Clin. exp. Immunol. (1988) 71, 329-335

Circadian rhythms in circulating T lymphocyte subtypes andplasma testosterone, total and free cortisol in five healthy men

F. A. LPVI*t, CHANTAL CANON*, Y. TOUITOU§, J. SULONT, M. MECHKOURI*, EMILIE DEMEYPONSARTT, J. P. TOUBOULt, J. M. VANNETZELt, IRENE MOWZOWICZ§, A. REINBERGt &

G. MATHE* *Institut de Cancerologie et d'Immunogenetique (CNRS UA04-1163), and SMST Hopital Paul-Brousse, Villejuif,France, tUnite de Chronobiologie et Chronopharmacologie (CNRS UA581), Fondation A. de Rothschild, Paris, France,

§Departement de Biochimie, FacuWtt de Medecine Pitie-Salpetriere, Paris, France, and ¶Departement de Clinique et PathologieMedicales, UniversiW de Liege, Liege, Belgium

(Acceptedfor publication 8 September 1987)

SUMMARYCircadian variations of circulating T lymphocyte subtypes and their possible relations with those ofendogenous cortisol or testosterone were investigated in five healthy young men. Venous blood(40 ml) was obtained every 4 h for 24 h from each subject in January, March, June, August andNovember. Leucocyte and differential counts were measured. Mononuclear cells were isolated onFicoll-Paque gradient, and samples were incubated with OKT3, OKT4 or OKT8 monoclonalantibodies for characterizing all T, T helper and T suppressor-cytotoxic lymphocytes respectively.The proportion of labelled lymphocytes was determined under an epifluorescence microscope and thecounts of circulating lymphocyte subsets (cells/mm3) computed. Total and free cortisol andtestosterone were also determined in the corresponding plasma samples. Results from analysis ofvariance and cosinor indicated statistically significant differences (P< 0-001) as a function of bothindividual subject and circadian sampling time for all variables. Circadian rhythms (with a period,T=24 h) were validated for total, T and T helper lymphocytes and for the T helper: T suppressor-cytotoxic ratio (P< 0-00 1), with double amplitudes (2A, total extent ofvariation accounted for by thefitted cosine function) ranging from 25% up to 50% of the 24 h mean (M), and acrophases ((D, time ofmaximum) localized near 0100 h. A rhythm with T_ 12 h characterized circulating T suppressor-cytotoxic lymphocytes (P < 0-001; 2A = 36% of M; (F = 0830 and 2030 h). Circadian rhythms werealso found for plasma cortisol (either total or free) and testosterone (P< 0-001). No correlation wasfound however between time-qualified data of these hormones and the immunological variablesherein investigated (162 pairs ofdata) whether or not a 4 h or an 8 h lag time was considered to allowfor hormonal actions to operate. This suggests that neither the circadian organization of the adrenalcortex nor that ofthe testis play a prominent role in the circadian time structure ofthe circulation ofTlymphocytes.

Keywords circadian rhythms lymphocyte hormonal regulation man

INTRODUCTION

Although circadian rhythms in circulating lymphocyte subtypeshave been documented in healthy human beings (Abo et al.,1981; Haus et al., 1983; Bertouch, Roberts-Thompson &Bradley, 1983; Ritchie et al., 1983; Levi et al., 1983, 1985;Miyawaki et al., 1983; Knapp & Pownall, 1984), some conflict-ing results were reported by different investigators, mostly with

Correspondence: F. A. LUvi, Institut de Cancerologie et d'Immuno-genetique, H6pital Paul-Brousse, 14-16 avenue Paul-Vaillant Coutur-ier, 94804-Villejuif, France.

regard to the T suppressor-cytotoxic and the T helper: Tsuppressor-cytotoxic ratio. Thus, no circadian rhythmicity hasbeen documented for the latter by Bertouch, Roberts-Thomp-son& Bradley (1983) or Ritchie et al. (1983), whereas we found alarge-amplitude circadian rhythm in preliminary studies (Levi etal., 1983; 1984). Since these investigations were performed atdifferent times of the year, seasonal changes in the circadianrhythmicity in T lymphocyte subsets might have accounted forsuch discrepancies. The circadian organization of the adreno-cortical function has been considered as the primary regulatorysystem which would account for circadian rhythms in immunefunctions (Tavadia et al., 1975; Cove-Smith et al., 1979; Abo et

329

F. A. Levi et al.

al., 1981; Kawate et al., 1981; Miyawaki et al., 1983). Nonethe-less other hormones have also been shown to exert a majorpharmacodynamic effect upon the immune system and may alsobe involved in its temporal regulation. For instance, thepresence of receptors for both androgens and oestrogens at thesurface of circulating lymphocytes may explain why thesehormones also appear to play an important immunoregulatoryrole (Hall & Goldstein, 1984; Grossman, 1985).

The present study aimed at documenting both the circadianand the circannual time structure of circulating T lymphocytessubsets and their possible regulation by endogenous cortisol ortestosterone secretions. In this report, we examine the overallcircadian organization of these variables and their possiblerelationship.

SUBJECTS AND METHODS

SubjectsFive apparently healthy male subjects volunteered for this study(median age 33 years; range: 24 to 36 years). They weresynchronized by daily activities and nocturnal rest. During thestudy they were recumbent from 2300 h to 0700 h; breakfast wastaken at -0730 h, lunch at _ 1245 h and dinner at -2030 h.With the use of both clinical and routine biological examin-ations, neither acute nor chronic infection was diagnosed, atleast during the month before the study and that after it. Onesubject had a past history of childhood asthma, and two wereheavy smokers (15 and 25 cigarettes per day) but with minimalsmoking or none ( < 5 cigarettes/24 h) during the study.

ProtocolBlood (40 ml) was drawn every 4 h for 24 h starting at 0830 h, inJanuary, March, June, August and November. Seven sampleswere obtained from each subject in January, March and August,and six in June and November. Because of the time-consumingprocedure needed for the separation of lymphocytes andlabelling, the group of five subjects was split into two subgroupsof two and three subjects. Each subgroup was studied I weekapart of each study-month, starting on a Monday in order tocontrol for possible weekly influences. For each sample, 13 ml ofblood were collected in standard EDTA-containing tubes andwere processed for an immediate haemogram determinationand subsequent hormonal analysis; 27 ml were collected in

syringes containing 3 ml of heparin and were processed for thedetermination of lymphocyte surface markers.

HaemogramThe total leucocyte count (cells per cubic millimetre of blood)was determined by an automatic heamocytometer (OrthoELT8) within 30 min ofcollection. Slides were made and stainedwith May-Grunwald-Giemsa. The proportions of lymphocytesand monocytes were determined microscopically, with 300 cellscounted per slide. The total number of circulating lymphocytesper cubic millimetre was calculated.

Lymphocyte surface marker determinationWithin 30 min of collection, each blood sample was processedfor isolation of mononuclear cells. Peripheral blood was

collected on heparin (Liquemine, Roche), and mononuclear

cells were separated on a Ficoll-paque gradient, then incubatedwith 5 p1 of OKT3, OKT4 or OKT8 monoclonal antibodies(Orthoclone, Aubervilliers, France) and processed as earlierdescribed (Levi et al., 1985). Two hundred cells were countedunder a Zeiss epifluorescence microscope. All counts wereperformed by the same investigator, the coefficient of variationfor this technique being 5% in her hands. Quality control foractivity was performed on each lot of monoclonal antibodies, inaddition to the fact that they were produced by the samehybridoma cell lines. The total number in each circulatinglymphocyte subtype was determined by multiplying the propor-tion of fluorescent cells by the number of circulating lympho-cytes.

Plasma hormone determinationTotal cortisol was measured by radioimmunoassay as pre-viously described (Sulon et al., 1978). The intra-assay variationwas 4-3 and 7-51X) and the interassay precision was 8-3 and 9-71yoat 12 and 35 pg/100 ml respectively. Plasma unbound cortisolwas measured with an equilibrium dialysis method (Demey-Ponsart et al., 1976). Plasma testosterone was assayed by RIA aspreviously described (Kutten et al., 1977) with a sensitivity of0-01 ng/ml of plasma. Intra-assay and inter-assay coefficients ofvariation were 6 and 8'S respectively.

Statistical analysesData were expressed both in their conventional units and aspercentages of the individual 24 h mean for each variable andtime point. The latter method was used in order to minimizeinter-individual differences occurring in 24 h mean values oflymphocyte subsets, as previously documented by Levi et al.(1985).

Means and one standard error of the mean were computedfor each time point, each variable, and on each study month.Time series were analysed both by analysis of variance(ANOVA) and by the cosinor method (De Prins, Cornelissen &Malbecq, 1986). The 2-way ANOVA considered two factors aspotential sources of variance: subject and circadian samplingtime. The cosinor method characterized a rhythm by theparameters of the fitted cosine function approximating all data.Periods, T-24 h and T-12 h, were considered a priori. Therhythm characteristics estimated by this linear least squaresmethod include the mesor (M: rhythm-adjusted mean), thedouble amplitude (2A: difference between minimum and maxi-mum of fitted cosine function), and the acrophase (1D: time ofmaximum in fitted cosine function, with midnight as D refer-ence). They are given with their 950, confidence limits. Arhythm was detected if the null amplitude hypothesis was

rejected with P < 0-05; however, A and .1 could be approximatedif 0 05 < P< 0.10. The cosinor method was applied to individualand pooled time series (Reinberg & Smolensky, 1983; De Prins,Cornelissen & Malbecq, 1986).

Multiple correlations were performed between the hormonalvariables and the lymphocytic ones. Since a lag time maycharacterize the actions exerted by such hormones on the countof total lymphocytes or that of their subsets, correlations were

also performed with a 4 h and an 8 h lag time (A 1D); e.g.hormonal values at 0800 h correlated with lymphocyte values at1200 h (A ID=4 h) or at 1600 (A4=8 h), etc.

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Circadian rhythms in T lymphocytes and plasma hormones

Table 1. Interindividual differences in 24 h mesors ofcirculating total and T lymphocytes and plasma cortisol and testosterone(intersubject differences were statistically validated for all variables by analysis of variance (P <0001))

Subject

Variable 1 2 3 4 5

Total lymphocytes (cells/mm3) 2690 + 111 * 2430 + 90 4540 + 170 2910 + 110 4420 + 220OKT3+ lymphocytes (cells/mm3) 1260+60 1230+ 70 2550+ 130 1500+60 2190+ 110OKT4+ lymphocytes (cells/mm3) 1080 + 65 920 + 50 1610 + 90 1080+60 1240 + 80OKT8+ lymphocytes (cells/mm3) 340 + 20 280 + 20 770 + 50 390+40 800 + 50OKT4+:OKT8+ 3 7+0 4 3-6+0 2 2 4+0 3 3 2+0 2 1-6+0-1Total cortisol (ng/ml) 0-230+0 016 0-338 +0 019 0 166+0 011 0 194+0 012 0-238+0-023Free cortisol (ng/ml) 0-012 +0-002 0-017+0 002 0-008+0-001 0-010+0-001 0-013 +0 002Testosterone (ng/ml) 343 + 17 319+ 18 350+ 15 510+ 19 539 +22

* One standard error of the mesor.

RESULTS

Two-way analysis of variance indicated an effect of both subjectand time of day for all variables including the three plasmahormones investigated (P < 0 00 1).

Inter-individual differences in the 24 h-mesorsIndividual circadian mesors of each variable are given in Table1. Inter-individual differences were validated with statisticalsignificance for all variables (P<0-001) (Table 1). Both theheavy smokers (Subjects 3 and 5) had the highest 24 h mean

values for total, all T, T helper and T suppressor-cytotoxiclymphocytes and the lowest mean values for the T helper:Tsuppressor-cytotoxic ratio. No trend was suggested for any ofthe plasma hormones in relation to smoking habits.

Inter-individual differences in the 24 h mesor led us toperform the cosinor analyses on data transformed as percent-ages of the individual's 24 h mean.

Circadian rhythms in lymphocyte-related variablesMean values of total lymphocyte count varied from 2650 cells/mm3 at 1230 h up to 3800 cells per mm3 at 0430 h. Lowest meanvalues in T, T helper and T suppressor cytotoxic lymphocyteswere also observed at 1230 h. Highest values were found at 0430h for T and T helper lymphocytes, but two peaks were found forT suppressor-cytotoxic lymphocytes, respectively at 0830 h andat 2030 h (Fig. 1). With regard to the T helper:T suppressor

cytotoxic ratio, lowest values (mean = 2 2) were found at 1230 hand highest ones at 0430 h (mean = 3 7) (Fig. 2). All circadianvariations were statistically validated by ANOVA (see legendsto figures).A rhythm with a period of z_ 24 h was further validated by

cosinor for total, OKT3+ and OKT4+ lymphocytes(P < 0-0001), but not for OKT8 + lymphocytes (P> 0 30) (Table2). The acrophases (maxima) of these rhythms were localizednear 0100 h. A rhythm with zT 12 h was also found for totallymphocytes, as a first harmonic of the fundamental 24 hrhythm. This was also the case for the OKT4+ :OKT8+ ratio,which was characterized by both a fundamental 24 h rhythmand a 12 h harmonic (P < 000001). The circadian acrophase was

2250r

20001

1750

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E

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1250[-

1000

750_

SC

TIt 0KT8

+

m1~ ~ ~ ~ ~ ~ ~ ~ ~ ,

0h0830 1230 1630 2030 0030 0430

Time (h)

Fig. 1. Plexograms ofcirculating T, T helper and T suppressor-cytotoxiclymphocytes along the 24 h scale. An effect of sampling time was

statistically validated by ANOVA (respectively F= 6 8, F= 6 5, F= 4-2;d.f.= 160; P<0-001). A circadian rhythm with a period, t-24 h, was

found for T and T helper lymphocytes by cosinor (P< 0 001). A rhythm,with t-12 h, was detected for T suppressor-cytotoxic lymphocytes(P< 0001).

331

F. A. Levi et al.

0

0

3.Q[

2.0J-r

0830 1230 1630 2030

Time (h)

0030 0430

Fig. 2. Plexogram of T helper:T suppressor cytotoxic ratio fromperipheral blood along the 24 h scale. A circadian rhythm was validatedby both an analysis of variance (F=49; P<0001) and cosinor(P<000 1).

also localized near 100 h. With regard to the OKT8+ subset, a

rhythm with r 12 h was demonstrated with acrophasesoccurring near 0830 h and 2030 h (P<0-0001).

Circadian rhythms in hormonal variablesBoth a circadian rhythm and a 12 h harmonic were statisticallyvalidated for plasma total and free cortisol as well as for plasmatestosterone. The circadian acrophases were respectivelylocalized at 0910 h, 0940 h and 1120 h (Table 2, Fig. 3) and the

double-amplitudes of these rhythms were respectively 166%,120% and 30% of the circadian-mesor.

Correlations between T tymphocyte subsets and plasma cortisoland testosteroneCorrelations were performed between the five lymphocyte-related variables and the three plasma hormones investigated.No statistically significant correlation was found whether the

data were expressed as raw values or as percentages of the

individual 24 h mean and whether hormonal values were

correlated with lymphocytic variables sampled at the same time

or 4 h or 8 h later (r 0-16; d.f.= 160; P>0 10).The temporal relationship of the circadian or circahemidian

(with =12 h) acrophases of both the immunological and the

hormonal variables investigated is summarized in Fig. 4.

DISCUSSION

Circadian rhythms were demonstrated for circulating total, T

and T helper lymphocytes with maxima localized in the first halfof the night and double-amplitudes exceeding 25% of the 24 hmesor. Such results confirm and extend those obtained in our

initial studies (Levi et al., 1983; 1985) and those reported byothers (Abo et al., 1981; Haus et al., 1983; Ritchie et al., 1983;Miyawaki et al., 1983; Knapp & Pownall, 1984).

No 24 h rhythm but a 12 h one was found for circulatingT suppressor-cytotoxic lymphocytes, with 2A equal to 36% ofthe 24 h mesor and acrophases (IF) localized near both 0830 hand 2030 h. With regard to this subset, a minor circadianvariation, if any, was reported by some (Ritchie et al., 1983;Miyawaki et al., 1983; Knapp & Pownall, 1984), whereas a

consistent rhythm was found earlier by us (Levi et al., 1983;1984) including a preliminary report by us on two subjectsstudied in late April. According to both of these investigations,the circadian maximum was localized near 2200 h, e.g. close to

our second maximum in the late evening.Such a 12 h rhythm may reflect a true circahemidian rhythm

in the circulating count of suppressor-cytotoxic lymphocytes. A12 h rhythm was documented for both DNA and RNA synthesisin circulating total lymphocytes (Carter et al., 1974; Kaplan et

al., 1976; S. Sanchez, W. Hrushesky & F. Levi, unpublished).Nonetheless, it was shown that both OKT4+ and HNK1 +

lymphocytes from peripheral blood may also express the T8antigen at their surface (Abo, Cooper & Balch, 1982; Blue et al.,1985). The observed 12 h rhythmicity in OKT8+ mononuclearcells may thus indicate that the T8 antigen is expressedpredominantly at 0830 h in a given subpopulation, and at 2030 hin another one. Double-labelling studies will be necessary toanswer this question.

The T helper:T suppressor cytotoxic ratio exhibited a

circadian rhythm with a pronounced double-amplitude averag-

ing 50% of the mesor and an acrophase localized at 0100 h. Thisfinding accords well with our preliminary report in two subjects(Levi et al., 1983).

Since no difference was statistically validated between valuesobtained 24 h apart at 0830 h for any of these variables an effectof repeated sampling is likely to be ruled out.

The circadian organization documented here constitutes an

average of five circannual stages. A seasonal modulation of thiscircadian time structure is discussed in a subsequent report (Leviet al., in press).

A circadian rhythm and an associated 12 h harmonic were

documented in the present study for plasma total and freecortisol and testosterone concentrations, with characteristicssimilar to those earlier reported (Reinberg et al., 1975; 1978;Touitou et al., 1982; 1983). The detection of this 12 h harmonic

most probably reflects the well known non-sinusoidality ofwave

form.No statistically significant correlation was found between

plasma cortisol (either free, or total) or testosterone and any of

the five immunological variables herein investigated despite 162

pairs of data were used for testing each correlation. Because

such hormones often need a lag time to exert their metabolic

actions, lag times of 4 and 8 h were also considered betweenplasma hormones and lymphocyte-related variables. No corre-

lation was found either between these two sets of variables.

332

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333Circadian rhythms in T lymphocytes and plasma hormones

Table 2. Circadian rhythm and 12 h component in circulating lymphocyte subsets, T helper:Tsuppressor-cytotoxic ratio, plasma cortisol and testosterone in five healthy young men (results from

cosinor analysis, with a period, T -24 h and 12 h)

2A+s.d.t +s.d.1Variable 24 h mesor + s.e.m. Period (h) P* (% mesor) (hours, min)

Lymphocytes (cells/mm3)Total 3380+95 24 <0-001 26+12 1-20+1 30

12 <0-01 14+12 6-00and 18-00+1-50OKT3+ 1740+60 24 <0-001 35+15 1 10+1-30

12 0-18OKT4+ 1190+40 24 <0-001 35+15 1-20+1 40

12 0-13OKT8+ 515+25 24 0 31 -

12 <0-001 36+20 8 30 and 2030+ 1 10

OKT4+:OKT8+ 2-9+0 1 24 <0 001 50+24 1-00+ 1 4012 <0-004 31 +22 3 40and 1540+ 1-40

Cortisol (ng/ml)Total 0-230+0-004 24 <0001 120+20 940+1 20

12 <0-001 64+28 8 10and20-10+0-50Free 0-012+0 001 24 <0-001 164+29 9 00+ 1-20

12 < 0 001 108+40 8 10 and 20-10+ 0 50Testosterone 410+12 24 <0-001 31+12 11 25+1 30

12 <0001 20+12 940and21-40+1-20

* P value from an F-test of the null amplitude rejection hypothesis.t Double-amplitude+ s.d.t Acrophase + s.d.

mean± s.e.m.

U-)uI _ _ _, I_____

500

E1-mIsw

-

0U)

0

U)

Variable period (h)Lymphocytes

Total (24,1OKT3 (24OKT4 (24OKT84 (12I

OKT4'/OKT8 (24,1

CortisolTotal (24,1;

Acrophase (± 95%/6 c.l.)

12)

12)

12)Free (24,12)

Testosterone (24,12)

300

10-~~~~~ _5 ,, ,

0830 1230 1630 2030 0030 0430Time (h)

Fig. 3. Plexograms of plasma concentrations in total (0) and free (0),ortisol and testosterone (es). An effect ofsampling time was statisticallyialidated by ANOVA (respectively, F= 18-4; F= 21-1, and F=6-4;P< 0-001). Both a circadian rhythm (with T=24 h) and a 12 h harmonicwere statistically validated by cosinor for all variables.

Fig. 4. Acrophase chart of circulating total lymphocytes, T subsets andplasma cortisol and testosterone in diurnally active healthy subjects.Data from all five study months were pooled. The periods detected foreach variable are indicated. Black dots indicate the location in time ofthe acrophase and the horizontal line its 95% confidence interval. Whena 24 h rhythm was detected, the circadian acrophase is the only one

shown. When a 12 h rhythm was detected alone (no 24 h rhythm), as wasthe case for T suppressor-cytotoxic cells, two acrophases are shown inthe 24 h scale. The level of statistical significance of these circadian orcircahemidian rhythms was <0-001.

0-40

0-30I

"I

-

0.0 0-201-

010F

r - r)r)L-..

334 F. A. Levi et al.

Other rhythms with a short period (so-called ultradian rhythms)are known to characterize both lymphocyte count and plasmacortisol and testosterone. Nonetheless, the circadian componentaccounts by far for the largest share of the temporal variabilityof all three variables (Carter et al., 1975; Guignard et al., 1980;Halberg et al., 1981). This further supports that a minor role canbe attributed to the circadian time-structure of plasma secre-tions of the adrenal cortex or the testis with regard to the controlof the circadian organization of circulating total, T3, T4 or T8lymphocytes and that of the T4: T8 ratio. Thus a coincidence intime between peak and trough values of two variables does notnecessarily imply any correlation and a fortiori any causalrelationship as was suspected for the T lymphocytes and plasmacortisol.

Nonetheless, it has to be emphasized that such a findingapplies to a physiological time-structure, and that qualitativelydifferent chronopharmacological effects may result from theexogenous administration of higher doses of these hormones fortherapeutic purposes..

ACKNOWLEDGMENTS

This work was supported in part by grant 6180 from the Associationpour la recherche sur le Cancer, BP3, 94800-Villejuif, France. We areindebted to M. Bennaceur, G. Debotte, E. Brugerie and A. Roulon foroutstanding technical assistance, and to N. Vriz and E. Couve for thefinal typing of this manuscript.

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Circadian rhythms in T lymphocytes andplasma hormones 335

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