Clinical and Experimental Hypertension, 30:720–731, 2008Copyright © Informa Healthcare USA, Inc.ISSN: 1064-1963 print / 1525-6006 onlineDOI: 10.1080/07420520802572333

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LCEH1064-19631525-6006Clinical and Experimental Hypertension, Vol. 30, No. 8, October 2008: pp. 1–23Clinical and Experimental Hypertension

Leucocyte Subset Redistribution in a Human Model of Physical Stress

Leucocyte Subset Redistribution and Physical StressF. Mignini et al. FIORENZO MIGNINI,1 ENEA TRAINI,1 DANIELE TOMASSONI,1 MARIO VITALI,2 AND VALENTINO STRECCIONI1

1Sezione di Anatomia Umana, Dipartimento di Medicina Sperimentale e SanitàPubblica, Università di Camerino, Camerino, Italy2Dipartimento di Medicina Clinica, Università di Roma “La Sapienza”, Roma Italy

This study has investigated, under controlled conditions, peripheral mononuclear cells(PMNC) subset redistribution in a human experimental stress model consisting ofcycloergometer activity in healthy male volunteers exposed to a stressful stimulus.After stressful stimuli, leucocyte subpopulations undergo a stereotyped redistributionpeculiar for each PMNC cytotype. PMNC subpopulations involved to a greater extentwere natural killer (NK) cells and lymphocytes T “memory” cells. The post-stressperiod was characterized by a decrease of the NK subpopulation. Our findings confirmthe view of a sensible functional reduction of immunocompetence in stress conditions.This brings to the opening, even if for a short time, an “immunological window.”. Thiswindow remains open throughout the time of the stimulus, probably representing thebasis of the progressive reduction of the competency of immune system. Catecholaminessupport the acute effects of stress influencing the anatomical redistribution of lymphocytesubpopulation and intermediating acute effects on PMNC. Cortisol, acting for longertime, contributes to create and maintain both the neutrocytosis and lymphopenia in thepost-stress period following lymphocytosis.

Keywords experimental stress, physical work, leucocytes, lymphocyte subsets, NKcells, immunological window

Introduction

Stress is an upset that arouses homeostasis. It was defined by Seyle as the organism’s non-specific response to very abnormal requests (1). Stressful agents are the etiological factorsthat induce stress. They are also defined as “stressors,” and may be physiological, environ-mental, or psychical. Examples of physiological stressors are surgical operations, injectionsof heterologue proteins, several diseases, anaesthesia, haemorrhage, physical efforts, andtrauma. Environmental stressors include long exposure to heat or cold, radiation, atmosphere

Submitted March 13, 2007; accepted April 24, 2007.Address correspondence to Prof. Fiorenzo Mignini, Dipartimento di Medicina Sperimentale e Sanità

Pubblica, Via Madonna delle Carceri, 9 62032 Camerino (MC), Italy; E-mail: fiorenzo.mignini@unicam.it

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Leucocyte Subset Redistribution and Physical Stress 721

pollution, and several chemical agents. Psychical stressors include harsh competitivenessbetween members of the same group, several extended conflicts, impact of new or opposing,intense emotions, grudge, frustration, feeling of inferiority, and repetitive jobs.

For the human body, overcoming stress corresponds to reaching a new homeostaticcondition. This condition may lead to the so-called “adjustment syndrome,” which is dividedinto the three phases listed below:

1. Alarm reaction. It begins as soon as the organism is exposed to an abnormal stimulation.In this phase, passive phenomena take place. They form the “shock” that anticipatesactive phenomena (counter shock), characterized by an exaggerated function of theadrenal cortex, leading to the overproduction of glucocorticoid and mineralcorticoidhormones.

2. Resistance phase. It is carried out against the original stressor if its action lasts for along time. In this phase, resistance to other stressors is reduced.

3. Exhaustion phase. Stimulus lasts for a given time and is accompanied by a progressivereduction of resistance to all the stressors. The time in which stressing stimulus lastsarouses the equilibrium reached by the organism during the second phase. This is followedby a new alarm phase. Hence, stress is an excessive response of the organism to anacute stimulus or it sets up when the stimulus is not exhausted in the due time.

In the last fifteen years, several studies have demonstrated that physical activity and stresscan alter immune function (2–5). Stressors of physical or psychological nature are veryhigh in number, and all of them can induce immuno-endocrine responses similar to thoseinduced by physical activity (6–10). Stressful physical activity represents a good experi-mental model, because it is quantifiable and repeatable (7,8). Such a model is easily realiz-able and allows one to understand, in controlled and repeatable conditions, therelationships between physical activity, stress, and immunological pathways. The presentstudy was designed to investigate peripheral blood mononuclear cells subset redistributionin an experimental stress model consisting of cycloergometer activity in healthy malevolunteers exposed to stress stimuli of pre-established intensity and time.

Material and Methods

Subjects

Healthy Volunteers. Twenty male trained subjects, not engaged in competitive sports, withage between 18 and 30 years (mean 27) and weight between 71 and 90 Kg (mean 79 Kg),were enrolled after informed consent. They were admitted into the study after personal andfamilial anamnesis and medical check-up. Exclusion criteria were acute or chronic infec-tions, allergy, acute or chronic use of medicines, tobacco use, and known hypersensitivity orallergy to pharmacological agents, including non-steroid anti-inflammatory drugs (NSADs).

Maximum Oxygen Consumption

Maximum oxygen consumption (VO2max) was determined for each subject before theexperiment by increasing progressively the rate of exercise up to the minimum strain. Theintensity of the workload was expressed as cardiac frequency. This parameter was chosendue to its relationship with oxygen consumption and because it is independent from sex,age, or athletic valor. The error in the appraisal of VO2max from the cardiac frequency is

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about 8%. The efficacy of this relation allows us to monitor strength of the work by thecardiac frequency and is valid for work made by arms or legs, in healthy subjects of normalor over weight as well as for cardiopathics and paraplegics (11–14).

Optimal cardiac frequency (FC) for training is assessed by the following formula:

with training frequency slightly higher than the 70% of the maximum frequency value(FCmax). The FCmax was evaluated for each subject participating to the investigation. Allsubjects were submitted every min for 10 min on the same cycloergometer to an increasedworkload, constantly monitoring the pulse by a cardiofrequencymeter (Vantage XL, Polar,New York, USA), linked to a computer. The FCmax was taken as the point of bending inthe curve obtained from the FC put in function of the increase of the workload. In thestress test, the point of 0.75, corresponding to the 75% of the FCmax, was used.

Stress Test

Stress test consisted of one hour at the cycloergometer, at an intensity greater than 75% of theFCmax, assessed as described above. In our stress model, the scale of strength (intensity) andthe duration of the muscular work were determined in some preliminary experiments, allow-ing us to classify muscular work as percentage to aerobic power as follows: light (<50%),sustained-moderate (50–65%), sustained-vigorous (65–85%), and maximal (85–100%).

Blood Sampling and Blood Analysis

Blood samples were taken from the antecubital vein one week before, within 10 min afterthe end of the muscular work, every hour in the three hours after exercise (recoveryperiod), and after 24 hours. Blood samples one week before the test were taken forassessing basal values at rest. To avoid interference with the subsequent analysis,subjects were requested to abstain from any exercise in the two days before the firstblood withdrawn. Volunteers were free to take water and carbohydrates before, during,and after the exercise. Blood samples were taken by vacutainer (Becton Dickinson,New York, USA) and centrifuged at 300 rpm at 4°C for 5 min. Plasma was then obtainedand put at -80°C until analysis. The first blood samples were taken from volunteersbetween 8:00 and 9:00 AM. On plasma samples cortisol levels were assayed by a directenzyme-linked immunosorbent assay (15), whereas hemoglobin and hematocrit weredetermined on samples of peripheral blood treated with K3EDTA with a Coulter JTautoanalyzer (Coulter Electronics, Hialeah, Florida, USA).

Separation of Lymphocytes from Blood Samples

Lymphocytes were separated by centrifugation in a density gradient of Fycoll-Hypaque(Pharmacia, Uppsala, Sweden). Samples were centrifuged at 1,800 rpm for 40 min.Lymphocytes, stratified in the middle, were separated, washed in RPMI, centrifuged at1,800 rpm for 20 min, and kept at 4°C in RPMI with 10% of PBS for no more than 12 h.Lymphocytes were stained with a Turk solution (80 ml of staining and 20 ml of cellularsuspension) and counted in a Burker camera. The best cellular concentration obtained was3 × 106 cells/ml.

FCtraining = FCrest + 0.6 (FCmax FCrest ), × −

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Immunofluorescence and Fluorescence Activated Cell Sorting (FACS) Analysis

Lymphocyte phenotypes were assessed by immunofluorescence and fluorescence activatedcell sorting (FACS) analysis using human monoclonal antibodies (Mab) for the CD3,CD4, CD8, CD19, and CD56 lymphocyte analyis. The CD8 antigen is present in bothT cells and natural killer (NK) cells. The number of CD8+ T cells was therefore evaluatedby subtracting the number of NK cells from the total of CD8 cells. Cellular suspensions(106 cells), maintained in a complete medium, were incubated with different Mabs for30 min at 4°C and washed three times with PBS . Relative intensity of cellular fluorescencewas evaluated, and the percentage of positive cells, determined on 10,000 events, was ana-lyzed with a FACScan Cytofluorimeter (Becton Dickinson, San Jose, California, USA).Fluorescence intensity was expressed on logaritmic scale with arbitrary units.

Isolation of NK Cells from PBMC

Separation of NK cells was made by negative selection using magnetic spheres (Dynal,OXOID S.p.A., MI, Italy) conjugated with the following specific primary monoclonalantibodies: CD14 for monocyte and macrophages, CD19 for B cells, and CD3 for CD3+ Tlymphocytes. To the blood cells of the white series, isolated on Fycoll, washed with PBS/FCS (2%) and re-suspendend at a concentration of 10–20 × 106 cells/ml, magnetic spheres(“dynabeads”) were added at a concentration of 10 × 106 spheres/ml. Samples were incu-bated for 1 hour at 2–4°C. Ten ml of PBS/FCS 2% were added and test tubes were put ona magnet for 2–3 min. The supernatant containing NK cells was transferred to anothertube, and the concentration of cells was adjusted to 5 × 106 cells/ml. Changes of plasmavolume were calculated on the basis of haemoglobin and haematocrit values (16) andcorrected accordingly.

Indomethacin Administration

Twenty male trained subjects were asked to take 75 mg/day of the NSAD indomethacin onthe 30th day after cycloergometer experiments. The drug was taken in the morning forthree days. This dose was chosen on the basis of pharmacokinetics studies (17).

Assay of E2 Prostaglandin (PGE2)

Plasmatic PGE2 was assayed by a competitive immunoenzymatic method (Biotrak,Pharmacia, Uppsala, Sweden). This method allows a direct measure of PGE2 on plasmawith a 100% recovery, eschewing the use and the following removal of the extractionproducts.

K-562 Cells Culture and Natural Cytotoxic Activity

Tumor K-562 cells (ATCC#243) were used as a target for cytotoxicity tests. The cell linewas maintained on logarithmic grow phase of a complete RPMI in a moist atmosphere at37°C and 5% CO2. Before the experiments, cell concentration was adjusted to 2 × 106

cells/ml. After determination of vitality (>90%), 2–10 × 106 cells were recovered on a25 ml tube. The labeling was made on pellet adding aliquots of 5mCi/ml of Na51CrO4containing 100 μCi of 51Cr. After incubation for 1h, at 37°C in an atmosphere of 5% CO2,cells were washed with 10 ml of RPMI-2 (2% FCS), and the supernatant was removed.

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The remaining cells were resuspended with 10 ml of complete RPMI, adjusting cellularconcentration at 5 × 104 cells/ml. Cytotoxic activity was determined by assessing 51Crrelease against tumoral target K-562. Labeled cell target was washed with RPMI (+10%FBS and antibiotics) and centrifuged at 1,400 rpm for 10 minutes, and the pellet wasresuspended in 2 ml of medium. Target cell concentrations and those of the effector wererespectively 5 × 104 and 5 × 106 cells/ml. The used effector/target rate was 25:1. Target andeffector were distributed in microtiter plates as protocol. After 4h of incubation at 37°C,plates were centrifuged for 5 min at 1,800 rpm, and the γ-emitting activity was determinedon the supernatant (Beckman LS 1801).

Statistics

Data were analyzed statistically by the Duncan’s Multiple Range, Mann-Whitney, and χ2

tests. Values in the text are shown as the mean ± SD (standard deviation). Duncan multiplerange was used to evaluate the significance of differences between the means of groups ofsamples, formed by the same number of elements. Mann-Whitney test was used to assess sig-nificance of differences of parameters examined between basal values and after the stressingmuscular work. Linear regression was calculated with the least squares. The relation forcebetween the variables was appraised by the Pearson’s (“r”) coefficient of correlation.To define the degree of correlation between the parameters, a level of p of 0.05 was accepted.

Results

Cortisol

After one hour of muscular work at an intensity not lower than 75% of FCmax, a significantincrease of cortisol plasma levels by about 46% was found soon after the end of the test(see Figure 1). This indicates that physical activity made was stressing for subjects inves-tigated. Cortisol plasma levels were not further changed in the four hours post-stress.

Figure 1. Stress-induced changes in plasma cortisol concentrations. Cortisol assay was performed aweek before the test (resting phase, A); soon after the end of stimulus (B); at the first (C), second(D), and third (E) hour of the recovery phase; and 24 hours later (F). Data are the means ± SE.*p < 0.01.

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Polymorphonucleated Blood Cells and Monocytes

The application of a stressing stimulus lead to an increase (p < 0.01) in the numberof polymorphonucleated blood cells (PBMC) by about 48% soon after the test.Leucocytosis, which was also present in the recovery period, was due to the post-stress increase of the number of polymorphonucleated cells (PMN) and of monocyte-macrophages. After 24 hours, the number of leucocytes returned to pre-test conditions(see Figure 2, panel A).

Physical stress significantly increased (p < 0.01) the number of PMN, which wereincreased by about 60% compared to the pre-test values. A progressive increase of PMNwas observed during the recovery time and reached a peak of a 95% increase compared toresting conditions after four hours from the end of the muscular work. Values returned topre-test conditions within 24 hours (see Figure 2, panel B).

The number of circulating monocytes (CD14+), which was unchanged at the end ofphysical work, significantly increased (p < 0.01) during the recovery period by 49% and44%, respectively, after the first and the second h after exercise. This increase was stillobserved at the third h (approximately 19%) after exercise. The number of monocytesreturned to pre-exercise values within 24 h from the end of muscular work (see Figure 2,panel B).

Figure 2. Stress-induced redistribution of total leucocytes (panel A); polymorpho nucleated cells(PNM; panel B) and CD14+ monocytes (panel C). Analysis was performed one week before the test(resting phase, A); soon after the end of stimulus (B); at the first (C), second (D), and third(E) hour of the recovery phase; and 24 hours later (F). Data are the means ± SE. *p < 0.05 vs. otherconditions.

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Lymphocytes

The number of total lymphocytes increased by about 39% compared to the pre-exercisevalues (p < 0.01) in the first 10 min after physical work. This increase was followedby a significant decrease (p < 0.01) under basal values (14%) in the first h, followedby a recovery to pre-exercise condition from the second h after exercise (see Figure 3,panel A).

The number of B cells (CD19+) was unchanged throughout the study (see Figure 3,panel B). Stress test increased by about 20% the number of T cells CD4+ (T helper/inducercells) (p < 0.01). In the first h post-stress, the number of T cells CD4+ significantlydecreased (17%) compared to resting values (p < 0.05), followed by a recovery to pre-exercise condition from the second h after exercise (see Figure 3, panel C).

The number of T-CD8+ (cytotoxic) lymphocytes increased by about 51% compared tothe pre-exercise values at the end of the test (p < 0.01), but returned to pre-test valuessince the 1st h post-test (see Figure 3, panel D).

During the test, the number of NK cells (CD16+/CD56+) was remarkably increased(by 93%, p < 0.01) compared to the pre-exercise conditions. In the recovery phase, theyquickly decreased (p < 0.05, p < 0.01) to values lower than the basal ones (19% and 22%,respectively, after 1 and 2 h). Within 24 hours, the number of NK cells returned to pre-testvalues (see Figure 4, panel A).

Figure 3. Stress-induced redistribution of total lymphocytes (panel A); CD19+ B lymphocytes(panel B); CD4+ T-Helper lymphocytes (panel C); and CD8+ Cytotoxic lymphocytes (panel 4).Analysis was performed one week before the test (resting phase, A); soon after the end of stimulus(B); at the first (C), second (D), and third (E) hour of the recovery phase; and 24 hours later (F). Dataare the means ± SE. *p < 0.01 vs. other conditions; **p < 0.05 vs. other conditions.

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Leucocyte Subset Redistribution and Physical Stress 727

NK-Mediated Cytotoxic Activity

The cytotoxic response of NK cells, under stress conditions, was different if the evaluationwas made on cellular bases or on a fixed number of PBMC, as usually done in clinicalroutine. With the clinical routine approach, in view of the stress-induced leucocyte redis-tribution, NK activity was significantly increased (by 32%, p < 0.05) compared to thebasal situation, whereas the analysis in cellular terms does not show significant differ-ences (see Figure 4, panel A).

The most relevant changes were found in the post-stress period, characterized by adecrease of NK-mediated cytotoxic activity and a subsequent normalization at 24 h afterstressful stimulus (see Figure 4, panel B). Administration of indomethacin abolishedmodifications of NK-mediated cytotoxic activity except the decreased activity at 3 h post-stress that remained unchanged (data not shown).

Plasma Prostaglandin E2 (PGE2)

Plasma concentrations of PGE2 showed a peak (p < 0.01) only at the 2nd h of the post-stress period. This increase of about 34% versus the pre-exercise value was not seen atother times evaluated (see Figure 5).

Discussion

The above data indicate that in a condition of controlled stress, experimentally inducedleucocyte subpopulations undergo a stereotyped redistribution typical for different cytotypesthat differed if evaluated during or after the stressing stimulus.

During physical activity, the adrenal medulla releases primarily epinephrine, whereassympathetic neuroeffector junctions release norepinephrine. As a consequence of thesetwo phenomena, arterial concentrations of the two catecholamines increase almost linearlywith the duration and intensity of the physical activity expressed as individual value ofVO2max (14). The localization of b-adrenoceptors on the surface of the T, B, and NK cellsof macrophage and PMN represents, in several species, the molecular basis for the recogni-tion of these cells as stress targets (6). It is also documented that the density of b-adrenoceptorschanges following activation and differentiation of lymphocytes (19,20).

Figure 4. Stress-induced redistribution of natural killer (panel A) and natural killer cytotoxic activ-ity (panel B). Analysis was performed one week before the test (resting phase, A); soon after the endof stimulus (B); at the first (C), second (D), and third (E) hour of the recovery phase; and 24 hourslater (F). Data are the means ± SE. *p < 0.01 vs. other conditions; **p < 0.05 vs. other conditions.

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The degree of cellular activation in response to catecholamines depends on the num-ber of adrenoceptors expressed on cellular surface of different PBMC subpopulations,which are known to bear different densities of adrenoceptors (20–22). In our study, NKcells were the most responsive cytotype to the stressing stimulus investigated. T-CD4+

lymphocytes were a less sensitive cytotype, whereas T-CD8+ lymphocytes showed anintermediate sensitivity. B-lymphocytes, which in our model did not show sensitivity tophysical stress, expressed a great density but a low affinity of −adrenoceptors comparedto T-helper lymphocytes (23). The lack of stress-induced modifications on B lymphocytesmay depend on molecular properties (density and affinity) of their −adrenoceptors, whichcould condition responsiveness and redistribution of lymphocytes under stress.

PMN leucocytes increase in number during the test with a typical extension of theevent during the recovery phase (post-stress). The number of total lymphocytes increasedduring the test but decreased progressively during the recovery phase, reaching valueslower than the basal ones. The increase in number of lymphocytes resulted from a redistri-bution in the bloodstream of several lymphocyte subpopulations, being primarily T-CD8+

lymphocytes and T-CD16+/CD65+ (NK cells), and to a lesser extent T-CD4+ lymphocytes,involved, whereas B lymphocytes were unchanged. The lowering of the CD4+/CD8+ ratioobserved probably accounts for the relative increase of T-CD8+ lymphocytes compared tothe T-CD4+ ones.

Stress leads to a quick mobilization of T-lymphocytes with a greater extent for T-memorylymphocytes, leaving the naive subpopulations almost unchanged. Cellular turnoverphenomena may be excluded because of evidence that T-CD8+ lymphocytes, when agingafter several cycles, have smaller telomers and leave the expression of the co-stimulantmolecule CD28 (18). As a response to stress, T-lymphocytes without CD28 (both CD8+ orCD4+) are quickly mobilized, and the length of their telomers is shorter than that ofcontrols (18).

The number of T-CD4+ and T-CD8+ increased with starting stress probably due onlyto a redistribution of activated cells. This is confirmed by clinical studies such as those on therepopulation of CD4+ after anti-HIV treatment, and by those of immunocompetent cells

Figure 5. Stress-induced changes of plasma prostaglandin PGE2 concentration. Analysis was per-formed one week before the test (resting phase, A); soon after the end of stimulus (B); at the first(C), second (D), and third (E) hour of the recovery phase; and 24 hours later (F). Data are the means± SE. *p < 0.05 vs. other conditions.

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kinetic after anticancer chemotherapy (19), or the repopulation of T-CD4+ and T-CD8+

lymphocytes after bone marrow transplant (20). It is important to consider that in spite ofthe global increase in number of lymphocyte subpopulations, the percent variation of theCD4+ decreased.

Several types of physical activity induce, during the test, an increase in the number ofcells mediating the cytotoxic response independent from major histocompatibility com-plex (MHC). This is true primarily for NK cells, the activity of which is also increased(test of the Cr51-release). If we consider the NK activity, evaluated during the test, in cel-lular terms, we observed that the activity does not change. Data on the topic are inconsis-tent, with some studies in agreement with our findings and others reporting a reduction ofthe NK activity during the test, evaluated in cellular terms (24–28). In view of these incon-sistencies, we have analyzed in detail this cytotype by analyzing NK cells during and afterthe stress test.

In the first experimental condition, the number of NK cells increased due to aleucocyte redistribution. During the recovery phase, a numerical and functional reduc-tion of NK, up to values lower than the basal ones, was noticeable. Functional down-regulation of NK activity seems to be related to the level of prostaglandins, theproduction of which from monocytes/macrophages reaches values significantly higherthan the basal ones during the resting time. This hypothesis is supported by in vitrostudies showing that NK activity is partially reduced by PGE2, PGE1, and PGD2 (29).Moreover, we have observed that post-stress reduction of cytotoxicity non-MHCis maintained, even if in cellular terms it is partially abolished in the presence ofthe NSAD indomethacin. In summary, the post-stress reduction of NK-mediatedcytotoxic activity is induced likely by a down-regulation operated by paracrine andendocrine factors, whereas cellular redistribution is probably secondary to prostaglandin-mediated phenomena.

The low functionality of NK cells (immunological window) found at the thirdhour of resting period may depend on stress-induced cell redistribution and partiallyon the action of cortisol. It has been reported that pharmacological doses of corticos-teroids inhibit adhesion of effector cells to their targets (30). Altogether, these studiessuggest that in vivo cortisol and prostaglandins may act in synergy, realizing the socalled “immunological window.” The numerical and functional reduction of NKcells was pointed out in this study only after the application of the stressing stimulus,during the resting phase, when a reduction of IFN-γ and IL-2 also occurs (data notshown).

Based on data available, it can be hypothesized that during the application of a stressingstimulus, blood is enriched on lymphocytes drafted from tissue pool (lungs, spleen, andlymphonodes). The number of cells reaching bloodstream depends on the intensity of thestimulus, but if its intensity is too strong or too long, lymphocyte concentration decreases.Several mechanisms are involved in this decrease, and a future topic of investigation willbe the analysis at a molecular level of mechanisms of the draft of mature cells and lym-phocyte redistribution from blood to other organs. To sum up, lymphopoenia after astressing physical activity is highly dependent on the combination of the intensity andlength of muscle work.

Declaration of Interest

The authors report no conflicts of interest. The authors alone are responsible for the con-tent and writing of the paper.

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