You are on page 1of 7

Clin. exp. Immunol.

(1988) 71, 329-335

Circadian rhythms in circulating T lymphocyte subtypes and plasma testosterone, total and free cortisol in five healthy men
LPVI*t, CHANTAL CANON*, Y. TOUITOU, J. SULONT, M. MECHKOURI*, EMILIE DEMEY PONSARTT, 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 Pathologie Medicales, UniversiW de Liege, Liege, Belgium
F. A.

(Acceptedfor publication 8 September 1987)

SUMMARY Circadian variations of circulating T lymphocyte subtypes and their possible relations with those of endogenous 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 and November. Leucocyte and differential counts were measured. Mononuclear cells were isolated on Ficoll-Paque gradient, and samples were incubated with OKT3, OKT4 or OKT8 monoclonal antibodies for characterizing all T, T helper and T suppressor-cytotoxic lymphocytes respectively. The proportion of labelled lymphocytes was determined under an epifluorescence microscope and the counts of circulating lymphocyte subsets (cells/mm3) computed. Total and free cortisol and testosterone were also determined in the corresponding plasma samples. Results from analysis of variance and cosinor indicated statistically significant differences (P< 0-001) as a function of both individual 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 suppressorcytotoxic ratio (P< 0-00 1), with double amplitudes (2A, total extent of variation accounted for by the fitted cosine function) ranging from 25% up to 50% of the 24 h mean (M), and acrophases ((D, time of maximum) localized near 0100 h. A rhythm with T_ 12 h characterized circulating T suppressorcytotoxic lymphocytes (P < 0-001; 2A = 36% of M; 0830 and 2030 h). Circadian rhythms were also found for plasma cortisol (either total or free) and testosterone (P< 0-001). No correlation was found however between time-qualified data of these hormones and the immunological variables herein investigated (162 pairs of data) whether or not a 4 h or an 8 h lag time was considered to allow for hormonal actions to operate. This suggests that neither the circadian organization of the adrenal cortex nor that of the testis play a prominent role in the circadian time structure of the circulation of T lymphocytes.
(F

Keywords circadian rhythms lymphocyte hormonal regulation

man

INTRODUCTION
Although circadian rhythms in circulating lymphocyte subtypes have 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 conflicting results were reported by different investigators, mostly with
Correspondence: F. A. LUvi, Institut de Cancerologie et d'Immunogenetique, H6pital Paul-Brousse, 14-16 avenue Paul-Vaillant Couturier, 94804-Villejuif, France.

different times of the year, seasonal changes in the circadian rhythmicity in T lymphocyte subsets might have accounted for such discrepancies. The circadian organization of the adrenocortical function has been considered as the primary regulatory system which would account for circadian rhythms in immune functions (Tavadia et al., 1975; Cove-Smith et al., 1979; Abo et

regard to the T suppressor-cytotoxic and the T helper: T suppressor-cytotoxic ratio. Thus, no circadian rhythmicity has been documented for the latter by Bertouch, Roberts-Thompson & Bradley (1983) or Ritchie et al. (1983), whereas we found a large-amplitude circadian rhythm in preliminary studies (Levi et al., 1983; 1984). Since these investigations were performed at

329

330

F. A. Levi et al.
cells were separated on a Ficoll-paque gradient, then incubated with 5 p1 of OKT3, OKT4 or OKT8 monoclonal antibodies (Orthoclone, Aubervilliers, France) and processed as earlier described (Levi et al., 1985). Two hundred cells were counted under a Zeiss epifluorescence microscope. All counts were performed by the same investigator, the coefficient of variation for this technique being 5% in her hands. Quality control for activity was performed on each lot of monoclonal antibodies, in addition to the fact that they were produced by the same hybridoma cell lines. The total number in each circulating lymphocyte subtype was determined by multiplying the proportion of fluorescent cells by the number of circulating lymphocytes.

al., 1981; Kawate et al., 1981; Miyawaki et al., 1983). Nonetheless other hormones have also been shown to exert a major pharmacodynamic effect upon the immune system and may also be involved in its temporal regulation. For instance, the presence of receptors for both androgens and oestrogens at the surface of circulating lymphocytes may explain why these hormones also appear to play an important immunoregulatory role (Hall & Goldstein, 1984; Grossman, 1985). The present study aimed at documenting both the circadian and the circannual time structure of circulating T lymphocytes subsets and their possible regulation by endogenous cortisol or testosterone secretions. In this report, we examine the overall circadian organization of these variables and their possible relationship.

SUBJECTS AND METHODS

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

Plasma hormone determination Total cortisol was measured by radioimmunoassay as previously described (Sulon et al., 1978). The intra-assay variation was 4-3 and 7-51X) and the interassay precision was 8-3 and 9-71yo at 12 and 35 pg/100 ml respectively. Plasma unbound cortisol was measured with an equilibrium dialysis method (DemeyPonsart et al., 1976). Plasma testosterone was assayed by RIA as previously described (Kutten et al., 1977) with a sensitivity of 0-01 ng/ml of plasma. Intra-assay and inter-assay coefficients of variation were 6 and 8'S respectively.
Statistical analyses Data were expressed both in their conventional units and as percentages of the individual 24 h mean for each variable and time point. The latter method was used in order to minimize inter-individual differences occurring in 24 h mean values of lymphocyte subsets, as previously documented by Levi et al. (1985). Means and one standard error of the mean were computed for 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 as potential sources of variance: subject and circadian sampling time. The cosinor method characterized a rhythm by the parameters of the fitted cosine function approximating all data. Periods, T-24 h and T-12 h, were considered a priori. The rhythm characteristics estimated by this linear least squares method include the mesor (M: rhythm-adjusted mean), the double amplitude (2A: difference between minimum and maximum of fitted cosine function), and the acrophase (1D: time of maximum in fitted cosine function, with midnight as D reference). They are given with their 950, confidence limits. A rhythm was detected if the null amplitude hypothesis was rejected with P < 0-05; however, A and .1 could be approximated if 0 05 < P < 0.10. The cosinor method was applied to individual and pooled time series (Reinberg & Smolensky, 1983; De Prins, Cornelissen & Malbecq, 1986). Multiple correlations were performed between the hormonal variables and the lymphocytic ones. Since a lag time may characterize the actions exerted by such hormones on the count of 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 at 1200 h (A ID=4 h) or at 1600 (A 4=8 h), etc.

Protocol Blood (40 ml) was drawn every 4 h for 24 h starting at 0830 h, in January, March, June, August and November. Seven samples were obtained from each subject in January, March and August, and six in June and November. Because of the time-consuming procedure needed for the separation of lymphocytes and labelling, the group of five subjects was split into two subgroups of two and three subjects. Each subgroup was studied I week apart of each study-month, starting on a Monday in order to control for possible weekly influences. For each sample, 13 ml of blood were collected in standard EDTA-containing tubes and were processed for an immediate haemogram determination and subsequent hormonal analysis; 27 ml were collected in syringes containing 3 ml of heparin and were processed for the determination of lymphocyte surface markers.
Haemogram The total leucocyte count (cells per cubic millimetre of blood) was determined by an automatic heamocytometer (Ortho ELT8) within 30 min of collection. Slides were made and stained with May-Grunwald-Giemsa. The proportions of lymphocytes and monocytes were determined microscopically, with 300 cells counted per slide. The total number of circulating lymphocytes per cubic millimetre was calculated.

Lymphocyte surface marker determination Within 30 min of collection, each blood sample was processed for isolation of mononuclear cells. Peripheral blood was collected on heparin (Liquemine, Roche), and mononuclear

Circadian rhythms in T lymphocytes and plasma hormones


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

331

Subject
Variable Total lymphocytes (cells/mm3) OKT3+ lymphocytes (cells/mm3) OKT4+ lymphocytes (cells/mm3) OKT8+ lymphocytes (cells/mm3) OKT4+:OKT8+ Total cortisol (ng/ml) Free cortisol (ng/ml) Testosterone (ng/ml)

1
2690 + 111 * 1260+60 1080 + 65 340 + 20 3 7+0 4 0-230+0 016 0-012 +0-002 343 + 17
*

2 2430 + 90 1230+ 70 920 + 50 280 + 20 3-6+0 2 0-338 +0 019 0-017+0 002 319+ 18

3 4540 + 170 2550+ 130 1610 + 90 770 + 50 2 4+0 3 0 166+0 011 0-008+0-001 350+ 15

4 2910 + 110 1500+60 1080+60 390+40 3 2+0 2 0 194+0 012

5
4420 + 220 2190+ 110 1240 + 80 800 + 50 1-6+0-1

0-238+0-023
0-013 +0 002 539 +22

0-010+0-001
510+ 19

One standard error of the mesor.

RESULTS

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

2250r
Inter-individual differences in the 24 h-mesors Individual circadian mesors of each variable are given in Table 1. Inter-individual differences were validated with statistical significance for all variables (P<0-001) (Table 1). Both the heavy smokers (Subjects 3 and 5) had the highest 24 h mean values for total, all T, T helper and T suppressor-cytotoxic lymphocytes and the lowest mean values for the T helper:T suppressor-cytotoxic ratio. No trend was suggested for any of the plasma hormones in relation to smoking habits. Inter-individual differences in the 24 h mesor led us to perform the cosinor analyses on data transformed as percentages of the individual's 24 h mean.
Circadian rhythms in lymphocyte-related variables Mean values of total lymphocyte count varied from 2650 cells/ mm3 at 1230 h up to 3800 cells per mm3 at 0430 h. Lowest mean values in T, T helper and T suppressor cytotoxic lymphocytes were also observed at 1230 h. Highest values were found at 0430 h for T and T helper lymphocytes, but two peaks were found for T suppressor-cytotoxic lymphocytes, respectively at 0830 h and at 2030 h (Fig. 1). With regard to the T helper: T suppressor cytotoxic ratio, lowest values (mean = 2 2) were found at 1230 h and highest ones at 0430 h (mean 3 7) (Fig. 2). All circadian variations were statistically validated by ANOVA (see legends to 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) (Table 2). The acrophases (maxima) of these rhythms were localized near 0100 h. A rhythm with zT 12 h was also found for total lymphocytes, as a first harmonic of the fundamental 24 h rhythm. This was also the case for the OKT4+ :OKT8+ ratio, which was characterized by both a fundamental 24 h rhythm and a 12 h harmonic (P < 000001). The circadian acrophase was
=

20001

<'-1'\~~-

1750

E E

1500~

ai ~~~~I/ A. .d~~~~~
OKT4

a)
a)

ci2
a

1250[-

C:
0

1000

750_

T
It
SC
+

0KT8
m1

0h
1630 2030 0030 0430 Time (h) Fig. 1. Plexograms of circulating T, T helper and T suppressor-cytotoxic lymphocytes 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).

0830

1230

332
4.OFr

F. A. Levi et al.
DISCUSSION

3.Q[

2.0J

Sri
0830

-r

000WZZZA I rz, ///Z16.


1230
2030 1630 Time (h)
0030

0430

Fig. 2. Plexogram of T helper: T suppressor cytotoxic ratio from peripheral blood along the 24 h scale. A circadian rhythm was validated by 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 12 h was demonstrated with acrophases rhythm with occurring near 0830 h and 2030 h (P<0-0001).
r

Circadian rhythms in hormonal variables Both a circadian rhythm and a 12 h harmonic were statistically validated for plasma total and free cortisol as well as for plasma testosterone. The circadian acrophases were respectively localized 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 cortisol and testosterone Correlations were performed between the five lymphocyterelated 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.

Circadian rhythms were demonstrated for circulating total, T and T helper lymphocytes with maxima localized in the first half of the night and double-amplitudes exceeding 25% of the 24 h mesor. Such results confirm and extend those obtained in our initial studies (Levi et al., 1983; 1985) and those reported by others (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 circulating T suppressor-cytotoxic lymphocytes, with 2A equal to 36% of the 24 h mesor and acrophases (IF) localized near both 0830 h and 2030 h. With regard to this subset, a minor circadian variation, 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 subjects studied 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. A 12 h rhythm was documented for both DNA and RNA synthesis in 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 T8 antigen at their surface (Abo, Cooper & Balch, 1982; Blue et al., 1985). The observed 12 h rhythmicity in OKT8+ mononuclear cells may thus indicate that the T8 antigen is expressed predominantly at 0830 h in a given subpopulation, and at 2030 h in another one. Double-labelling studies will be necessary to answer this question. The T helper: T suppressor cytotoxic ratio exhibited a circadian rhythm with a pronounced double-amplitude averaging 50% of the mesor and an acrophase localized at 0100 h. This finding accords well with our preliminary report in two subjects (Levi et al., 1983). Since no difference was statistically validated between values obtained 24 h apart at 0830 h for any of these variables an effect of repeated sampling is likely to be ruled out. The circadian organization documented here constitutes an average of five circannual stages. A seasonal modulation of this circadian time structure is discussed in a subsequent report (Levi et al., in press). A circadian rhythm and an associated 12 h harmonic were documented in the present study for plasma total and free cortisol and testosterone concentrations, with characteristics similar 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 of wave 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 between plasma hormones and lymphocyte-related variables. No correlation was found either between these two sets of variables.

Circadian rhythms in T lymphocytes and plasma hormones


Table 2. Circadian rhythm and 12 h component in circulating lymphocyte subsets, T helper:T suppressor-cytotoxic ratio, plasma cortisol and testosterone in five healthy young men (results from cosinor analysis, with a period, T -24 h and 12 h)

333

2A+s.d.t
Variable
24 h mesor + s.e.m. Period (h)
P* <0-001 <0-01 <0-001 0-18 <0-001 0-13 0 31 (% mesor) 26+12

+s.d.1
(hours, min)
1-20+1 30 6-00and 18-00+1-50 1 10+1-30
1-20+1 40
8 30 and 2030+ 1 10

Lymphocytes Total

(cells/mm3) 3380+95
1740+60
1190+40 515+25 2-9+0 1

OKT3+
OKT4+
OKT8+

24 12 24 12 24 12 24 12 24 12 24 12 24 12 24 12

14+12
35+15 35+15

<0-001
<0 001

36+20
50+24 31 +22

OKT4+:OKT8+
Cortisol Total
Free

<0-004
<0001 <0-001 <0-001 < 0 001 <0-001 <0001

1-00+ 1 40 3 40and 1540+ 1-40


940+1 20 8 10and20-10+0-50 9 00+ 1-20 8 10 and 20-10 + 0 50 11 25+1 30 940and21-40+1-20

(ng/ml) 0-230+0-004 0-012+0 001


410+12

120+20 64+28 164+29

Testosterone

108+40 31+12 20+12

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

500

0-40
Variable period (h)

Lymphocytes Total
0-30I
E
"I

Acrophase ( 95%/6 c.l.)

(24,1 12)
(24 (24

1Is
w

OKT3 OKT4

OKT84

(12I
(24,1 12)

.0 0

0-201mean

U)

OKT4'/OKT8
Cortisol Total Free Testosterone

U)

(24,1;12) (24,12)

010F
r r)r) L-.. U-)uI
-

s.e.m.

(24,12)

300
_

10-~~~~~ , _

_5 ,, ,
_ I_____

0830
Fig.

1230

1630 2030 0030 0430 Time (h)

3. Plexograms of plasma concentrations in total (0) and free (0) ,ortisol and testosterone (es). An effect of sampling time was statistically ialidated 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 harmonic were statistically validated by cosinor for all variables.

Fig. 4. Acrophase chart of circulating total lymphocytes, T subsets and plasma cortisol and testosterone in diurnally active healthy subjects. Data from all five study months were pooled. The periods detected for each variable are indicated. Black dots indicate the location in time of the acrophase and the horizontal line its 95% confidence interval. When a 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 was the case for T suppressor-cytotoxic cells, two acrophases are shown in the 24 h scale. The level of statistical significance of these circadian or circahemidian rhythms was <0-001.

334

F. A. Levi et al.
piandrosterone A-4 and rostenedione, testosterone and dihydrotestosterone. Acta Endocr. 94, 536.
HALBERG, F., CORNELISSEN, G., SOTHERN, R., WALLACH, L., HALBERG, E., AHLGREN, A., KUZEL, M., RADKE, A., BARBOSA, J., GOETZ, F., BUCKLEY, J., MANDEL, J., SCHUMAN, L., HAUS, E., LAKATUA, D., SACKETT, L., BERG, H., WENDT, H., KAWASAKI, T., UENO, M., UEZONO, K., MATSUOKA, M., OMAE, T., TARQUINI, R., CAGNONI, M., GARCIA-SAINZ, M., PEREZ-VEGA, E., WILSON, D., GRIFFITHS, D., DONATI, L., TATTI, P., VASTA, M., LOCATELLI, J., CAMAGNA, A., LOUTO, R., TRITSCH, G. & WETTERBERG, L. (1981) International geographic studies of oncologic interest on chronobiologic variables. In: Neoplasms-Comparative Pathology of Growth in Animals, Plants and Man (ed. by H.E. Kaiser), p. 563. Williams & Wilkins, Baltimore. HALL, N.R. & GOLDSTEIN, A.L. (1984) Endocrine regulation of host immunity. The role ofsteroids and thymosin. In: Immune Modulation, Agents and Their Mechanisms (ed. by R.L. Fenichel & M.A. Chirigos), p. 533. Immunology Series Vol. 25. Marcel Dekker, New York. HAUS, E., LAKATUA, D., SWOYER, J. & SACKETT-LUNDEEN, L. (1983) Chronobiology in hematology and immunology. Am. J. Anat. 168, 467. KAPLAN, M.S., BYERS, V.S., LEVIN, A.S., GERMAN, D.F., FUDENBERG, H.H. & LECAM, L.N. (1976). Circadian rhythm of stimulated lymphocyte blastogenesis. A 24 hour cycle in the mixed leucocyte culture reaction and with SKSD stimulation. J. Allergy clin. Immunol. 58, 180. KAWATE, T., ABO, T., HINNMA, S. & KUMAGAI, K. (1981) Studies on the bioperiodicity of the immune response. II. Covariations of murine T and B cells and a role of corticosteroid. J. Immunol. 126, 1364. KNAPP, M.S. & POWNALL, R. (1984) Lymphocytes are rhythmic: is this important? Brit med. J. 289, 1328. KUTTEN, F., MOWSZOWICZ, I., SCHAISON, G. & MAUVAIS-JARVIS, P. (1977) Androgen production and skin metabolism. J. Endocr. 75, 83. LEvI, F., CANON, C., BLUM, J.P., REINBERG, A. & MATHt, G. (1983) Large-amplitude circadian rhythm in helper, suppressor ratio of peripheral blood lymphocytes. Lancet ii, 462. LEvI, F., CANON, C., BLUM, J.P., MISSET, J.L., MECHKOURI, M., BENNACEUR, M. & MATHt, G. (1984) Circadian rhythms in 6 circulating lymphocyte subtypes in healthy men. Ann. Rev. Chronopharmacology 1, 131. LEVI, F., CANON, C., BLUM, J.P., MECHKOURI, M., REINBERG, A. & MATHt, G. (1985) Circadian and/or circahemidian rhythms in nine lymphocyte-related variables from peripheral blood of healthy subjects. J. Immunol. 134, 217. LtVI, F., CANON, C., TouITOU, Y., SULON, J., REINBERG, A. & MATHt, G. Seasonal modulation of circadian rhythmicity in circulating T lymphocyte subtypes from healthy subjects. J. clin. Invest. (in press). MIYAWAKI, T., TAGA, K., NAGAOKI, T., SEKI, H., SUZUKI, Y. & TANIGUCHI, N. (1983) Circadian changes of lymphocyte subsets in human peripheral blood. Clin. exp. Immunol. 55, 618. REINBERG, A., LAGOGUEY, M., CHAUFFOURNIER, J.M. & CESSELIN, F. (1975). Circannual and circadian rhythms in plasma testosterone in five healthy young Parisian males. Acta Endocr. 80, 732. REINBERG, A., LAGOGUEY, M., CESSELIN, F., TOUITOU, Y., LEGRAND, J.C., DELASELLE, A., ANTREASSIAN, J. & LAGOGUEY, A. (1978) Circadian and circannual rhythms in plasma hormones and other variables of five healthy young human males. Acta Endocr. 88, 417. REINBERG, A. & SMOLENSKY, M. (1983) Biological Rhythms and Medicine. Cellular, Metabolic, Physiopathologic and Pharmacologic Aspects. pp. 305. Springer Verlag, New York. RITCHIE, W.S., OSWALD, I., MICKLEM, H.S., BOYD, J.E., ELTON, R.A., JAZWINSKA, E. & JAMES, K. (1 983) Circadian variation of lymphocyte subpopulations: a study with monoclonal antibodies. Br. med. J. 286, 1773. SULON, J., DEMEY-PONSART, E., BEAUDOUIN, P. & SODOYEZ, J.C. (1978) Radio-immuno-assay of cortisone, cortisol and corticostrone: their application to human cord and maternal plasma. J. Steroid Biochem. 9,671.

Other rhythms with a short period (so-called ultradian rhythms) are known to characterize both lymphocyte count and plasma cortisol and testosterone. Nonetheless, the circadian component accounts by far for the largest share of the temporal variability of all three variables (Carter et al., 1975; Guignard et al., 1980; Halberg et al., 1981). This further supports that a minor role can be attributed to the circadian time-structure of plasma secretions of the adrenal cortex or the testis with regard to the control of the circadian organization of circulating total, T3, T4 or T8 lymphocytes and that of the T4: T8 ratio. Thus a coincidence in time between peak and trough values of two variables does not necessarily imply any correlation and a fortiori any causal relationship as was suspected for the T lymphocytes and plasma cortisol. Nonetheless, it has to be emphasized that such a finding applies to a physiological time-structure, and that qualitatively different chronopharmacological effects may result from the exogenous administration of higher doses of these hormones for therapeutic purposes..

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

REFERENCES
ABO, T., KAWATE, T., ITOH, K. & KUMAGAI, K. (1981) Studies on the bioperiodicity of the immune response. I. Circadian rhythms of human T, B, and K cell traffic in the peripheral blood. J. Immunol. 126, 1360. ABO, T., COOPER, M.D. & BALCH, C.M. (1982) Characterization of HNK- I + (Leu7) human lymphocytes. I. Two distinct phenotypes of human NK cells with different cytotoxic capability. J. Immunol. 129, 1752. BERTOUCH, J.V., ROBERTS-THOMPSON, P. & BRADLEY, J. (1983) Diurnal variation of lymphocyte subsets identified by monoclonal antibodies. Brit. med. J. 286, 1171. BLUE, M.L., DALEY, J.F., LEVINE, H. & SCHLOSSMAN, S.F. (1985) Coexpression of T4 and T8 on peripheral blood T cells demonstrated by two-color fluoresence flow cytometry. J. Immunol. 134, 2281. CARTER, J.B., BARR, G.D., LEVIN, A.S., BYERS, V.S., PONCE, B. & FUDENBERG, H.H. (1975) Standardization of tissue culture conditions for spontaneous thymidine-2-'4C incorporation by unstimulated normal human peripheral lymphocytes: circadian rhythm of DNA synthesis. J. Allergy clin. Immunol. 56, 191. COVE-SMITH, J.R., POWNALL, R., KABLER, T. & KNAPP, M.S. (1979) Circadian variations in cell-mediated immune response in man and their response to prednisolone. Chronopharmacol. 19, 369. DE PRINS, J., CORNELISSEN, G. & MALBECQ, W. (1986) Statistical procedures in chronobiology and chronopharmacology. Ann. Rev. Chronopharmacology 2, 27. DEMEY-PONSART, E., FOIDART, J.M., HENDRICKX, J.C. & SODOYEZ, J.C. (1976) Effect of serum dilution on binding of cortisol to thermolabile and thermostable serum proteins. J. Steroid Biochem. 8, 1091. GROSSMAN, C.J. (1985) Interactions between the gonadal steroids and the immune system. Science 227, 257. GUIGNARD, M., PESQUIES, P., SERRURIER, B., MERINO, D. & REINBERG, A. (1980) Circadian rhythm in plasma levels of cortisol, dehydroe-

Circadian rhythms in T lymphocytes and plasma hormones


TAVADIA, H.B., FLEMING, K., HUME, P.D. & SIMPSON, H.W. (1975) Circadian rhythmicity of human plasma cortisol and PHA-induced lymphocyte transformation. Clin. Exp. Immunol. 22, 190. TOUITOU, Y., SULON, J., BOGDAN, A., TouITou, C., REINBERG, A., BECK, H., SODOYEZ, J.C., DEMEY-PONSART, E. & VAN CANWENBERGHE, H. (1982) Adrenal circadian system in young and elderly human subjects: a comparative study. J. Endocr. 93, 201.

335

TouITOU, Y., SULON, J., BOGDAN, A., REINBERG, A., SoDoYEz, J.C. & DEMEY-PONSART, E. (1983) Adrenocortical hormones, ageing and mental condition: seasonal and circadian rhythms of plasma 8 hydroxy- 1 I-deoxycorticosterone, total and free cortisol and urinary corticosteroids. J. Endocr. 96, 53.

You might also like