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Presents abstracts of recently published studies related to psychosocial factors, stress, and cardiovascular heath. "Relationships Between Cardiovascular and Immunological Changes in an Experimental Stress Model," by R.J. Benschop, G.L. Godaert et al; "Pressor reactions to psychological stress and prediction of future blood pressure: data from the Whitehall II Study," by D. Carroll, G.D. Smith, D. Sheffield, M.J. Shipley and M.G. Marmot; "Identification of a cortical site for stress-induced cardiovascular dysfunction," by D.F. Cechetto; Others.

Previously we demonstrated that stressors varying on the dimension of mental effort and controllability have distinctive effects on cardiovascular, endocrine and immune system responses. The purpose of the present study was to relate individual differences in physiological stress responsivity to task appraisal and stress-induced mood changes (issue 1), trait characteristics (issue 2) and daily hassles (issue 3). Appraisal and mood changes did not mediate the differential effects of the stressors. The trait characteristics, aggression and external locus of control and daily hassles moderated the effect of the stressor on physiological parameters, especially immune parameters. Moreover, the moderation effect was different in the high versus the low effort stress task. High aggression, high external locus of control and more daily hassles were associated with increased reactivity in the low effort condition and decreased reactivity in the high effort condition, which is suggested to reflect less differentiated responding to changing task demands and hence, less flexibility in the immune system.

Objective: Two important determinants of physiological stress responses have been identified, uncontrollability of the stressor and amount of effort involved in coping with the stressor. In the present experiment, we tried to identify the specific contributions of effort and uncontrollability to immune system responses to stress., Methods: In a 2 x 2 design, effort and uncontrollability were manipulated independently of each other. Subjects participated in one of four experimental conditions, and their endocrine, immune, and sympathetic nervous system responses to the task were assessed., Results: Effort had a stimulating effect on enumerative immunological parameters (CD8 and CD16+ cells) and on natural killer cell activity. The effect occurred immediately after the stressor and was transient. Regression models indicated that this effort effect may have been mediated by activation of the sympathetic nervous system. Uncontrollability influenced in vitro production of the cytokine interleukin-6, leading to decreased production 15 and 30 minutes after the stressor. Uncontrollability also led to an increased level of cortisol, but no evidence was found that the decrease in cytokine production was mediated by cortisol release., Conclusion: The results suggest that two major stressor characteristics, effort and uncontrollability, may have differential effects on the immune system.

Objective: Although stressor uncontrollability has been shown to suppress immune responses in animals and for human subjects, the results have been inconsistent. We reanalyzed results of our previous study regarding stress-related immune deviation in man, to establish whether perceived uncontrollability of an acute stressor acts as a co-determinant in the observed changes in immunological parameters., Method: Three types of cognitive reactions to an acute interpersonal stressor were assessed: "motivation," "uncontrollability," and "guiltiness." Stress-induced changes in the number of several types of immune cells in peripheral blood and proliferative responses of lymphocytes to antigens and mitogens were assessed., Results: In comparison with control subjects and with subjects perceiving high control over the experimental stress situation, the subject perceiving low control showed a stressor-induced decrease in the number of T helper cells. Reversely, subjects perceiving high control showed an increase in the number of B cells as opposed to the other two groups. The effects of perceived uncontrollability could not be accounted for by mood changes, but they were related to previously experienced life stress., Conclusions: Perceived uncontrollability of an acute stressor can have immuno-modulating effects over and above those of the stressor per se.

The objective of the study was to investigate the unique and interactive effects of the controllability of a task and mental effort required by that task on cardiovascular and endocrine reactivity, when both were manipulated independently. A 2 x 2 factorial design was used, with two levels of mental effort and two levels of control. Twenty-four healthy male subjects participated in each experimental condition. Heart rate, blood pressure, catecholamine and cortisol responses were determined. High effort lead to greater increases in heart rate, blood pressure and norepinephrine levels. Uncontrollability lead to higher cortisol, blood pressure and norepinephrine responses. In addition, there was an effort x control interaction effect on the diastolic blood pressure response. In conclusion, effort has clear sympathetic effects, whereas control influences both the sympathetic nervous system and the release of cortisol. Having control seems to be most beneficial in high effort situations, at least with respect to sympathetic reactivity.

In a previous study it was shown that antibody formation after vaccination with a low-dose recombinant DNA (rDNA) hepatitis B vaccine was negatively influenced by psychological stress. The present study was designed to assess whether the same inverse relation between HBs-antibody levels and psychological stress could be observed, while administering the standard, and thus higher, dose of vaccine. Volunteers (n = 68) scoring extremely low or high on a combination of questionnaires measuring daily problems and psychoneurotic symptoms were selected for participation. Antibody levels were determined 2, 6, and 7 months after the first vaccination. Questionnaires were completed before entering the study and at month 6. In contrast to the previous study, psychological stress was not found to be related to the antibody levels at any timepoint. These results suggest that, under certain conditions, stress-induced immunomodulation in vivo might be dependent on antigen dose.

To investigate the relationships between cardiovascular variables (SBP, DBP, and HR) and circulating natural killer (NK) cell numbers, 70 male volunteers were subjected to a rest condition (N = 30) or a stressful laboratory task (N = 40). At baseline, no significant relationships could be demonstrated between the number of NK cells and the cardiovascular variables. Analysis of covariance showed that the stressor induced increases in the number of NK cells, SBP, DBP, and HR. Changes in Nk cell numbers were highly correlated to changes in cardiovascular variables in both the task and the no-task group. These results indicate that there is no relationship between the number of circulating NK cells and cardiovascular levels per se, but that changes in these variables, either stress-induced or under rest conditions, are regulated by a common mechanism.

We examined whether habitual defense and coping affect the response of hormones (ACTH. cortisol, prolactin. endorphins, and noradrenaline) and immune parameters (numbers of T cells. B cells. natural killer [NK] cells, and proliferative responses to mitogens or antigens) to an acute laboratory stressor (i.e., solving a 3-dimensional puzzle and explaining it to a confederate) in 86 male high school teachers. Defense and coping were assessed by Kragh's tachistoscopic Defense Mechanism Test (a measure of perceptual defense) and by 4 questionnaire-based coping styles assessing instrumental mastery-oriented coping, emotion-focused coping, cognitive defense, and defensive hostility. The laboratory stressor per se caused a relative increase in immunological (in particular NK cells) and endocrine (cortisol, prolactin) parameters. Defense and coping, however, significantly codetermined the response to the stressor. In particular, instrumental mastery-oriented coping and perceptual defense were related to stress-induced changes in numbers of B cells and in the pituitary-adrenal hormones. The results indicate that the impact of a mild psychological stressor on the immune and endocrine system depends to a considerable extent on the specific ways people deal with stressors.

Stress-induced brain-mediated immunoregulation is effected by two pathways: autonomic outflow and (neuro)endocrine outflow. Particular attention is given to the interaction-effects of chronic an acute stress. Recent data have established that cells of the immune system produce neuro-peptides and hormones. In concert with cytokines released by these immune cells the brain can be informed on the nature of ongoing immune activity. The significance of conditioning of immune responses is discussed.

1. The circulation of natural killer (NK) cells in vivo is influenced by physical exercise, mental stress, and infusion of beta-adrenoceptor agonists. We have previously presented in vitro data, showing that beta 2-adrenoceptor agonists induce detachment of NK cells from endothelial cells (EC), supporting the hypothesis that NK cells can be recruited from the marginating pool in blood vessels. 2. Because NK cells as well as EC express beta 2-adrenoceptors, the present study was conducted to investigate whether stimulation of the beta-adrenoceptors on NK cells, EC or both cell types is required to induce detachment from EC. 3. Cells were pretreated (15 min) with a selective beta 2-adrenoceptor antagonist, GR81706, at various concentrations. The duration of beta-adrenoceptor blockade was tested by determining the adenosine 3',5'-cyclic monophosphate (cyclic AMP) production induced by terbutaline (a beta 2-adrenoceptor specific agonist). This receptor-mediated response was effectively inhibited for at least 4 h, whereas the cyclic AMP production in response to forskolin (a direct activator of adenylate-cyclase) was not affected. 4. Functional adhesion assays were then performed to determine the role of beta-adrenoceptors on the different cell types involved (NK and EC) in catecholamine-induced detachment. Peripheral blood mononuclear cells were allowed to adhere for 1 h to monolayers of unstimulated EC in the presence or absence of cyclic AMP inducing agents, and the percentage of NK cells in the adhering lymphocyte fraction was determined by flow cytometry. 5. Both adrenaline (10(-5) M) and forskolin (10(-5) M) caused detachment of NK cells from EC. After blockade of the P2-adrenoceptors on NK cells by pretreatment with GR81706 (10-6 M), the effect of adrenaline on NK cells adhesion was pretented; after blockade of the beta2-adrenoceptors on EC, NK cell adhesion was still significantly reduced by adrenaline. In all cases, forskolin caused detachment of NKcells.6. To establish further that stimulation of beta-adrenoceptors on NK cells is sufficient to cause detachment,we showed that adrenaline also reduced adhesion of NK cells to monolayers of Chinese hamster ovary cells, which do not express beta-adrenoceptors.7. Together, these results show that stimulation of beta2-adrenoceptors on NK cells negatively influences their capacity to adhere to EC, and that beta2-adrenoceptors on EC play a negligible role in this phenomenon.

This study investigated the effects of self-reported life stress and locus of control on reactivity of several immune parameters to a mild and short-lasting interpersonal stressor. Subjects were 86 male teachers aged 24 to 55 years. Immune reactivity was defined as changes in numbers of monocytes. T-lymphocytes and subsets, HLA-DR+ cells, and NK cells as well as changes in (in vitro) proliferative responses of peripheral blood lymphocytes to the antigens PHA and PWM. Multiple regression analysis was used to study the interaction effects of life stress and locus of control by experimental condition on immune reactivity. Life stress, but not locus of control, influences reactivity of the immunological parameters to the stressor. In particular, high numbers of daily hassles were associated with stressor-induced decreases in numbers of T cells and NK cells in peripheral blood. On the other hand, numbers of HLA-DR+ cells in high life stress scorers decreased only slightly during the stressor, whereas they increased in the control condition. The findings suggest that accumulated life stress is related to reactivity of immunological parameters to subsequent experimental stress. Possible physiological explanations and implications of these effects are discussed.

Background: Acute mental stress evokes responses in the cardiovascular and the immune systems. In particular, the subset of natural killer (NK) cells is found to be responsive to mental stress. The role of beta-adrenergic mechanisms in these processes in the subject of this investigation., Methods and Results: Healthy male volunteers (n = 31) were subjected to two consecutive mental tasks. Subjects were randomly assigned to a beta-blocker (propranolol 40 mg) or a placebo group. The capsules were ingested 1 hour before the tasks. The tasks evoked sympathetic responses, as indicated by an increase in heart rate and a decrease in the preejection period. These effects were abolished under beta-blockade, indicating that effective beta-blockade was achieved. In the immune system, significant increases were found for the number of NK cells and NK cell activity in the placebo group; these increases were absent in the propranolol group. In addition, an increase in all lymphocyte subsets was observed in subjects who had ingested propranolol. This increase, however, was also observed in subjects who had received propranolol but had not performed the tasks, indicating that these non-subset-specific increases in lymphocytes were a side effect of the beta-blocker., Conclusions: Mental stress induces activation of the sympathetic nervous system, with concomitant increases in the number of NK cells in the circulation. These changes were inhibited by propranolol, indicating that stress-induced increases in the number and activity of NK cells in the circulation are controlled by a beta-adrenergic mechanism.

This study deals with the effect of chronic stress on physiological responsiveness to an acute psychological stressor in male high school teachers. Chronic stress was operationalized as the self-reported number of everyday problems. Twenty-seven subjects reporting extremely low or high numbers of everyday problems were exposed to an acute psychological stressor, and changes in immunologic, endocrine, and cardiovascular parameters were monitored. The stressor included a learning process followed by a teaching session in which a confederate to the researchers was involved. Twenty subjects served as controls. The stressor had no effect on the endocrine variables measured but induced increases in heart rate and blood pressure, which were similar in both chronic stress groups. Analysis of subsets of blood lymphocytes revealed differences in natural killer (NK) and T cell responses in the low and the high stress groups. It is concluded that 1) immunologic responsiveness to an acute psychological stressor is related to problems experienced in daily life, and that 2) chronic stress differentially modifies the sensitivity of biological systems to mild acute stressors.

Physical exercise, mental stress, or infusion of beta-adrenergic agonists result in an increase in the number of natural killer (NK) cells in the peripheral circulation. In view of the specific migration pattern of NK cells in vivo, it has been suggested that these cells may be released from the marginating pool in blood vessels. In the present report, the in vitro effect of catecholamines on the adhesion of NK cells to unstimulated human endothelial cells (EC) was characterized. Peripheral blood mononuclear cells were allowed to adhere to monolayers of EC, after which the adherent lymphocyte fraction was analyzed phenotypically by flow cytometry. NK cells were found to adhere preferentially to EC, a process that was reversed by the addition of various adrenergic agonists. Catecholamines selectively affected adhesion of NK cells and had no effect on T cell adhesion to EC, as was determined by the use of purified cell populations. Detachment of NK cells from EC could be achieved by short incubations (5 min) with epinephrine (EPI) and was concentration-dependent, with an ED50 of 2 x 10(-10)M. Using a panel of alpha- and beta-adrenergic agonists and antagonists, we show that the detachment of NK cells is mediated via beta 2-adrenergic receptors. In line with the lower affinity for beta 2-adrenergic receptors, norepinephrine was less effective than EPI in inducing detachment of NK cells from EC. Direct activation of adenylate-cyclase with forskolin gave similar results as observed with EPI, indicating that signaling through cAMP is necessary to induce detachment of NK cells from EC. The results of the present study lend support to the hypothesis that catecholamines, via beta 2-adrenergic receptors, can induce recruitment of NK cells from the marginating pool to the circulating pool, by changing the adhesive interactions between NK cells and EC.

The present study focused on the relationship between psychological stress and immune reaction to a novel antigen. Participants completed questionnaires on daily hassles, psychoneurotic complaints, coping style, and loneliness, 2 and 6 months after the first of a series injections with a low dose recombinant DNA hepatitis B vaccine. Antibody response was determined 7 months after the first vaccination. Based on the psychological questionnaires two different stress measures were calculated: a Stress Index score-month-2 and a Stress Index score-month-6 indicating stress levels experienced at the beginning and at the end of the study respectively. Antibody levels were found to be negatively related with the Stress Index score-month-2. Although the influence of psychological stress reported on month 6 tended to be in the same direction, this effect was not significant. Coping styles and loneliness were not associated with antibody formation. These results suggest that antibody formation to rDNA hepatitis B vaccine is negatively influenced by psychological stress.

The present report investigated whether percentages of circulating natural killer (NK) cells and NK cell activity (NKCA) are associated with psychological variables. Subjects (n = 95) were selected, based on a combination of low or high scores on questionnaires on daily hassles and self-reported symptoms, to create four extreme groups. NK cell percentages were different between two of the four groups, only when the analysis was not controlled for gender, life style and endocrine parameters. No evidence was found for a relationship between group membership and NKCA. NKCA, however, was found to differ between men and women and to be associated with percentages of NK cells and intracellular levels of cAMP. Furthermore, the hypothesis was tested, that hormone-induced changes in NKCA in vitro are dependent on the individual's current stress profile. To investigate this issue, NKCA was measured after cells had been incubated with hydrocortisone (10(-6) or 10(-7) M) or the beta-adrenergic agonist isoprenaline (10(-5) or 10(-7) M) in vitro. Changes in NKCA were found to be related to plasma adrenaline levels, but no evidence was found for involvement of psychological variables. It is concluded that, in the current setting, there is no association between the combination of scores on the two psychological questionnaires, and NKCA or hormone-induced changes therein.

In the present study, the relationship between psychological variables and hydrocortisone (HC)-induced immunoglobulin (Ig) production in vitro was investigated. Ninety-five human volunteers were selected based on their extreme (low or high) scores on a daily hassles and a symptoms questionnaire. Four groups were composed: (1) few hassles, few symptoms; (2) many hassles, few symptoms; (3) few hassles, many symptoms; and (4) many hassles, many symptoms. Incubating peripheral blood mononuclear cells (PBMC) for 2 weeks with HC (concentrations ranging from 10(-8) to 10(-6) M), resulted in a concentration-dependent rise in IgM and IgG secretion. In vitro IgM as well as IgG secretion were found to be related to plasma Ig levels. Plasma cortisol levels were positively associated with HC-induced IgG secretion. Furthermore, Ig secretion was found to depend on psychological profile, indicating a differential sensitivity of PBMC to HC for the four groups.

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.

There is now a substantial body of data available indicating that the brain, the nervous system, and the immune system are functionally connected. Consequently, environmental conditions of a psychosocial or physical nature may influence the body's defence. The magnitude of the change in immune reactivity is partly determined by the individual's evaluation of the psychological or physical stimulus. This is illustrated by pharmacological modification of perception resulting in more pronounced stress-induced immunomodulation. In man, it has been demonstrated that recently experienced life stress, particularly daily hassles, can codetermine the effect of an experimental stressor applied for a short period of time on the immune system. An intriguing finding relates to the synthesis and secretion of hormones and neuropeptides by immune cells. These chemical messengers act as autocrine or paracrine immunoregulatory molecules on the one hand, and as messengers for communication with the brain and peripheral nervous system on the other. Animal research indicates that this dialogue is of biological significance.

Fifty male subjects (aged 24 to 55 years) were subjected to a mild and potentially uncontrollable interpersonal stress situation. They were asked to solve a difficult puzzle. Subsequently they were requested to explain their solution to "another subject," actually a confederate to the researchers. The confederate frustrated the subjects' explanation efforts. Care was taken that neither solving nor explaining of the puzzle was successful. The experimental situation induced mild psychological strain as documented by mood changes in the experimental group when compared with a control group of 36 male subjects. Peripheral blood was drawn by an indwelling catheter just before, directly after, 15 minutes after, and 30 minutes after the stress situation. Numbers of leukocytes, lymphocytes, monocytes, T-cell subsets, natural killer (NK) cells, and B-cells were determined. As functional assays we used in vitro proliferative responses of T- and B-cells to mitogenic stimulation (PHA and PWM) and to an antigen cocktail. The potential influences of health- and biobehavioral variables were taken into account in the analyses, as well as incidental differences in initial mood or immunological baseline. The results replicated and expanded on previous research. In contrast to controls, experimental subjects showed a significant increase in numbers of NK cells after the stress-period, returning to baseline values after 15 minutes of rest. A similar effect was shown on T-suppressor/cytotoxic cells and, inversely, on T-helper/suppressor ratio, but these effects could be attributed to changes in the numbers of CD8+CD57+ cells. No effects were observed on proliferation. From the results we conclude that the effects of a short lasting mild psychological stressor are mainly restricted to cells of the NK cell population.

Psychosocial factors can influence immune functions: the brain intermediates between the outside world and the inside of the body. The functional interaction between the central nervous system and the immune system is effected by 2 different pathways: the nervous "wiring system" innervating lymphoid tissues and the "soluble connection" via the neuroendocrine system. The paper reviews data from the literature regarding changes in susceptibility to infections due to stressful conditions. Emphasis is given to information regarding infection of the oral cavity and the upper respiratory tract.

Lymphocytes are now recognized as an extrapituitary source of neuropeptides, such as the opioid peptide beta-endorphin. In the present paper the intracellular signalling pathways involved in regulation of the secretion of immunoreactive (ir) beta-endorphin by human peripheral blood mononuclear cells are described. Activation of protein kinase-C with a phorbol ester rapidly induces secretion of ir-beta-endorphin by T-cells as well as by the non-T cell fraction. Stimulation of protein kinase-A by the addition of (Bu)2cAMP to T-cells or non-T-cells can also induce the secretion of ir-beta-endorphin by these cells. Investigation of the effect of different mitogens or antigen on ir-beta-endorphin secretion by lymphocytes revealed that the nature of the stimulus determines the kinetics of the response and the responding cell type. Induction of ir-beta-endorphin secretion by T-cells after stimulation with a T-cell mitogen is relatively fast; it can be observed within 3 h. In contrast, the response of the non-T-cell fraction to, for example, stimulation with (Bu)2cAMP can only be observed after 18 h of culture. Evidence will be presented that the fast response is mediated via protein kinase-C activation. The response observed after 18 h can be mediated via protein kinase-C as well as protein kinase-A activation.

In the present paper we demonstrate that one of the early effects of the opioid peptide beta-endorphin on human peripheral blood mononuclear cells is the induction of a change in the intracellular cAMP level. However, the effect of beta-endorphin on cAMP levels is not uniform; increases as well as decreases in cAMP level are observed. It appears that beta-endorphin is a true modulator of intracellular cAMP level: the peptide will increase cAMP levels in cells with a low baseline level. In contrast, beta-endorphin tends to decrease cAMP levels is cells with a high cAMP concentration. Moreover, beta-endorphin modulates the rise in cAMP induced by beta-adrenergic activation. The effect of beta-endorphin on cAMP level correlates negatively with the magnitude of the change in cAMP level induced by beta-adrenergic activation.

The immune system and the neuroendocrine system have been shown to be functionally interactive. The neuroendocrine system can modulate the immune response and immune mediators can influence the neuroendocrine system. The present paper focuses on the capacity of lymphocytes to produce and secrete neuroendocrine substances. Lymphocytes can secrete the neuropeptide beta-endorphin in response to activation with mitogen or antigen. Moreover, mediators that are involved in the adaptation to stress have also been shown to induce the release of immunoreactive-beta-endorphin by lymphocytes. It is shown here that stimulation of human peripheral blood mononuclear cells with the beta-adrenergic agonist isoprenaline induces beta-endorphin secretion. The effect of isoprenaline can be mimicked by elevation of the intracellular concentration of cAMP with forskolin or (Bu)2cAMP. Inhibition of cAMP-dependent protein kinase PKA by the antagonist N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide abrogates isoprenaline-induced secretion of immunoreactive-beta-endorphin by peripheral blood mononuclear cells. The present data give evidence that, beta-adrenergic activation activation of lymphocytes stimulates the secretion of ir-beta-endorphin via a protein kinase A-dependent mechanism. Both beta-adrenergic agonists as well as beta-endorphin have been shown to modulate the immune response. The data presented here are indicative for a role of beta-endorphin in the modulation of the immune response after beta-adrenergic activation.

Cells of the immune system can be stimulated to secrete POMC-derived peptides such as beta-endorphin and ACTH. Recently, it has been reported that CRF induces beta-endorphin secretion by human peripheral blood mononuclear cells in vitro (1). It has been shown that interleukin-1 (IL-1) mediates the CRF-induced secretion of beta-endorphin by lymphocytes in vitro. In the present report it is demonstrated that sc administration of CRF to rats can also induced beta-endorphin secretion by lymphocytes from spleen and mesenteric lymph nodes. Moreover, this CRF-induced secretion of beta-endorphin coincides with enhanced secretion of IL-1 by macrophages. Previously, we reported that IL-1 can activate CRF neurons in the hypothalamus of the brain. Our data indicate the existence of an intricate relationship between CRF and IL-1, peptides that can be viewed as playing a pivotal role in the interaction between the central nervous system and the immune system.

Corticotropin-releasing factor (CRF), a 41-aminoacid neuropeptide, can induce lymphocytes to production of beta-endorphin (beta E). Furthermore, the neuropeptide Arginine-Vasopressin (AVP) can enhance CRF-induced production of beta E. We have demonstrated that CRF acts by stimulating monocytes to production of the cytokine interleukin-1 (IL-1). IL-1 can in its turn activate the lymphocytes to secretion of beta E. Here we demonstrate that the glucocorticoid analogue dexamethasone is capable of modulating CRF-induced beta E secretion by lymphocytes. It appeared that dexamethasone can inhibit secretion of lymphocyte-derived beta E. The mechanism by which dexamethasone exerts its inhibitory activity is by blocking CRF-induced production of IL-1, thereby preventing induction of beta E secretion by B cells. These results support the concept that peptide hormones and glucocorticoids are mediating a reciprocal modulation of neuroendocrine and immunological activities.

The exact function of receptors for C3b (CR1), which are present on B lymphocytes, is not clear. The present studies were performed to determine the influence of heterologous anti-CR1 on the production of IgM, IgG, and IgA by human peripheral B lymphocytes in vitro. Anti-CR1, raised in rabbits by immunization with purified erythrocyte CR1, was rendered immunospecific and was converted to F(ab')2 and Fab' by enzyme digestion. Pre-immune F(ab')2 and Fab' served as controls. Normal human blood T lymphocytes and T cell-depleted mononuclear cells were cultured for 6 days in RPMI medium and 10% heated fetal calf serum with a low dose (7 micrograms/ml) of pokeweed mitogen (PWM) alone or with PWM and F(ab')2-anti-CR1. In control experiments the effects of F(ab')2-anti-CR1 without PWM and of pre-immune F(ab')2, Fab'-anti-CR1 and pre-immune Fab' in combination with PWM were also tested. When PWM and F(ab')2-anti-CR1 were present simultaneously, 4.3 X 10(3), 2.5 X 10(3), and 1.8 X 10(3) ng/ml of IgM, IgG, and IgA were synthesized, respectively, in cultures containing 4.5 X 10(5) mononuclear blood cells. All other combinations resulted in synthesis of less than 250 ng/ml of each class of Ig. The presence of purified CR1 in the cultures containing PWM and F(ab')2-anti-CR1 caused a dose-dependent decrease of Ig synthesis. By culturing B cells isolated by E-C3b rosetting with T cells and monocytes, the anti-CR1 responding population was demonstrated to be mainly in the CR1-rich B cell fraction. Fab'-anti-CR1 at high doses was also able to stimulate Ig production in the presence of low concentrations of PWM. These data suggest that triggering of CR1 on B cells results in modulation of antibody production and that triggering of CR1 with more than one antibody is required for an optimal response.

Human cord blood mononuclear cells (CBMC) were stimulated in vitro with a number of T cell-dependent antigens. Antigen-induced B cell activation was measured applying a plaque-forming cell assay for the detection of antigen-specific IgM-secreting B cells. With the exception of diphtheria toxoid, the antigens ovalbumin, sheep red blood cells, Helix pomatia hemocyanin, burro red blood cells, and tetanus toxoid elicited an IgM-plaque-forming cell response in cultures of CBMC to levels obtained for peripheral blood mononuclear cells (PBMC) from adult controls. However, for each antigen used, the antigen dose optimal for the induction of a response was consistently found to be a hundred to a thousand times lower than the concentration of the corresponding antigen optimal for adult PBMC. Longitudinal studies on PBMC obtained from infants between 2 and 30 months of age revealed that a shift of the antigen dose toward concentrations needed to induce plaque-forming cells in cultures of adult PBMC occurs at approximately age 8 months. Our data indicate that various antigens can be used for the in vitro analysis of antigen-specific B cell activation and regulatory T cell functions in studies concerning the ontogeny of the humoral immune response in humans.

We have studied patients with various immunodeficiencies for the occurrence of blood lymphocytes bearing six different surface markers: the affinity to sheep erythrocytes (Es) to identify T cells, the presence of surface-bound immunoglobulins (sIg) to distinguish B cells, the affinity to mouse erythrocytes (Em) as a second B cell marker, and the receptors for the Fc part of IgM (IgM-FcR), the Fc part of IgG (IgG-FcR) and for complement (CR). IgG-FcR bearing lymphocytes were present in normal proportions and the same was found for CR-positive lymphocytes. None of the patients with congenital agammaglobulinaemia had sIg-bearing or Em-binding B lymphocytes. Four patients with ataxia teleangiectasia had low B cells and 3 out of 4 also had low proportions of IgM-FcR-bearing (T) cells. A child with partial DiGeorge syndrome did not have a grossly abnormal marker pattern although there appeared to be a shift in the T/B cell ratio in favour of the B cells. In a patient with selective cellular immunodeficiency associated with a purine nucleoside phosphorylase deficiency, who was followed during reconstitution therapy with plasma and red cells, a positive change in the marker pattern was seen. Similar observations were made in a child with combined immunodeficiency during treatment. The findings are being discussed in the light of the current knowledge of the functions and ontogeny of lymphocyte subsets.

The application of the fluorochrome 4-acetamido-4'-isothiocyanato stilbene-2,2'-disulphonic acid (SITS) in immunofluorescence was studied. The optimal excitation wave length is 350 nm, and optimal fluorescence is obtained at 420 nm. After purification of the commercial compound, conjugation is performed in a strong buffer at pH 9.0-9.5. SITS conjugates were very satisfactory for immunofluorescence studies of the cytoplasmic antigens of cell preparations, but their blue emission was difficult to distinguish from autofluorescence in sections of human tissues. Good results with immunofluorescence on membrane bound antigens were obtained by using an ultra-violet laser beam as light source. SITS can be used simultaneously with FITC and TRITC conjugates thus making it possible to show three antigens in one preparation.

The purpose of our study was to determine if normal human B cells can be activated to autoantibody production using an antigen-specific system. For this purpose we investigated the in vitro antibody response to the autoantigen thyroglobulin (Tg) employing soluble or insolubilized Tg (i-Tg) and a B cell growth and differentiation factor (BDGF) to stimulate lymphocytes from healthy individuals. Similar experiments were carried out with the xenoantigen ovalbumin (OVA). The presence of Tg and OVA-reactive B cells was demonstrable by stimulating lymphocytes from tonsil, spleen and blood with a combination of pokeweed mitogen and formalinized Staphylococcus aureus: mitogen stimulation resulted in the generation of IgM anti-Tg and IgM anti-OVA antibody-forming cells (AFC) as detected in a spot enzyme-linked immunosorbent assay. Soluble antigen failed to induce autoantibody production. However, i-Tg or i-OVA did activate normal tonsil and spleen B cells. Differentiation of these activated B cells to IgM AFC required the presence of BGDF, derived from a human T hybrid clone. Preincubation experiments with the particulate autoantigen show that a specific activation signal is provided by the antigen which subsequently renders the B cells responsive to BGDF. i-Tg-dependent stimulation of B lymphocytes could be inhibited by adding free Tg to the cultures; the same applied to i-OVA stimulation. We conclude that the normal human B cell repertoire contains B cells that can be activated to autoantibody production by the autoantigen Tg if the necessary T cell signals are provided. Thus, these B cells are not in an inherently anergic state. Similar mechanisms seem to play a role in the activation of B cells responding to i-OVA and i-Tg.

The usefulness of cryopreserved sheep erythrocytes (unsensitized or coated with fluorescein, IgG-type antibody or complement) for marker tests on human blood lymphocytes was studied. It appears that the rosetting properties or freshly prepared or frozen-stored erythrocytes are not significantly different. The method may be used to provide homogeneous stocks of sensitized erythrocytes for lymphocyte rosetting technique.

In this study, six surface marker tests were performed on lymphocytes from normal individuals of three age groups: neonates, children from 0 to 2 years of age, and adults. Determined were the proportions of T cells binding sheep red blood cells, of B cells carrying surface immunoglobulins or binding mouse red blood cells, and of lymphocytes bearing receptors for IgM, for IgG or for complement. The T cell percentage appeared to increase with the age; the percentage of B cells was highest in the children's group, as determined by both marker tests. Neonates had a much lower proportion of IgM-receptor bearing cells than older individuals. The variation in the percentages of T cells and of IgM-receptor bearing cells in the young age groups was relatively large as compared to adults. The significance of these observations is discussed; they may be of value for the proper evaluation of results obtained in diagnostical tests on neonates and young children.

We examined bone marrow from myeloma patients for the presence of cells with the characteristics of the clonogenic cell in the myeloma stem cell assay. We identified a novel type of cell that contained cytoplasmic immunoglobulin of the relevant idiotype located in a cytoplasmic spot. This "spotted" Ig could be located in the rough endoplasmic reticulum. Spotted cells are highly proliferative, as evidenced by the nuclear staining with the antibody Ki67, and were found in the bone marrow from most of the myeloma patients studied. This type of cell was also present in patients with immunocytomas, in some cases of benign monoclonal gammopathy, and in patients in the state of polyclonal hypergammaglobulinemia. IgG subclass distribution of so-called spotted cells and plasma cells, found in a patient with pseudo biclonal gammopathy, indicates that spotted cells are intermediate between B cells and plasma cells. Spotted cells express the B cell-associated antigens HB4 and HB6 but do not express other B cluster of differentiation antigens or plasmacytoid antigens tested.

Earlier studies on antisera against Fab of pooled human IgG and IgA myeloma proteins disclosed the presence of class-specific Fd antibodies, the demonstration of which required interaction of heavy and light chains. To extend our knowledge about the antigenic structure of the Fd fragment of human immunoglobulins, antisera were prepared in rabbits against gamma chains of pooled IgG and of four IgG1 myeloma proteins, and Fd gamma fragment and a cyanogen bromide-produced CH1 preparation of an IgG1 myeloma protein, and an Fd' alpha fragment of an IgA1 myeloma protein. No antigenic determinants exclusively confined to the CH1 region of intact human IgG could be demonstrated with these antisera. The antigenic structure of CH1 intact immunoglobulins may thus only be defined by light-chain-dependent determinants.

The presence of Fc receptors (FcR) on rabbit peripheral blood leukocytes is demonstrated using rosette formation with an ox erythrocyte-antibody (EoxA) complex. The receptor is specific for the Fc fragment of IgG (neither IgM nor F (ab')2 anti-Eox mediates rosette formation) that is antigen-bound (aggregated rabbit IgG inhibits the rosette formation only transiently). The receptor is species-specific: guinea pig IgG/Eox, goat IgG/Eox and sheep IgG/Eox complexes do not show rosette formation, and goat IgG aggregates do not inhibit rosette formation. The origin of the target erythrocytes is of importance. Sheep erythrocytes are not useful, and within Eox large differences between donors were found. Rosette formation was only inhibited by pretreatment of the rosette-forming cells with homologous immune complexes, whereas the size of the antigen greatly influenced the degree of inhibition. The rabbit FcR is pronase-resistant, unlike the human and murine RcR. The interaction of IgG and the FcR is not inhibited by isolated C gamma 3 domains. Further evidence for the requirement of the whole Fc region was obtained in experiments where inhibition of the rosette formation was observed using antisera directed to the C gamma 3 and the C gamma 2 domain, respectively. Anti-Fab antiserum did not inhibit rosette formation. Results are discussed in relation to the mechanism of allotypic suppression.

Serum levels of IgM, IgG, IgA, IgD, and IgE were determined in serum samples of 270 healthy Dutch children (aged 4-13 years) and of 30 healthy Dutch adults, the amounts being expressed in International Units per millilitre. Special attention is given to the IgD and IgE results, since the IgM, IgG, and IgA levels in mg per 100 ml of these sera and their implications have already been reported. In the children's sera the occurrence of relatively high IgD and IgE levels was frequently observed, whereas the adult group did not show excessive variation in this respect. The mean IgD levels found for adult males and females are 21 I.U./ml and 24 I.U./ml, respectively; the mean IgE levels for the same groups are 68 I.U./ml and 88 I.U./ml, respectively. The mean IgD and IgE levels in the children of each year group were usually higher than those of each of the juvenile groups and the mean level of the adult group was not statistically significant. A statistically significant influence of sex and season on the IgD and IgE levels could not be demonstrated in this material either. Three of the 270 children's sera showed an exceptionally low IgA content. In two of these cases the serum was sampled and studied a second time after an interval of four years, when the IgA deficiency proved to be still present. The IgE levels in the sera of these healthy IgA-deficient children were normal, whereas the presence of IgD could not be demonstrated.

In the present study the activation of purified human T lymphocytes by the calcium ionophore A23187 was analysed in the light of current concepts of receptor-linked inositol lipid metabolism. It was found that A23187 was only slightly mitogenic, with a narrow optimum at 400-500 nM. The proliferation could be blocked by anti-Tac ascites at 10(-3) dilution, suggesting an interleukin 2 (IL-2)-dependent pathway of activation. However, an unexpectedly large proportion of A23187-stimulated cells expressed the IL-2 receptor. Reculturing the cells with exogenous IL-2 after removal of A23187 resulted in strongly enhanced proliferation. Phorbol myristic acetate (PMA) at non-mitogenic concentrations exerted an extremely strong synergistic effect on A23187-induced cell proliferation, which was, again, mediated via an IL-2-dependent pathway. Supernatants of A23187-stimulated T cells did not contain detectable amounts of IL-2. Combination of PMA and A23187 resulted in considerable IL-2 production. It is concluded that A23187 induces the expression of IL-2 receptors without concurrent stimulation of IL-2 production, thus allowing only low levels of proliferation. Addition of exogenous IL-2 or of PMA restores the imbalance between the occurrence of IL-2 and its receptor and results in high rates of proliferation.

Ox erythrocytes, sensitized with IgM-type rabbit antibodies, were found to form rosettes with human blood lymphocytes. Granulocytes, monocytes, and macrophages appeared to lack this property. The rosette formation could be inhibited with human IgM and its Fc fragments. IgM-binding lymphocytes were also present in cell suspensions isolated from human tonsils and thymuses and among blood lymphocytes cultured in vitro. The outcome of combined marker tests and cell separation experiments disclosed that the rosetting lymphocytes belong to the T-lymphocyte population.

An enzyme-linked immunosorbent assay (spot-ELISA) for individual immunoglobulin secreting cells, which became recently available, was applied to the enumeration of human B lymphocytes secreting specific antibodies of thyroglobulin. Polyclonally activated B cells from patients with auto-immune thyroid disease are incubated in thyroglobulin coated plates. After removal of the cells specific antibodies are visualized by means of an immunoenzyme technique employing agarose to localize converted substrate. Individual specific antibody secreting cells are counted as blue spots using an inverted microscope. Numbers and isotype of spots correlate well with the amount and isotype of secreted antibody as detected with a conventional ELISA. This easy to perform, complement-independent technique offers a useful alternative to conventional plaque forming cell assays.