2g-\\-9lc Lymphocytic 5' -Ectonucleotidase: A marker of psychological stress-induced immune suppression Jane Blake-Mortimer B.A. (Hons). M.Psych. (Adelaide) Thesis submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Department of Psychology The University of Adelaide July, 1996 Table of Contents Page List of Tables vlll List of Figures x Abstract xll Author's Statement xiv Acknowledgements xv Chapter 1: General Introduction 1 1.1 General overview 1 1.2 Definition of stress 2 1.3 Historical development of the field of 2 P sychoneuroimmunology t.4 The immune system: An overview 6 1.4.r Factors involved in the immune response 6 r.4.2 Innate immunity: The immediate response 6 r.4.3 Acquired immunity 9 r.4.4 T helper cell activation 10 1.4.5 The cell mediated response i0 t.4.6 The humoral response 11 1.5 The interactions between the neuroendocrine 15 and lymphoid systems 1.6 Methodological and conceptual issues 2T 1.6 i Defi nition of depresston 21 1 6.2 Characteristics of studies that have bedn 22 conducted to investigate the relationship between depression and immunity (i) Experimental design 22 (ii) Heterogeneityofdiagnosticclassification 22 (iii) Sampling 23 (iv) Severity of depression and hospitalisation 24 status ll Table of Contents Cont'd Page 1.6.3 Characteristics of studies that have been 24 conducted to investigate the relationship between psychological stress and immunity (i) Definition of stress and conceptual 24 problems (ii) Experimental design 25 (iii) Sampling 25 (iv) Lack of standardisation in the 26 stressors used and psychological measures (v) Methodological limitations of PNI 26 tests 1.7 A critique of the current tests commonly used 27 in PNI in humans t.7 .I Enumeration studies 27 t.7.2 Lectin or mitogenic responses 34 r.7.3 Natural killer cell activity 4I r.7 .4 Interleukins, enzymes, proteinases, acute phase 45 proteins and DNA expression during major depression 1.7 .5 Antibo dy (immuno globulin) measures 45 1.8 The effects of stress on humoral and cell-mediated 50 immunity in animals 1.8. I The learned helplessness model of depression in 50 animals 1.9 Summary 51 Chapter 2: Lymphocytic 5'-ectonucleotidase: A marker 52 of stress-induced immune suppression in humans 2.1 Introduction: Rationale for selecting 52 lymphoq'tic 5' -ectonucleotidase 22 Experiment 1 53 2.2.1 Method 54 (Ð Subjects 54 (ii) Materials 55 (iii) Procedures 55 lll Table of Contents Cont'd Page 222 Results 57 (i) Demographic data 57 (iÐ Psychological variables 57 (iii) NT results 63 (iv) Relationship between NT and 66 psychological variables 2.3 Experiment 2 68 2.3.r Method 68 (Ð Subjects 68 (ii) Materials 69 (iii) Procedures 69 2.3.2 Results 70 (i) Demographic data 70 (ii) Psychological variables 72 (iii) NT results 73 2.3.3 Discussion 77 Chapter 3: Evidence for free radical-mediated reduction 80 of lymphocytic 5'-ectonucleotidase during stress 3.1 Introduction 80 3.2 Experiment 3 80 3.2.1 Method 81 (i) Subjects 81 (ii) Materials for laboratory tests 82 (iii) Psychological tests 82 (iv) General procedures 82 (u) Procedure for ascorbate assay 82 3.2.2 Results 83 (i) Demographic data 83 (iÐ Psychological variables 85 (iii) NT activities 86 (i") Ascorbate results 86 (v) Relationship between NT, psychological 89 variables, ascorbate and blood cell counts lv Table of Contents Cont'd Paee J.J Experiment 4: In vitro study on NBT 9l 3.3.1 Materials and method 91 (i) Reagents 91 (iD Procedure 92 (iiÐ Inhibitor concentrations used in oxyradical 92 tests (iv) In vitro study on NBT: Effects on NBT and 93 xanthine oxidase 3.4 Experiment 5: In vitro study on NT 97 3.4.1 Method 97 (Ð Subjects 97 (iÐ Materials 97 (iii) Procedure 97 3.4.2 Results of in vitro study on NT 98 (Ð Psychological data 98 (ii) NT and antioxidant data 99 3.5 Discussion 101 Chapter 4: The effect of depression in an animal 704 model on 5'-ectonucleotidase, antibody production and tissue ascorbate stores 41 Introduction i04 4.2 Experiment 6 r04 4.2.1 Method 105 (i) Subjects 105 (ii) Pretreatment aPParatus 106 (iiD Test task apparatus 108 (i") Pretreatment procedure 110 (v) Test task procedure 11i (vi) Materialsforimmunological,antioxidant tt2 and antibody assays (vii) Procedure for ascorbate assay 112 (viii) Procedure for rat antibody assay t12 (i") NT assay in rats 1i3 4 2.2 Results 115 (i) Behavioural variables 115 (ii) NT results 119 (iii) Antibody results 119 (iu) Ascorbate results 122 4.2.3 Discussion t24 v Table of Contents Cont'd Page Chapter 5: General conclusions and directions t26 for future research 5.1 A potential mechanism 126 5.2 Future directions for research into 129 this mechanism (Ð Future directions for human research 129 (iÐ Future directions for animal research 131 5.3 Limitations of the experiments reported in this 132 thesis 5.4 Summary t33 List of Appendices 135 Appendices 136 References 151 vl List of Tables Page Table 1 1 Factors involved in the immune response and their functions 7 Table 7.2 The source and function of the major cytokines in the 9 immune response Table 1.3 Summary of studies of enumeration studies and major 30 depression Table 1.4 Summary of studies of enumeration and chronic stress 31 Table 1.5 Summary of studies of enumeration and acute stress 32 Table 1.6 Summary of studies of enumeration and acute laboratory 33 StreSS Table 1.7 Summary of studies of lectin responses and major depression 36 Table 1.8 Summary of studies of lectin responses and chronic stress 37 Table 1.9 Summary of studies of lectin responses and acute stress 38 Table 1.10 Summary of studies of NKCA and major depression 42 Table 1.1 I Summary of studies of NKCA and chronic stress 43 Table I.I2 Summary of studies ofNKCA and acute stress 44 Table 1.13 Summary of studies of psychological stress, personality 47 characteristics and serum antibody levels Table 1.14 Summary of studies of psychological stress, personality 48 characteristics and salivary IgA Table 1. 15 Summary of studies of psychological stress and antibody 49 titres Table 2.1 Experiment 1: Demographic data for the non-clinical study 59 Table 2.2 Experiment 1: Means and standard deviations of the POMS 60 variables for the groups in this study Table2.3 Experiment 1: Means and standard deviations of the 6l STAI-trait scores for the groups in this study Table 2.4 Experiment 1: Correlations of NT values with POMS 66 variables Table 2.5 Experiment 2:Demographic data for the clinical study 71 vll List of Tables Page Table2.6 Experiment 2: Means and standard deviations of 72 psychological variables for the groups in this study Table 3.1 Experiment 3:Demographic data 84 Table3.2 Experiment 3: Means and standard deviations for 85 psychological variables for the groups in this study Table 3.3 Experiment 4: Inhibitor studies on NBT reduction 95 by the superoxide anion Table 3.4 Experiment 5: Scores of psychological variables 98 for subject A and subject B vlll List of Figures Page Figure 1.1 Antigen processing and T helper cell activation Figure 1.2 The cell mediated immune response Figure 1.3 The humoraVantibody response Figure !.4 The pathways through which behaviour, the nervous system and the immune system interact Figure 1.5 A homeostatic mechanism operating between the immune system and the hypothalamic pituitary axis Figure 2.1 Experiment i. The group by time interaction of the Total Mood Disturbance scores Figure2.2 Experiment 1: Means and standard deviations for the non-clinical study Figre 2.3 Experiment 1: The group by time interaction of NT activities Figure 2.4 Experiment 1: The correlation between NT and Total Mood Disturbance scores Figure 2.5 Experiment 2. Means and standard deviations of NT activities in the clinical study Figure 2.6 Experiment 2: The correlation between NT and Beck Depression Inventory scores Figure 2.7 Experiment 2. The correlation between NT and State Trait Anxiety Inventory scores Figure 3.1 Experiment 3:Means and standard deviations ofNT activities for the groups in this study Frgure 3.2 Experiment 3: Means and standard deviations of ascorbate values Figure 3.3 Experiment 3: The correlation between NT and ascorbate values Figure 3.4 Experiment 4: The rate of reduction ofNBT to formazin by the superoxide anion Figure 3.5 Experiment 4: A dose response curve indicating the individual and combined effects of zinc and ascorbate on inhibiting the reduction of NBT by the superoxide anion lx List of Figures Page Figure 3.6 Experiment 5: The effect of the superoxide anion on NT in 100 subject A and subject B Figure 4.1 Experiment 6: The apparatus used in the pretreatment task t07 Figure 4.2 Experiment 6: The apparatus used in the test task 109 Figure 4.3 Experiment 6: Complement-mediated lysis of SRBC in the 114 presence of serum containing antibodies against SRBC Figure 4.4 Experiment 6: Mean shuttle escape latencies and standard 116 deviations for FR-1 and FR-2 trials in the acute condition Figure 4.5 Experiment 6: Mean shuttle escape latencies and standard 1i8 deviations for FR-1 and FR-2 trials in the chronic condition Figure 4.6 Experiment 6: Means and standard deviations of NT t20 activities for the groups in this study Figure 4.7 Experiment 6. Means and standard deviations of antibody T2T responses to SRBC for the groups in this study Figure 4.8 Experiment 6: Means and standard deviations of t23 ascorbate levels for the for the groups in this study Figure 5.1 Two homeostatic mechanisms (circle A and circle B) operating 728 to control immunity x ABSTRACT Lymphocytic 5'-Ectonucleotidase: A marker of psychological stress-induced immune suppression The aim of the studies reported in this thesis was to find a mechanism to explain the well known phenomenon of stress-induced immunosuppression.