agent such as Poly I:C, or abolishment of NK cell activity using antibodies directed towards NK cells. Studies of tumour-infiltrating lymphocytes will also provide data as to what is occuring in vivo at the tumour site. These studies are currently in progress in our laboratory. It would be interesting to determine what subsets of lymphocytes are present at the tumour site as well as their activity, and if they are altered between the various housing groups. Studies have shown that biologically significant alterations in numbers of lymphocyte subsets may occur without changes in total number of lymphocytes (Stein, et al., 1991). The relationship between the neuroendocrine and immune systems is well documented (Besedovski, et al., 1985; Blalock, 1989; Plaut, 1987; Rabin, 1989), with NK cells included in this network (Sibinga & Goldstein, 1988; Kraut & Greenberg, 1986). Measurement of neuroendocrine peptide hormones both in vivo and in vitro would reveal more of this complex pathway. It also must be considered that the psychosocial stress of housing is affecting tumour development in ways other than via the immune system. The growth of some tumours is directly affected by hormones, such as prolactin, or by opioid peptides that are released by stress (Welsch & Nagasawa, 1977; Zagon & McLaughlin, 1986). The SCI 15 tumour itself is known to respond to androgens and glucocorticoids (Omukai et al., 1987; King & Yates, 1980) and these hormones are known to be released during times of stress (Armario & Castellanos, 1984; Christian & Davis, 1964; Frankel & Ryan, 100 1981). It is possible that changes in plasma levels of these hormones may be responsible for the altered tumour growth rates observed in this model. Previously, it was found that at 3 wk post-injection, levels of testosterone, dihydrotestosterone and corticosterone are not significantly different among mice from the 4 housing conditions. However, as with the immune system, functionally important changes in the endocrine system may occur within the first wk post tumour-cell or vehicle injection and rehousing, and return to normal by 3 wk (Frankel & Ryan, 1981; Peng, et al., 1989). This hypothesis is currently being tested using peripheral blood taken from the mice used in this NK cell timecourse study. In summary, data from the spleen cell proliferation assay reveals that, at 3 wk, differential effects of housing on tumour-cell injected animals is not observed, although overall proliferative activity is suppressed compared to vehicle injected controls. 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