Journal of Clinical Immunology, Vol. 13, No.

2, 1993

Changes of Natural Killer Cells During Acute Psychological Stress

MANFRED SCHEDLOWSKI, 1"4 ROLAND JACOBS, 2 GERTRUD STRATMANN, 2 STEFAN RICHTER, 3 ANDREAS HADICKE, 3 UWE TEWES, I THOMAS O. F. WAGNER, 3 and REINHOLD E. SCHMIDT 2

Accepted: October 23, 1992

Emotional stress is often followed by increased susceptibility to infections. One major role in the immediate immune response to infection is played by natural killer (NK) cells. This study was designed to establish whether acute psychological stress influences cellular immune functions and to elucidate the role of endocrine parameters as potent mediators of stress induced alterations of the immune system. Forty-five first-time tandem parachutists were examined continuously for their plasma concentrations of cortisol and catecholamines from 120 min before to 60 min after jumping. Lymphocyte subsets, NK activity, and ADCC were determined 2 hr before, immediately after, and 1 hr after jumping. There was a significant increase in sympathetic-adrenal hormones during (adrenaline, noradrenaline) and shortly after jumping (cortisol). Lymphocyte subsets and the functional capacity of NK cells revealed an increase immediately after jumping followed by a decrease significantly below starting values 1 hr later. These changes were significantly correlated to plasma concentrations of noradrenaline. Thus, quick mobilization of NK cells is suggested as one major mechanism for this effective adaptation of the immune system to stress situations.
KEY WORDS:

Stress; natural killer cells; catecholamines;cortisol; parachutejumping. INTRODUCTION Stress has been suggested for a long time to be associated with a decrease in natural killer (NK) 1Division of Medical Psychology, Medizinische Hochschule Hannover, Posffach61 01 80, 3000 Hannover61, FRG. 2Divisionof Immunology,MedizinischeHochsehuleHannover, Postfach 6l 01 80, 3000 Hannover61, FRG. 3Division of Endocrinology, Medizinische Hochschule Hannover, Postfach61 0t 80, 3000 Hannover61, FRG. 4To whom correspondence should be addressed at Abteilung Medizinische Psychologie, Medizinische Hochschule Hanhover, Postfach61 01 80, 3000 Hannover61, Germany. 119

activity. There is also accumulating evidence from psychoneuroimmunological research (1) that stress situations such as examination (2, 3) or bereavement (4) may alter a wide range of immunological functions, especially N K cell function. Moreover, increased incidences of viral infections or cancer have been reported in stressed populations (5, 6). N K cells are believed to act early in the immune response, before specificity can be generated, and represent approximately 12-15% of peripheral blood lymphocytes (7-9). They mediate first-line defense by direct cytotoxicity against various types of target cells without apparent prior immunization (10). More recently they have been characterized phenotypically as coexpressing the cell surface antigens CD16 and CD56 and lacking the CD3/TCR complex (11). N K cells have also been demonstrated to play an important role in the immune surveillance against tumors and virally infected cells (9, 10). Other reports have shown that N K cells represent a subset of peripheral blood lymphocytes which quickly respond to activation signals such as interleukin-2 (IL-2) via the respective cytokine receptors in vivo and in vitro (7, 12). In vitro stimulation of N K cells with high concentrations of adrenaline causes a decrease in N K function. In contrast, lower concentrations appear to increase N K activity (13). In vivo administration in humans also induces an increase in N K activity with a rapid decrease during follow-up (t4, 15). Other in vivo data in humans during physical stress reported an increase in N K activity after exercise (16-i8). These observations together with data demonstrating that lymphocytes express receptors for a variety of hormones (19) suggest that various neuroendocrine parameters may influence N K activity in

0271-9142/93/0300.0119507.00/0 1993 Plenum Publishing Corporation

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peripheral blood. Whether psychological stress is influencing peripheral blood lymphocytes and, in particular, NK cells and which hormones are involved have not been established yet, since the manipulation of the sympathetic-adrenal system in vivo has remained difficult. In order to analyze physiological parameters such as hormones and alterations in the cellular immune system in an acute psychological stress situation, a model with a high degree of experimental control was chosen (20-22). First-time tandem parachutists before, during, and after jumping were continuously monitored for their heart rate and plasma concentrations of the sympathetic-adrenal hormones cortisol and catecholamines. At three defined time points lymphocytes and their respective subsets were characterized for cell surface antigens and their functional capacity. MATERIALS AND METHODS

tubes containing glutathione. Blood flow was adjusted to 0.8 ml/min. Every 10 min the tubes were changed and samples for the endocrine variables were fractionated. The samples were centrifuged at 4C and stored frozen at -70C until assayed. In addition, blood samples were drawn to determine immunological parameters: 2 hr before the jump (baseline), immediately after, and 1 hr after the jump. All analyses were performed immediately after the last sample was collected.

Endocrine Analyses
All samples of each subject were analyzed in the same assay. Cortisol was determined by radioimmunoassay (Biermann GmbH, Bad Nauheim, Germany). Catecholamine plasma levels were analyzed by high-pressure liquid chromatography (HPLC, electrochemical detector).

Subjects and Procedure

Forty-five male subjects aged 19-39 years (mean = 25.4 years) participated in this study after giving their informed consent. All subjects had passed an intensive health examination before entrance. Subjects with drug or alcohol abuse, medication, or infections within the last 2 weeks were excluded. All subjects performed tandem jumps, i.e., the novice was secured in front of an experienced tandem master. Jumps were performed between 10.00 and 12.00 hr. Two to three hours before jumping the catheter was inserted and baseline samples were drawn. Boarding time was 30 min before exit. During ascent of the plane to exit altitude (3500 m) subjects sat in the plane without being physically active. Jumpers had 45 sec free-fall time until the parachute was released and approximately 5 min under the open parachute until landing. Subjects completed the State Trait Anxiety Inventory (STAI) (23) to determine the state anxiety at baseline and immediately before exit. Heart rate was continuously recorded with Ag-AgCI electrodes in the lead II configuration by using a portable recording system (24). For continuous blood sampling a catheter (Certofix, Braun Melsungen, Melsungen, Germany) was inserted into a brachialis vein. The catheter was connected to heparinized silicon tubing (1 mm +). Blood was continuously drawn by a small portable pump (Fresenius, Homburg, Germany) and collected in lithium heparin

Monoclonal Antibodies
The monoclonal antibodies used have been described in detail previously (25): CD2 (Leu5b), CD3 (Leu4), CD8 (Leu2a), CD16 (Leullb), CD56 (Leul9 PE), and CD25 were purchased from Becton Dickinson (Heidelberg, Germany). CD3 (OKT3), CD4 (OKT4), and CD8 (OKT 8) were from Ortho (Neckargemfind, Germany). CD2R (Tll.3), CD26, and CD56 (NKH-1A) were generously provided by Drs. S . F . Schlossmann, E. L. Reinherz, and J. Ritz (Dana Farber Cancer Institute, Boston, MA). Tu27 (anti-p75) was generously given by Dr. K. Sugamura (Department of Microbiology, Tohuko University School of Medicine, Sendai, Japan).

Phenotypic Analyses
Phenotypic analyses were performed by indirect immunofluoreseence using fluorescein-conjugated goat anti-mouse F(ab') Ig (GM-FITC, Dianova, Hamburg, Germany). For two-color analyses directly labeled antibodies were utilized. The staining procedure has been described in detail previously (26). Briefly, 1 to 3 105 cellsAvell were incubated with murine monoclonal antibodies at an optimal dilution for 30 min. Nonspecific binding was eliminated by mixing the samples with a 1:5 solution of a commercial human IgG (Intraglobin, Biotest, Frankfurt, Germany). Samples were incubated for another 30 min with GM-FITC and washed three times, and 10,000 cells
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121

were then analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany).

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Cell Lines
Several continuously growing cell lines were used. K562 is an erythromyeloid human line, L1210 a murine lymphoma cell line.

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Cytotoxicity Assays
All cytotoxicity assays were performed in triplicates at various effector to target (E/T) ratios using V-bottom microtiter plates with 5 103 5aCrlabeled target cells per well as previously described in detail (26). Medium for cytotoxicity assays was RPMI 1640 supplemented with 5% FCS and 1% penicillin-streptomycin. Specific cytotoxicity was measured following 4 hr of incubation at 37C by determining the 51Cr release. Spontaneous release was determined by incubating target cells without effector cells. Maximal release was obtained by lysing target cells with the detergent NP40. The specific lysis was calculated as described (27). Lytic units (Lid) were calculated as described in detail previously (28). The calculation of LU for NK activity was based on the number of CD56 + cells, and that for ADCC on the number of CD16 + cells.

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1800

Statistical Analyses
Statistical analyses of the data were performed by using ANOVAs with repeated measures. All repeated-measures analyses of variance were corrected for nonsphericity by the G e i s e r - G r e e n h o u s e method (29). For each repeated measure we report the uncorrected degrees of freedom and the epsiloncorrected P value. Pearson correlations were performed to determine the correlations between the physiological and the immunological parameters.
RESULTS

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Fig. 1. Heart rate frequencies (A) and plasma levels of cortisot (B), adrenaline (C), and noradrenaline (D) before, during, and after the jump at 10-rain intervals from 120 rain before to 60 man after the jump. Data are expressed as mean -+ standard deviation ( ~ ) .

State anxiety of the subjects was within normal range at baseline (M = 34 - 7.4) and significantly increased immediately before exit (M = 45 __. 9.4) (df = 1, F = 63.43, P < 0.0001). The heart rate as representative parameter for cardiovascular reactivity was recorded throughout 3 hr, i.e., during preparation, at exit, and after landing (Fig. 1A). Heart rate increased before boarding (30 min before exit) and reached 152 beats/min during exit. The

frequency normalized 20 min after the jump (df = 18, F = 79.92, P < 0.0001). In parallel to the heart rate, plasma concentrations of cortisol and catecholamines from 120 min before to 60 min after the jump were determined at 10-rain intervals (Figs. 1B-D). Adrenaline peaked at 400 ng/L synchronously with the heart rate during the jump itself (df = 18, F = 60.03, P < 0.0001) (Fig. 1C). Cortisol and noradrenaline peak levels appeared slightly delayed

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SCHEDLOWSKI ET AL.

compared to adrenaline levels (Figs. 1B and D). Peak values for cortisol were 23 ~g/dl and did not reach baseline levels within 1 hr after exit (dr = 18, F = 17.72, P < 0.0001). In contrast, noradrenaline (df = 18, F = 19.00, P < 0.0001) and adrenaline quickly decreased within 20 min after exit and reached normal plasma concentrations. No significant alteration could be observed for plasma levels of dopamine 120 min before to 60 min after the jump (df = 18, F = 1.27, ns) (data not shown). When peripheral blood lymphocytes and their respective subsets were measured before, immediately after, and 1 hr after jumping, a significant increase in CD2 + cells (T and N K cells) (df = 2, F = 20.48, P < 0.0001) and, also, in CD3 (df = 2, F = 10.92, P < 0.0001), CD4 (df = 18, F = 5.86, P < 0.01), and CD8 (dr = 18, F = 28.64, P < 0.0001) lymphocytes was observed (Fig. 2A). The T lymphocytes 1 hr after jumping were significantly decreased compared to values 2 hr before exit (P < 0.01). The most impressive changes were found for N K cells. Immediately after the jump the absolute number of CD16 cells increased by more than 100%. One hour after jumping a decrease below prejump values (280 N K cells/l~l) was observed (df = 2, F = 41.58, P < 0.0001). Similar numbers were obtained when CD56 + cells (df = 2, F = 36.56, P < 0.0001) were determined (Fig. 2B). In parallel to these enormous changes of the N K cells, alterations of activation antigens occurred (Fig. 2C). The number of p75 lymphocytes increased significantly immediately after the jump and dropped thereafter (dr = 2, F = 39.34, P < 0.000t). The pattern was completely parallel to the changes of N K cells, p75 is known to be expressed mainly on N K cells (11). Using two-color fluorescence it was demonstrated that nearly all CD56 N K cells express p75 IL2R13 chain (Fig. 3). In addition, there was a significant change in CD26 cells (df = 2, F = 5.49, P < 0.01), while statistical analysis revealed no significant differences for CD25 and CD2R + cells. N K activity and ADCC were determined in parallel to the phenotypic studies to test the N K lymphocytes for their functional capacity. Significant changes for both types of cytotoxic activities were observed, demonstrating that the increase in absolute N K cell numbers and the relatively enhanced frequency in peripheral blood was paralleled by an increase in specific cytotoxicity (NK and ADCC at an E:T ratio of 15:1: df = 2, F = 64.77, P < 0.0001, and df = 2, F --- 51.01, P < 0.0001) (Fig. 4). When we then transformed these cytotoxic activities into lytic units, the

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differences were even more significant (Table IA). But clearly the lytic activity per celt revealed dramatic increases and decreases as well (Table IB). In addition, covariance analysis with repeated measures, with the absolute numbers of N K cells (CD16 + or CD56 ) as the covariate, were performed. After controlling for N K cell numbers the alterations in N K

Journal o f Clinical Immunology, Vol. 13, No. 2, 1993

CHANGES OF NK CELLS DURING STRESS

123

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activity and ADCC over time remained significant (data not shown). In order to analyze which of the psychological, cardiovascular, and endocrine variables studied corresponded to the observed alterations in immune functions, Pearson correlations of anxiety measures, heart rate, endocrine variables, and immunological parameters were calculated. There were no significant correlations between the anxiety measures, heart rate, cortisol, and immunological variables. However, catecholamines, especially noradrenaline plasma levels at 10-min intervals, correlated significantly with CD8 + cells and NKcell numbers (CD16 + and CD56 +) and functions
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(NK activity) immediately after the jump and 1 hr after the jump. The correlation coefficients for each time point are given in Tables II and III. In contrast, the plasma concentrations of adrenaline significantly correlated only in the sample 30 min before the jump with the CD56 + cells immediately after jumping (r = 0.37, P < 0.05) and adrenaline plasma levels collected 50 rain after the jump correlated significantly with CD16 + cells (i- = 0.39, P < 0.05), CD56 + cells (r = 0.37, P < 0.05), and N K activity (r = 0.34, P = 0.05) 1 hr after jumping. DISCUSSION It has been well documented that physical exercise induces an increase in N K cells and their

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I. N K Cytotoxicity and ADCC as Lyric units (LU) (A) and L U per Single Cell for N K Activity and ADCC (B) at Three Time Points: 2 hr Before the Jump (I), Immediately After the Jump (II), and 1 hr After the Jump (III) A

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L U per single cell NK ADCC 0.0006 0.0025 II 0.0019 0.0830 III 0.0005 0.0022

Fig. 4. N K cytotoxicity and ADCC before ( ~ 1 ) , immediately after (12;51), and 1 hr after ( I l l ) the jump. Different effector-totarget cell ratios were utilized and specific lysis was calculated. Data are presented as mean -+ standard error.

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Table II. Matrix of Significant Pearson Correlation Coefficients (r) B e t w e e n Plasma Noradrenaline at 10-rain Intervals Prior to and Including Exit, on the O n e H a n d , and C D 8 + , C D 1 6 + , and C D 5 6 + Cells and N K Activity, on the Other, Immediately After the J u m p Interval prior to and including Exit (min) 120 110 t00 90 80 70 60 50 40 30 20 10 Exit aNonsignificant. *P < 0.05. **P < 0.01. Immediately after j u m p CD8 __a 0.42* 0.43** 0.44** 0.38* 0.37* 0.40* 0.35* 0.44** 0.46** 0.47** 0.35* -CD16 __ 0.42** 0.46** 0.49** 0.45** 0.46** 0.42** 0.39* 0.42** 0.42** 0.45** 0.35* -CD56 __ 0.39* 0.42* 0.46** -0.34* 0.38* -0.41"* 0.40** 0.39* 0.33* -NK activity __ 0.62** 0.57** 0.59** 0.39* 0.43** 0.49** -0.48** 0.42** 0.36* 0.41"* 0.33*

functions. For psychological stress, though, possible alterations of the immune system and the hormones involved are not clear (30). A parachute jump has been employed previously as a model of well-controlled intensive psychological stress showing that the increase in cardiovascular activity and hormonal response is caused principally by the psychophysiological arousal prior to and during a jump (20-22). The present study using this model demonstrated that emotional strain induces significant changes not only of various hormones but also of leukocytes and their subsets. An increase in granulocytes and a significant change in absolute numbers of lymphocytes were observed. Among the
Table I l L Matrix of Significant Pearson Correlation Coefficients (r) B e t w e e n P l a s m a Noradrenaline at 10-min Intervals After the J u m p and C D 8 + , C D 1 6 + , and C D 5 6 + Cells and N K Activity 1 hr After the J u m p 1 hr after j u m p CD8 0.39* 0.39* 0.50** 0.45** 0.52** 0.47 CD16 J -0.45** 0.46** 0.43** 0.47** CD56 0.33* 0.38* 0.47** 0.41"* 0.49** 0.47** NK activity --0.32* -0.35* 0.35*

Interval after j u m p (rain) 10 20 30 40 50 60

aNonsignificant. *P < 0.05. **P < 0.01.

various circulating lymphocyte subsets, the most significant increases were determined for CD8 + cells and, in particular, for N K cells (Fig. 2). The latter quickly more than double in number in the stress situation, drop thereafter, and subsequently return to levels significantly below those obtained 2 hr before jumping in the same individuals. We have to assume that also the alteration of CD8 + cells is due mainly to N K cells, since 30-50% of N K cells also express low-density CD8 antigen. In addition, examination of activation antigens revealed a significantly enhanced expression of the intermediate-affinity IL-2 receptor p75, which reflects the NK-cell increase. Functional studies demonstrated that increased numbers of N K cells are accompanied by significantly enhanced N K and ADCC activity. The decrease in NK-cell numbers after the jump is paralleled by a functional loss of activity (Fig. 4). But additional calculation of lytic units and covariance analysis suggest that the functional changes observed not only reflect the alterations in NK-cell numbers, but also occur on a per-cell basis. Studies in individuals during physical exercise have also reported increased N K cells and activity (15-18) followed by a decrease after exercise (17, 3i). Here we demonstrate that psychological stress also alters the distribution of circulating lymphocytes to the same extent. In contrast to the phenotypic changes observed in increased NK cells after bone marrow transplantation or in in vivo IL-2 application (32, 33), the surface antigen characteristics of the enhanced NK cells immediately after the jump were not changed to high-density CD56 expression, suggesting that at least one major mechanism is quick mobilization of N K lymphocytes from marginal pools in the spleen and the lung. The enhanced secretion of cortisol, adrenaline, and noradrenaline represents the sympatheticadrenal activation during psychophysiological arousal. While suppressive in vitro effects of glucocorticoids on NK-cell functions have been described (34), in our study there is no correlation between plasma levels of cortisol and lymphocyte subpopulations or NK-cell functions at any time. However, the changes in NK cells and activity at most time points before and after the jump are significantly correlated with preceding plasma levels of noradrenaline (Tables II and III). This analysis is, of course, restricted to the first hour after jumping and may therefore be biased toward shortterm effects caused by noradrenaline.

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For physical exercise it has been postulated that catecholamines may cause increased N K activity by direct interaction with these lymphocytes. This hypothesis is supported by the observation that in vivo administration of adrenaline in man increased both NK-cell numbers and activity (14, 15, 35). On N K cells and T cells, 13-adrenergic receptors have been identified, with the highest receptor density on N K cells (36-38). However, it is still controversial whether the observed increases in lymphocyte subsets are mediated via t3t or [~2 adrenergic receptors on these lymphocytes. While one study found no changes in the distribution of T and N K cells after infusion of the [31-selective agonist noradrenaline (37), another study reported a significant increase in NK activity after noradrenaline administration and a subsequent decrease 30 rain later (39). Correlations between exercise-induced increases in circulating lymphocytes and adrenaline (38) and the noradrenaline-associated increase in T and N K cells after moderate exercise (31) provide further evidence for an adrenergic stimulation of lymphocytes via 131 and 1~2 receptors. According to other studies, NK-cell trafficking could be due to vascular contraction in the spleen and other marginal pools for N K cells, which may be induced predonfinantly by noradrenaline (40). We suggest that the observed changes of cell numbers in our study most likely must be attributed to cell mobilization from respective reservoirs such as spleen and lung (37). However, this mechanism by itself explains the increase in lytic activity only in part. Certainly a significant part of the enhancement in cytotoxicity is due to the increased lytic activity of the individual cells (Tables IA and B). Alternatively, some of the other sympathetic-adrenal hormones and neuropeptides (41, 42) or their interaction with cytokines such as IFN-~/ and TNF-a might be responsible. Other studies reported no association between catecholamirle levels and increased N K activity during brief psychological stress (43). Moreover, it needs to be considered that the exerciseinduced increase in N K activity was attenuated by prior administration of naloxone, an opioid antagonist (44). Cytokines and hormones might also alter expression of adhesion structures on N K or endothelial cells, allowing fast NK-cell release and most likely a fast pooling thereafter (45). In contrast to T and B cells, even from animal studies there is not much known on migration and recirculation of NK cells (46). Further research is needed to clarify these mechanisms. The biological meaning of the increase

and subsequent decrease in circulating lymphocytes, particularly of N K cells after acute stress, may be interpreted as an adaptation to environmental stimuli. Since evidence is provided here for a decrease in NK cells after psychological stress, the time kinetics of these changes are of further interest. In summary, these data suggest that short-term psychological stress leads to an enhanced secretion of sympathetic-adrenal hormones, with a rapid increase and subsequent decrease in N K cell numbers and their respective functions. These changes are significantly correlated with plasma levels of noradrenaline, indicating a central nervous-immune system interaction during phases of intense emotional stress. ACKNOWLEDGMENTS This work was supported by the VolkswagenStiftung Germany, Grant 1/66 077, and by funds of the Deutsche Forschungsgemeinschaft DFG Schm 596/3-2. We thank Drs. Helmuth Deicher and Reinhard Pabst for critically reading the manuscript and Jens Alker and Frank Pr~hl for excellent technical assistance.
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Journal o f Clinical Immunology, VoL 13, No. 2, 1993