Clin. exp. Immunol. (1975) 19, 343-346.

CIRCADIAN RHYTHMS IN CELL MIGRATION IN VITRO, AND ITS EFFECT ON ANTIGENINDUCED MIGRATION INHIBITION ELIZABETH RICHENS, K. A. BROWN, A. J. COLLINS AND P. A. BACON Pharmacology Department, University of Bath, and Arthritis and Rheumatism Council Research Department, Royal National Hospitalfor Rheumatic Diseases, Bath (Received 11 July 1974) SUM MARY

White cell migration from capillary tubes has been studied using blood taken from man and rats over a 24-hr period. When the blood white cell count was lowest in both species, the migration areas were maximal. In man, this variation did not affect the migration index obtained in response to a specific antigen. INTRODUCTION Circadian variations in the production of many biochemical substances and cell populations in vitro are well documented. Examples are found in the rhythms of corticosteroid excretion (Weitzman et al., 1968) and peripheral blood leucocyte count (Sharp, 1960). In view of the known effects of cortisol on both cell numbers and cell function in vitro (Halberg et al., 1959; Stevenson, 1973), a circadian variation in cell migration in vitro might be predicted. This is both of theoretical interest, and relevant to the performance of the leucocyte migration inhibition test (Bendixen & S0borg, 1969), which is commonly used as a simple in vitro correlate of cellular immunity in humans (Federlin et al., 1971). This study investigates the variation in the leucocyte migration inhibition test (LMT) using peripheral blood cells taken sequentially throughout the day. Results are presented from experiments in both man and rats. The wide fluctuations in the areas occupied by cells migrating from capillary tubes in vitro in the absence of antigen are shown, together with the migration inhibition values obtained with the use of specific antigen: both are related to white cell and cortisol levels.

MATERIALS AND METHODS Human studies were performed on seven volunteers, sensitized to dinitrochlorobenzene (DNCB) (2000 yug) dissolved in acetone, by a single cutaneous application. Animal studies were carried out using male Wistar rats of 250 mg body weight (Fisons Pharmaceuticals, Loughborough). The animals were light/dark stabilized on a 6 a.m. to 6 p.m. light phase for Correspondence: Dr A. J. Collins, School of Pharmacy and Pharmacology, University of Bath, Bath. K

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12 days before the experiment commenced. Rats were used in groups of five for each determination of cell migration. Leucocytes washed three times were adjusted to 5 x 107 cells/ml in Eagle's medium, containing 15% foetal calf serum (MEM). Nine-microlitre capillary tubes were filled with cell suspension and centrifuged at 200 g for 5 min. A length of tube containing the cell pellet was placed in migration chambers containing MEM with or without antigen. Antigen (250 pg/ml) was added to the chambers as DNCB coupled to human serum albumin (HSA). The area of migration was measured by projection and planimetry after 21 hr incubation.

RESULTS The results of seven experiments with human blood taken over a 24-hr period, examining cortisol levels, white cell numbers, migration areas and migration inhibition, are shown in

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FIG. 1. Sequential measurements of (a) plasma cortisol, (b) total white cell numbers, (c) white cell migration area, and (d) the migration inhibition index (MI), from seven human volunteers over a 24-hr period. Vertical bars indicate standard error in the mean.

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Fig. 1. The peak level of cortisol was observed at the 06.00 hr measurement, with a gradual fall towards evening. In contrast, the spontaneous migration areas were maximal at 09.00 hr. The largest increase in migration area was 60% over the basal level at midnight. The total white cell counts showed an inverse relationship to the migration area with the lowest count and the peak migration area coincident at 09.00 hr. The fluctuation in migration area was not reflected in the migration inhibition values (Fig. Id), which showed no significant circadian rhythm. 251-

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FIG. 2. Sequential measurements of (a) plasma corticosterone, (b) total white cell count and (c) white cell migration areas, from a group of fifteen rats, taken over a 24-hr period. Blood was collected by cardiac puncture. Vertical bars indicate standard error of the mean.

The results of the rat circadian studies are shown in Fig. 2. Here again an inverse correlation is seen between total white cell count and spontaneous migration areas. However, in contrast to the humans, the timing is reversed. In the nocturnal rat the leucocyte numbers are lowest, and the migration areas peak, in the dark phase corresponding to midnight. The cortisterone levels in the rat show a double curve with a peak level early in the dark phase but another increase in the light phase.

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DISCUSSION These observations show that considerable fluctuations take place in both cortisol levels and in the numbers of peripheral cells concerned in immune inflammatory responses. It is interesting that when the peripheral blood white cell numbers are at their lowest, these cells show maximal migration in spite of equal cell numbers in the capillary tube. The peak in migration areas lags approximately 3 hr after the peak in cortisol levels in humans. It is known that cortisol stimulates polymorph migration in vitro (Stevenson, 1973). Corticosteroids also have a direct effect on leucocytes in vivo, leading to both a neutrophil leucocytosis (Kimball et al., 1973) and a depression of subpopulations of human lymphocytes (Fauci & Dale, 1974). The variation in migration areas obtained with rat leucocytes showed a different time course from that seen in humans, although in both species there was an inverse relation to total white cell numbers. The peak migration pattern coincides with the phase of the greatest physical activity of these nocturnal animals. Recently it has been shown that in humans exercise alters the ratio of circulating T and B lymphocytes (Steel, Evans & Smith, 1974). The data here suggest that this ratio varies in both species throughout the 24-hr period in response to physical activity. It is possible that T cells, responsible for the antigen-specific inhibition of the LMT, show a relative increase as the total leucocyte count decreases. This would counteract the increased polymorph migration potential seen at lower cell numbers. This study shows that when the numbers of leucocytes added to the capillary tube are kept constant, the in vitro immunological response as assessed by the inhibition of leucocyte migration does not change significantly with the time of day despite circadian fluctuations in spontaneous migration. These findings are of importance for the use of the LMT as a practical test of cellular immunity. ACKNOWLEDG M ENT

We are grateful to Aspro Nicholas Ltd for support for this work. REFERENCES BENDIXEN, G. & S0BORG, M. (1969) A leucocyte migration technique for in vitro detection of cellular (delayed type) hypersensitivity in man. Dan. med. Bull. 16, 1. FAUCI, A.S. & DALE, D.C. (1974) The effect of in vivo hydrocortisone on sub-populations of human lymphocytes. J. clin. Invest. 53, 240. HALBERG, F., HALBERG, E., BARNUM, C.P. & BITTNER, J.J. (1959) Photoperiodism and Related Phenomena in Plants and Animals (ed. by R. B. Withrow), p. 803. A.A.A.S., Washington. FEDERLIN, K., MAINI, R.N., RUSSELL, A.S. & DUMOND, D.C. (1971) A micromethod for peripheral leucocyte migration in tuberculin sensitivity. J. clin. Path. 24, 533. KIMBALL, M.R., SHELDON, M.W., TALAL, N., PLOTZ, P.H. & DECKER, J.L. (1973) Marrow granulocyte reserve in the rheumatic diseases. Arthr. and Rheum. 16, 345. SHARP, G.W.G. (1960) Reversal of diurnal leucocyte variations in man. J. Endocr. 21, 107. STEEL, C.M., EVANS, J. & SMITH, M.A. (1974). Physiological variation in circulating B cell :T cell ratio in man. Nature (Lond.), 247, 387. STEVENSON, R.D. (1973) Hydrocortisone and the migration of human leucocytes: an indirect effect mediated by mononuclear cells. Clin. exp. Immunol. 14, 417. WEITZMAN, E.D., GOLDMACHER, D., KRIPKE, D., MACGREGOR, P., KREAM, J. & HELLMAN, L. (1968). Reversal of sleep-waking cycle: effect on sleep stage pattern and certain neuroendocrine rhythms. Trans. Amer. neurol. Assoc. 93, 153.

Circadian rhythms in cell migration in vitro, and its effect on antigen-induced migration inhibition.

White cell migration from capillary tubes has been studied using blood taken from man and rats over a 24-hr period. When the blood white cell count wa...
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