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J Appl Physiol 90: 1736-1743, 2001;
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Vol. 90, Issue 5, 1736-1743, May 2001

Simulated microgravity, psychic stress, and immune cells in men: observations during 120-day 6° HDT

A. Choukèr1, M. Thiel1, V. Baranov2, D. Meshkov2, A. Kotov2, K. Peter1, K. Messmer3, and F. Christ1

1 Clinic of Anaesthesiology and 3 Institute of Surgical Research, University of Munich, 81366 Munich, Germany; and 2 Institute of Biomedical Problems, 123007 Moscow, Russia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Because 6° head-down tilt (HDT) is an established method to mimic low gravity on earth, the aim of the present study was to determine the effects of 120-day HDT on psychic stress and peripheral blood immune cells in six healthy male volunteers. Psychological state was assessed by a current stress test, and cortisol was measured in saliva. During HDT, all volunteers developed psychic stress, and the diurnal rhythm of cortisol secretion was significantly altered. In addition, urine excretion of dopamine and norepinephrine increased. The innate part of the immune response was activated, as evidenced by the increase in the expression of beta 2-integrins on polymorphonuclear leukocytes and a rise in the number of circulating natural killer (NK) cell lymphocytes. The ratio of T-helper to T-cytotoxic and T-suppressor cells decreased, whereas no changes in T and B lymphocytes were observed. Plasma levels of interleukin-6 increased significantly and returned to basal levels after the end of the HDT period. Thus 6° HDT appears to be a valid model to induce psychic stress and neuroendocrine-related changes in the immune system, changes that might also be encountered by astronauts and cosmonauts during long-duration spaceflights.

stress; cortisol; neutrophils; lymphocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALREADY DURING SHORT-TERM spaceflights, the immune response might be affected by several adverse effects due to psychic stress, radiation, malnutrition, and microgravity (4). The compromise of the immune system, however, is of increasing importance regarding the longer duration of planned spaceflights and interplanetary missions. Former studies on the effects of microgravity on the human immune system have been carried out pre- and postflight or in ground-based studies under the conditions of head-down tilt (HDT).

In numerous studies, HDT at 6° was considered a reliable model to mimic the effects of microgravity on cerebral and venous hemodynamics (8, 18, 35), cardiovascular functions and reflexes (14, 16, 26, 27), thermoregulatory responses (6), as well as the hematopoetic system (7). However, in most of these studies, HDT was applied only for a few days or weeks, and neuroendocrine changes were described during HDT for not longer than 7 days (23). In our study, six healthy male volunteers were subjected to 120 days in the 6° HDT position. During this time period, we evaluated the effects of long-term hypokinesia with respect to psychic stress, the secretion of stress hormones, and changes in peripheral white blood cell (WBC) populations.

In the present study, we tested the hypothesis that 6° HDT for 120 days, like microgravity, causes psychic stress that might affect the circadian rhythm of cortisol secretion as well as the unspecific and the specific part of the immune system.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

After approval by the local ethics committee and receipt of informed consent, six healthy male volunteers [age 30.8 ± 7.5 (SE) yr, weight 79.5 ± 7.1 kg, height 180.7 ± 5.2 cm] were subjected to permanent bed rest for 120 days at 6° HDT. The study was carried out at the Institute of Biomedical Problems in Moscow, Russia. To obtain constant experimental conditions and to avoid imbalances in the sleep-awake rhythm, lights were switched off between 2200-2300 and 0600-0700, whereas normal daylight illumination was present for the rest of the day. During the HDT period, the subjects were not allowed to sit or to leave their bed; physical activities were limited to a very low state. The daily routine procedures (washing, etc.) were standardized and were kept constant during the entire study period.

Study protocol. According to the study protocol, measurements were taken before the start of the HDT period (Pre), at the 65th day (D65) and 105th day during HDT (D105), and 6 days after the end of the bed rest (Post). However, because of logistic problems, psychological well-being and cortisol secretion could not be determined before the start of the HDT period. Therefore, we determined psychological well-being and cortisol secretion in groups of healthy volunteers matched for age and body weight with the groups subjected to HDT.

Current stress test. The current stress test (CST) was developed and validated by German psychologists (2, 20) and is commercially available under the product name, Actual Strain Short Questionaire, Kurzfragebogen zur aktuellen Beanspruchung (Beltz Testzentrale European Test Publisher Group, Göttingen, Germany). The CST was designed to determine an individual's psychological state repeatedly under the conditions of acute and chronic stress. The test is composed of six pairs of contradictory feelings of increasing intensities among which the subject has to decide. From the sum of the values, the final CST score is calculated and may range from 1 (i.e., no stress) to 6 (i.e., maximal stress). In contrast to other anxiety evaluation questionnaires, e.g., the state-trait anxiety inventory (STAI) (24), the self-estimating CST test is a very short, one-page paper test that is performed in <1 min and is, therefore, easily applicable to the present study. Because of the composition of the questionnaire, the test person usually cannot remember how he decided the last time. Therefore, there is no carryover effect, which is a strict prerequisite for the application of the test in longitudinal studies. As published previously, the CST is validated by comparison with STAI (2), which is considered as one of the most commonly used scales for the measurement of state anxiety. The correlation between CST and STAI was reported to be 0.7-0.9 (2, 25).

Cortisol, blood and urine sampling, transportation, and determination of parameters. After acquisition of cortisol and urine samples, specimens were kept frozen until the analyses were performed in our laboratories in Munich, Germany.

To enable the various parameters to be determined in blood specimens, different preservation procedures were applied. For measurement of beta 2-integrins, blood specimens were stored in ice-cold water immediately after acquisition until determination within the next 12 h in Munich, whereas for evaluation of lymphocyte subsets, whole blood samples were kept at room temperature according to the instructions of the manufacturer. Plasma was removed from whole blood after centrifugation at the Institute of Biomedical Problems in Moscow and kept frozen until assessment of cytokines a few weeks later in Munich, Germany.

Cortisol. Because glucocorticoids are known to be periodically secreted in response to a variety of environmental and hormonal stimuli (e.g., psychic stress and physical exercise), which alone and/or together with cortisol might affect the immune system, free cortisol was determined in saliva samples collected in the morning (8 AM) and in the evening (8 PM). Saliva was collected by having the subject chew on a cotton swab for 30-45 s; the swab was then stored in a SALIVETTE device tube (Sarstedt, Nürnbrecht, Germany). Samples were frozen, and free-cortisol concentrations were quantified by a commercially available ELISA according to the instruction of the manufacturer (Orion Diagnostica, Espoo, Finland). To evaluate possible changes in the circadian rhythm of cortisol secretion, the ratio between the morning and evening (m/e) cortisol values was calculated.

Catecholamines. Epinephrine, norepinephrine, and dopamine were determined in urine samples collected for a period of 24 h in a container prefilled with hydrochloric acid (10%). Acidified urine samples (pH 3-4) were thereafter analyzed by HPLC (Chromosystems, Martinsried, Germany), and the amounts of catecholamines secreted during the 24-h period (µg/24 h pooled urine) were calculated.

Cell counts. Lymphocytes, monocytes, and polymorphonuclear leukocytes (PMNL) were electronically enumerated in a EDTA anticoagulated whole blood specimen (Coulter STKS, Coulter Electronics, Luton, UK).

beta 2-Integrins. The expression of adhesion molecules (beta 2-integrins, CD18) was determined in an ice-cold, heparinized (10 IE/ml; VETREN, Byk Gulden, Konstanz, Germany), whole blood specimen by flow cytometry, as previously described (28). After incubation of aliquots of cell suspensions with saturating concentrations of FITC-labeled monoclonal antibody IB4 (K. Arfors, Experimental Medicine, Princeton, NJ), which specifically binds to beta 2-integrins, fluorescence intensities were determined on a linear scale by using a Becton Dickinson FACScan (Becton Dickinson, San Jose, CA). The results are expressed as relative fluorescence units.

Lymphocyte subpopulations. To demonstrate possible changes in the specific part of the immune system, the percentages of functionally different populations of lymphocytes were determined. For this purpose, the expression of cell surface differentiation antigens of blood lymphocytes was measured: the cell differentiation antigen CD3 on all T lymphocytes, CD4 on T-helper lymphocytes, and CD8 on T-suppressor and T-cytotoxic lymphocytes. CD19 is expressed only on B lymphocytes, and CD56 in combination with CD16 is expressed on a specific subgroup of lymphocytes, the NK cells. All of these cell-surface antigens were detected by a commercially available two-color direct immunofluorescence reagent kit (Simultest IMK-Lymphocyte, Becton Dickinson). Monoclonal fluorochrome-labeled antibody reagents were added to a whole blood specimen (EDTA) and bind specifically to antigens on the surface of leukocytes.

Specificity of binding was controlled by isotype-matched control antibodies with specificity against antigens not found on human leukocytes. After staining, WBCs were fixed, and red blood cells were lysed by BD Lysing Solution (Becton Dickinson) before analysis by flow cytometry.

Neutrophils, monocytes, and lymphocytes were discriminated in terms of forward (FSC) and right angle (side) light scatter (SSC) characteristics. A minimum of 4,000 lymphocytes was gated in the FSC/SSC dot plots, and relative percentages of FITC/phycoerythrin double-labeled lymphocyte subpopulations were determined by FACScan software (version 2.1, Research Software) (see Fig. 1).


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  		Fig. 1.   Representative dot plots for analyses of relative percentages of lymphocyte subpopulations in 1 subject at day 105 of head-down tilt (D105). A: FITC and phycoerythrin (PE) double-labeled lymphocytes were discriminated from neutrophils and monocytes by forward (FSC) and side scatter (SSC) light characteristics. Relative percentage of, e.g., T-helper cells among all lymphocytes was determined after a gate was set on lymphocytes in the FSC-SSC dot plot. Based on the results obtained for nonspecific binding of FITC- and PE-labeled isotype-matched control antibodies by gated lymphocytes, 4 quadrants (Q1-Q4) were defined in the dot-plot display of fluorescence channels 1 (FL1) and 2 (FL2) (B-D). FL1 and FL2 detect light emitted by FITC and PE, respectively. In the case of analyses of CD4+/CD3+ helper and T cells, whole blood was incubated with PE-labeled anti-CD4 and FITC-labeled anti-CD3 monoclonal antibody. Thus helper T cells are identified as higher red-orange (PE) fluorescence 2 and high yellow-green (FITC) fluorescence 1 positive cells (Q2 in B). Lymphocytes stained by nonspecific binding of FITC and PE-labeled isotype-matched control antibodies were located in Q3 (not shown). C and D: FL1-FL2 dot plots represent analyses of anti-CD8+ (suppressor/cytotoxic)/CD3+ T cells (Q2 in D), whereas low yellow-green and high red-orange fluorescence represent anti-CD16+CD56+/CD3 natural killer cells (Q1 in D), respectively.

Because the different fluorochromes emit light at different wavelengths when excited [488 nm, argon-ion laser, FITC-labeled leukocytes emit yellow-green light (515 nm), whereas phycoerythrin-stained cells emit red-orange light (580 nm)], fluorescence compensation was set on the instrument, and detector sensitivity was adjusted by using CaliBRITE calibration beads (Becton Dickinson).

Because lymphocyte surface antigens and beta 2-integrins on neutrophils could not be measured in Moscow, blood samples were transported either at 20°C (for immunophenotyping of lymphocytes) or at 0°C (for determination of beta 2-integrins) and analyzed within 12 h after the aquisition of the blood specimen. Additional control experiments were performed that did not show any alteration of cell surface antigens because of transportation (data not shown).

Superoxide anions. The production of superoxide anions (O<UP><SUB>2</SUB><SUP>−</SUP></UP>) was determined in whole blood samples by photometric measurement of the reduction of cytochrome c, occurring spontaneously or after stimulation of PMNL with the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). For that purpose, 1 ml of heparinized whole blood was aliquoted into four plastic tubes prefilled with 1.4 ml of prewarmed (37°C) Hanks' buffered salt solution, cytochrome c (0.625 mg/ml), and cytochalasin B (2.5 µg/ml). One tube had no further additives, whereas the other three contained, additionally, superoxide dismutase (SOD; 50 U/ml) or fMLP (10-6 M) in the absence and presence of SOD. Blood was transferred into these tubes simultaneously with a Multipette device (Eppendorf, Hamburg, Germany), avoiding any lag time among the samples. The reaction mixtures were incubated at 37°C for 15 min. Thereafter, reactions of cells were stopped by putting vials on ice, and cellular components were spun down (5 min, 600 g, 4°C), the supernatants were transferred to a 96-well microtiter plate, and the absorbances were determined (single reading) in quadruplicates on a multichannel automated photometer (Dynatec MRX 7000, Dynatec Laboratories, Alexandria, VA). Plate readings were performed at 550 nm and were fitted with a 630-nm interference filter; the pathlength in the microplate containing 180 µl fluid was 0.43 cm. From the difference between the absorbances determined in the samples, e.g., stimulated with fMLP in the absence and in the presence of SOD, the fMLP-stimulated amount of O<UP><SUB>2</SUB><SUP>−</SUP></UP> was calculated. The results are expressed as calculated values of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production per 1 × 106 granulocytes. All chemicals used were of analytic grade, and the concentrations given are final concentrations (Sigma Chemical, Deisenhofen, Germany).

Cytokines. EDTA-anticoagulated blood specimens were centrifuged at 600 g for 5 min in a cooled centrifuge (4°C), and plasma was stored at -80°C until determination of cytokines. Quantitative analysis of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha ), IL-1beta , and interferon-gamma was done by a commercially available ELISA (Combokit, Genzyme, Alzenau, Germany).

Statistics. The results presented here are part of a large study that was coordinated by the Institute of Biomedical Problems in Moscow. Because of several limitations, it was not possible to study more than six individuals. In agreement with the design of other studies on the effects of HDT in men (23), statistical analysis was used to answer whether the null hypothesis (no changes occur because of 120-day HDT) could be rejected or not. To this end, data were tested for normal distribution by one-sample Kolmogorov-Smirnov test and compared with the Pre values of the same subjects by repeated-measures ANOVA with adjustment for multiple comparisons by Bonferroni. In the case of CST score and cortisol measurements, values of D65 were used as reference for statistical comparison. Two-tailed level of significance was set at P < 0.05. Data are presented as means ± SE. All statistical analyses were performed by SPSS 8.0 program (SPSS, Chicago, IL).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CST. Because of logistics problems, it was not possible to determine CST scores at Pre. To provide an idea of normal values for psychological well-being, CST score values were determined in an age- and body weight-matched group of healthy men. The CST values of the group not subjected to HDT (Table 1, Pre values marked by asterisks) were well in the normal range (2.4-2.8) reported (20). The same was true for CST scores taken in individuals at D65. With progress of the HDT period, averaged scores increased to a maximum value of 3.7 at D105. CST values remained elevated even at Post. However, these changes did not reach the level of statistical significance (Table 1).

                              
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  		Table 1.   CST score, saliva cortisol, and urine catecholamine secretion

Cortisol. As with the determination of CST, saliva cortisol secretion could be assessed only from D65 onward. The values determined in the group not subjected to HDT and the values taken at D65 showed normal physiological variations in cortisol secretion with higher levels in the morning (15-20 nmol/l) followed by a decline down to 4-6 nmol/l in the evening. After D65, morning cortisol concentration exhibited only a transient increase (D105). In contrast, evening concentrations of cortisol steadily increased up to threefold, even when the bed rest was finished and remobilization occurred (Post). As a result, the m/e cortisol saliva concentrations were in the normal range (3-5) at D65 but were significantly decreased at D105 and Post. Thus the amplitude of the circadian rhythm of cortisol secretion significantly decreased during HDT and was almost lost at Post (Table 1).

Catecholamines. Twenty-four-hour urine secretion of epinephrine remained unchanged during HDT. Its precursors, dopamine and norepinephrine, however, increased at D65, declined toward levels still above Pre values, and finally increased again at Post (Table 1).

Cell counts. Cell counts of WBC, PMNL, monocytes, and lymphocytes were determined at the same time as the other blood parameter. There was no significant variation during the entire observation period (Table 2).

                              
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  		Table 2.   Cell counts, expression of beta 2-integrins (CD18), superoxide anion production, lymphocyte subpopulations, and interleukin-6 plasma concentration

beta 2-Integrins. Expression of beta 2-integrins on peripheral blood neutrophils almost doubled at D65 and returned back to the Pre levels after hypokinesia was finished (Post) (Table 2).

O<UP><SUB>2</SUB><SUP>−</SUP></UP> production. Production of O<UP><SUB>2</SUB><SUP>−</SUP></UP> by fMLP-stimulated phagocytes showed a tendency to increase at the end (D105) and at Post (Table 2). These changes, however, were not statistically significant. Spontaneous release of O<UP><SUB>2</SUB><SUP>−</SUP></UP> did not change during the entire observation period (data not shown).

Lymphocyte subpopulations. Immunophenotyping of circulating lymphocytes demonstrated changes neither in the absolute and relative numbers of lymphocytes among all WBCs (data not shown) nor in the respective numbers of T (CD3+) or B (CD19+) cells among lymphocytes. Analysis of the percentages of T-cell subpopulations indicated a small decrease in CD4+ cells and a slight increase in CD8+ cells. However, when the CD4-to-CD8 ratio was calculated, the ratio significantly decreased during the bed-rest period. Interestingly, CD16+/CD56+ cells, which are considered to represent NK cells, continuously increased during HDT. After remobilization (Post), percentages of NK cells rapidly decreased but still remained at higher levels compared with cells observed before the start of hypokinesia (Pre) (Table 2).

Cytokines. From all of the cytokines studied (IL-6, TNF-alpha , IL-1beta , and interferon-gamma ), only plasma concentrations of IL-6 increased during simulated microgravity and returned back to initial values at Post (Table 2). Data are not shown for the other cytokines.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Development of stress during HDT. During spaceflights, astronauts and cosmonauts encounter numerous unfamiliar conditions, like microgravity, radiation, confinement, malnutrition, and alterations of the day-night cycle. From a phylogenetic point of view, environmental changes with possible adverse effects are not restricted to spaceflights but have been the driving force for the development of the human body's ability to mount an adaptive stress response. Although adaptive stress responses are mainly directed at enabling the individual to survive, the mechanisms involved may also cause side effects, like immunosuppression. In the case of spaceflights, such unwanted effects of adaptive stress responses on the immune system may become of prime importance during long-duration missions.

Using the ground-based model of 120-day simulated microgravity by hypokinesia in the 6° HDT position, we investigated the possible development of psychic stress, alterations of the regulation of stress hormones, as well as the relationship between such changes and the function and distribution of peripheral WBCs of the immune system.

HDT was considered an appropriate model to simulate low gravity, because, in former studies, it was shown to cause hemodynamic effects resembling closely those induced by microgravity (1, 16, 18, 26, 27). Besides adaptive changes of the cardiovascular system, permanent bed rest in the 6° HDT position is also a reliable and accepted experimental procedure to mimic the effects of microgravity on various other organ systems, including the neuromuscular apparatus (10, 15), thermoregulation (6), and the circadian rhythm (23).

Psychic stress, which might be caused by the prolonged period of 6° HDT, was determined with the CST. The CST was developed to determine psychological well-being repeatedly during longitudinal studies (20). Although we were not able to obtain CST score values at Pre, the values observed at D65 were ~3, which is in or near the normal range (2, 20). In addition, D65 CST values were not different from those determined in an age- and body weight-matched group of healthy men not subjected to HDT. After D65, self-estimated discomfort continuously increased, as also reflected by the rise in the CST values determined at D105. Interestingly, 6 days after remobilization and return of the volunteers to normal daily activities (Post), CST score values still remained elevated compared with those at D65. However, these changes did not reach the level of statistical significance (Table 1). In agreement with the hypothesis that stress activates cerebral regions, leading to subsequent alterations in the secretion of stress hormones such as cortisol and catecholamines, we observed changes in the diurnal rhythm of cortisol secretion. In our study, cortisol was determined in the saliva specimen for several reasons. First, it represents a noninvasive method. Second, determination of cortisol in saliva allows the detection of the protein-unbound free cortisol, because only this form can enter saliva and is not affected by the saliva flow rate (12). Third, it is the unbound, free cortisol that can reach target cells and their receptors (19) and hence reflects the biologically active cortisol that is responsible for the induction of physiological or pathophysiological effects. As with the determination of CST score values, it was not possible to take saliva cortisol measurements before the start of the hypokinesia experiments. Nonetheless, the cortisol values determined at D65 and the m/e values were in the normal range and comparable to values obtained in a group of healthy men not subjected to HDT (Table 1). Under physiological conditions, cortisol levels show a diurnal rhythm because of three- to fivefold higher levels in the morning than in the evening (5, 17). HDT for >65 days induced a much stronger increase in the evening saliva cortisol concentrations compared with the changes in the morning values. As a result, the m/e values significantly decreased, thereby most likely indicating the loss of the circadian rhythm of cortisol secretion. In consequence, these data may indicate that chronic stress leads to alterations in the regulation of the hypothalamic-pituitary-adrenocortical axis, ending up in the suppression of its circadian regulation.

In contrast to the loss of the circadian rhythm of cortisol secretion after D65, urine excretion of catecholamines first increased for dopamine and norepinephrine at D65 and returned thereafter to lower concentrations, although amounts of catecholamines secreted at D105 and Post were significantly elevated in part above Pre values (Table 1). Unexpectedly, the amounts of secreted epinephrine remained unchanged. In general, catecholamines are considered as short-term-acting substances, enabling acute adaption to stress, whereas corticosteroids are considered to allow the host to adapt to repetitive, chronic stress (32). In agreement with catecholamines mediating acute stress responses and corticosteroids conferring rather long-term adaption are reports on the kinetics of the activation of the sympathoadrenal axis with the latter usually preceding the stimulation of the hypothalamic-adrenocortical axis (32).

This sequence of events fits well with that observed in our study: first an increase in catecholamine secretion and then a rise in cortisol secretion (Table 1).

Taken together, the increase in CST score values, the rise in the secretion of urine catecholamines, as well as the loss of the circadian rhythm of cortisol secretion demonstrate that HDT at 6° for 120 days causes psychic stress.

HDT and peripheral WBCs. The unspecific, innate part as well as the specific part of the immune system play an important role in the protection of the host against microorganisms invading tissues as well as the circulating blood (9). In our study, circulating immune cells were collected by venipuncture to characterize changes in immunocompetent cells by enumerative and functional assays. Because peripheral blood cells represent only those cells that are at best on the transit from the bone marrow or other regions of the body to sites where immune responses take place, caution has to be taken in the interpretation of data obtained from WBCs alone. Bearing these limitations in mind, our results demonstrate that prolonged periods of HDT might result in the activation of the innate part of the immune system. This view is mainly based on the observation of an increase in the expression of beta 2-integrin adhesion molecules on circulating neutrophils and on the rise of the relative and absolute numbers of CD56+/CD16+ lymphocytes.

beta 2-Integrins are prestored in intracellular organelles, which, by fusion with the cell membrane, cause rapid expression of these adhesion molecules on the neutrophils' outer cell membrane. Besides mediating adhesion and emigration of neutrophils to and through the vascular endothelium, the major beta 2-integrin (CD18/CD11b), being numerically upregulated, also represents the complement receptor III. As evidenced by nature's experiment of genetic deficiency in beta 2-integrin adhesion receptors (leukocyte adhesion deficiency syndrome type I), the lack or the dysfunction of beta 2-integrins is followed by severe bacterial infections (11).

CD56+/CD16+ cells are considered to represent NK lymphocytes, which, without previous sensitization by specific antigens, can kill, e.g., virus-infected cells or tumor cells in a myosin heavy chain-independent manner (30).

Concerning the kinetic of the changes in the expression of beta 2-integrins and the number of NK cells, activation of neutrophils reached its peak on D65, i.e., before D105 when the rise in CD56+/CD16+ cells reached its maximum (Table 2). One may speculate that these signs of activation of the innate part of the immune system can be considered as a compensatory reaction to the changes in the percentages among specific lymphocyte subsets. According to our results, the ratio of T-helper (CD4) to T-cytotoxic and T-suppressor (CD8) cells was significantly decreased already at D65 and kept low throughout the whole observation period compared with the respective ratio determined before the start of the hypokinesia period (Table 2). Because a fall in CD4+ cells, and hence in the ratio to CD8+ cells, is usually observed in states of immunosuppression, the decrease in CD4 to CD8 might reflect the compromise of the specific part of the immune system during prolonged periods of HDT. In addition to immunophenotyping of functionally distinct lymphocyte subsets, however, one wishes to have studied more directly the functional parameters of the specific and innate immune systems by appropriate in vitro or in vivo assays (e.g., lymphocyte proliferation after challenge by polyclonal or specific stimuli and cytotoxic activities of neutrophils and NK cells, respectively).

In an attempt to interpret the coincidence of the development of psychic stress and the functional and enumerative alterations of leukocyte subsets in peripheral blood, it is worthwhile to briefly consider data published on stress-induced changes in the immune system. A meta-analysis of 38 peer-reviewed publications on the effects of short-term or long-term stressors on immunity (9) indicated that short periods of stress were positively correlated to the concentration of WBCs and negatively related to the number of circulating B and T lymphocytes, as well as to the blood concentrations of T-helper and T-suppressor cells. In addition, catecholamine secretion due to short-term stressors was shown to be associated with a transient increase in NK cells. In imitating acute stress, infusion of adrenaline was followed by a pronounced increase in NK cells (C56+/CD16+ cell fraction). Noradrenaline also caused a rise in NK cells, although to an extent lower than that induced by adrenaline (3). These observations were confirmed and extended by investigations on the effects of acute psychological stress. It was shown that acute mental or emotional stress predominantly increased the number of NK cells (3).

The regulation of NK cells is not yet fully understood. However, the rise in NK cells in the circulation observed after intravenous administration of catecholamines may well be due to the mobilization of these cells from the extravascular space or to the release from the intravascular marginal pool (3). Thus one might explain the observed rise in the number of NK cells (Table 2) by the increase in secretion of catecholamines (Table 1), although the kinetics of both changes appear not to fit together.

In contrast to situations of acute stress, social stress, which is supposed to exert its effects for longer periods of time, was shown to correlate negatively with the proliferative responses of lymphocytes to polyclonal T-cell mitogens like phytohemagglutinin and concavalin A. In addition, the activity of NK cells decreased (9). A likely explanation for such effects is the enhanced release of corticosteroids, which might also modulate the interacting network of T-helper (CD4+) and T-suppressor (CD8+) lymphocytes. In this regard, it is worth noting that corticosteroids can induce alterations in the lymphocyte subpopulations of CD4+ and CD8+ cells, leading to a decrease in the ratio of CD4+ to CD8+ cells (33). Similar changes were observed during severe stress (9). Because in our study evening saliva concentrations increased two- to threefold during HDT (without significant changes in morning concentrations), one is tempted to speculate that the decrease in the ratio of CD4+ to CD8+ cells was caused in part by the enhanced evening cortisol secretion. However, the kinetics of both the reduction of CD4/CD8 (Table 2) and the rise in evening cortisol values (Table 1) again do not fit.

Therefore, it would seem appropriate to consider also, besides catecholamines and cortisol, other factors with known immunomodulatory effects (e.g., vasopressin, dehydroepiandrosterone, prolactin, and growth hormone) (9). This situation becomes further complicated by the interplay of the effects of stress-dependent hormones, immune-competent cells, and cytokines.

Interestingly, we could observe increasing concentrations of IL-6 during HDT (Table 2). IL-6 is known to be secreted mainly at inflamed sites but also during stress. In addition, it seems to be positively controlled by catecholamines (21, 29). This cytokine, which is produced by several cells, including immune cells, has pleiotropic effects in the regulation of the immune response (13). Notably, it regulates the acute-phase response of the body to various forms of cellular stress. Moreover, it was shown to play an important role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis as well. IL-6 is a potent stimulus for the secretion of growth hormone and thyroid-stimulating hormone (29). In a transgenic mouse model with increased astrocytic expression and release of the cytokine IL-6, the HPA axis was upregulated, leading to increased plasma concentrations of cortisol during immobilization stress (22). These observations are in agreement with the known stimulation of the HPA axis after acute administration of IL-6 (31). On the other hand, cortisol is a well-documented downregulator of IL-6 (34). However, when data from Table 1 are compared with those from Table 2, changes in plasma concentrations of IL-6 were best correlated with the duration of the bed-rest period but hardly with changes in diurnal cortisol secretion or daily urine catecholamine secretion. However, because plasma concentrations of TNF-alpha and IL-1 were not altered during the entire observation period, the rise in IL-6 appears to be related rather to psychic stress than to nonspecific inflammatory reactions.

Summary. HDT at 6° for 120 days was associated with the development of psychic stress as indicated by a self-estimating stress test, by the increase in the evening secretion of cortisol, the subsequent loss of the diurnal rhythm, as well as the rise in the urine excretion of catecholamines. Functional and enumerative evaluation of peripheral WBCs indicated activation of the innate part of the immune system as suggested by the enhanced expression of beta 2-integrin adhesion molecules on circulating neutrophils and a rise in the relative and absolute number of NK cells. In contrast, relative numbers of CD4+ T lymphocytes slightly decreased, whereas CD8+ T lymphocytes increased. As a result, the ratio of CD4+ to CD8+ cells decreased, which might be associated with a compromise of the specific immune system. In parallel to the onset and duration of the hypokinesia period, plasma concentrations of IL-6 increased, whereas other inflammatory cytokines (TNF-alpha and IL-1) remained unchanged.

These results demonstrate that the long duration of HDT at 6° is a reliable approach to inducing psychic stress and hence appears to be useful for the study of neuroendocrine-related changes of the immune system. In view of long-duration flights on space stations or to other planets, such as Mars, there is a need for additional experiments by which the biological significance of spaceflight-induced changes of immune parameters can be investigated further. In this respect, HDT appears to be a helpful ground-based surrogate model.


    ACKNOWLEDGEMENTS

This study was supported by Deutschen Agentur für Raumfahrt-angelegenheiten Grant 50-WB9654.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Thiel, Clinic of Anaesthesiology, Ludwig-Maximilians-Univ. Munich, 81366 Munich, Germany (E-mail: manfred.thiel{at}ana.med.uni-muenchen.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9 March 2000; accepted in final form 11 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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J APPL PHYSIOL 90(5):1736-1743
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


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