BRITISH MEDICAL JOURNAL

VOLUME 286

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CLINICAL

RESEARCH

Circadian variation of lymphocyte subpopulations: with monoclonal antibodies

study

ALASTAIR W S RITCHIE, IAN OSWALD, H SPEDDING MICKLEM, JANICE E BOYD, ROBERT A ELTON, ELIZABETH JAZWINSKA, KEITH JAMES
Abstract
Use of monoclonal antibodies to identify subpopulations of circulating lymphocytes in healthy adults showed pronounced circadian variations in total T cells, the two major T cell subsets, and HLA-DR+ lymphocytes. When the results for the T cell subsets were expressed as a ratio (helper:suppressor) no significant rhythmic variation was observed. Lymphocytes bearing a surface antigen identified by the HNK-1 antibody (a population containing the natural killer and antibody dependent killer activity) did not show significant rhythmic variation. There was an inverse relation between plasma cortisol concentration and numbers of T and B cells. These observations have therapeutic implications and should be considered in the course of immunological monitoring.

the development and regulation of the immune study was made possible by the development of a characterised monoclonal antibodies, which in conjunction with flow cytometry permit rapid, precise enumeration of functionally distinct lymphocyte subpopulations.1 2As a consequence of these technological developments lymphocyte subpopulations are under intensive study in a wide variety of diseases. For example, alterations of the relative proportions of the T "helper" and T "suppressor/cytotoxic" populations in peripheral blood of recipients of renal allografts correlate with rejection episodes.3 Since importance is being attached to relatively small changes in the ratio of T helpers to suppressors we considered it important to establish a baseline by investigating physiological variation of the T cell subsets and other subpopulations with a role in the immune response.
TABLE I-Panel of monoclonal antibodies used

play

a part in response. This range of well

Introduction
Circadian variation of biological phenomena is often ignored by both doctors and scientists. In this report we describe wide circadian variation of some of the lymphocyte subsets, which

Monoclonal antibody

Cell population identified

Anti-Leu-4
Anti-Leu-3a Anti-Leu-2a Anti-Leu-2b f Anti-HLA-DR
HNK-1 (Anti-Leu-7)

Helper subset of T cells Suppressor/cytotoxic subset of T cells B cells and activated T cells (monocytes*) NK and K cells

Department of Surgery, University Medical School, Edinburgh

* Monocytes were excluded from analysis using 90 light scatter variables (see text).

Department of Psychiatry, University of Edinburgh IAN OSWALD, DSC, FRCPSYCH, professor ELIZABETH JAZWINSKA, BSC, postgraduate student Department of Zoology, University of Edinburgh H SPEDDING MICKLEM, MA, DPHIL, reader
Medical Computing and Statistics Unit, University of Edinburgh ROBERT A ELTON, BA, PHD, lecturer

Methods Peripheral blood samples were obtained at intervals of three hours from 10 healthy volunteers (mean age 51, range 45-58). Within the constraints of the blood sampling the subjects performed normal daily activities. They slept in a sleep research laboratory, of which they all had previous experience.
Total and differential white cell counts were performed on each blood sample. Plasma was separated and stored frozen for subsequent analysis of cortisol concentrations. Mononuclear cells were isolated on Ficoll Hypaque and then reacted with a panel of monoclonal antibodies to lymphocyte surface antigens (table I). Mouse monoclonal antibodies

Correspondence

to: Mr A W S Ritchie.

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either conjugated with fluorescein isothiocyanate direct or were visualised with a second, antimouse antibody conjugated with fluorescein isothiocyanate. All analyses were performed on unfixed cell preparations with a fluorescence activated cell sorter (FACS IV, Becton Dickinson FACS Systems, Sunnyvale, California). Ten thousand cells were analysed for each antibody, the number of fluorescent cells being expressed as a percentage of the total lymphocytes. Non-lymphocytic cells contaminating the preparations were excluded from analysis using scatter gates set on the 90 light scatter profile.3a The proportions of cells that were fluorescent on analysis with the fluorescence activated cell sorter were used in conjunction with the total and differential white cell counts to calculate absolute numbers of cells in each subpopulation. Statistical analyses of the percentages and absolute numbers of cells were performed using the methods of Nelson et al,4 which entailed fitting sine curves to the data from individual subjects and testing whether the variables of these curves showed consistent patterns of circadian variation.
were

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360

35i
0

320 30

280
25Cell number 2 0x109/1
1.50~~~~~~~~~

240

200
88
\ 0 8

Plasma

,'

/~/ //

cortisol
160 (nmol/l) 120

80
10-

o----o * *

Plasma cortisol concentration Total lymphocytes

40

0800 1100 1400 1700 2000 2300 0200 0500 08C00 Time of day

Results
Variations of all the lymphocyte subpopulations were noted over 24 hours of study. The variations were significant (p <005) for absolute numbers of Leu- 1+, Leu-4+ (total T cells), Leu-3a+ (T helper subset), Leu-2a+, Leu-2b+ (T suppressor/cytotoxic subset), and HLA-DR+ (B cells and activated T cells) lymphocytes. In contrast, HNK-1+ cells did not exhibit significant circadian variation. Figure 1 shows the variation in the Leu-3a+ subset of cells, with peak levels occurring at night. When expressed as percentages of the total number of lymphocytes Leu-3a+ and HNK-1+ cells showed significant
variation.

FIG 2-Comparison of circadian variations of total lymphocytes and plasma cortisol concentration. (Each point represents mean value in 10 subjects.) Conversion: SI to traditional units-Cortisol: 1 nmol/l 36-3 ng/100 ml.

TABLE ii-Circadian variation of lymphocyte subpopulations contrasted with plasma cortisol variation (data derived from best fitting sine curves)
Mesor Amplitude

Peak time
2222 2232 2156 2305

Significance of Circadian variation (,,) circadian rhythm

Plasma cortisol concentration showed the expected large amplitude rhythm and varied inversely with the numbers of T and B cells (fig 2). The ratio of T helper to suppressor cells (Leu-3a+ :Leu-2b+) showed only minor variations over time and did not exhibit significant rhythmic variation. Table II summarises the data for each subset.

Results expressed as absolute number of cells x 108/1

Leu-2b4

16 0 11 7 43 35 25 66 2 47 4 17 9 14 5 110

43 34

0-9

10

001 p<.005 p 0 05
<

p < 005

p<0
NS

Results expressed as percentage of total lymphocytes

Leu-3a4

5-1
28

2339
1115

22 51

Leu-2b+ HLA-DR+

HNK-18
Discussion

NS pc<005 NS NS

p.001
NS

Other results

There is now considerable evidence that the magnitude of the immune response varies with the time of day.5 The findings of this study may in part explain the variations by demonstrating changes in the number of circulating cells available to encounter and process antigen.
Circadian variation of total lymphocytes,6 surface Ig+ cells,7

Helper: suppressor ratio Plasma cortisol

2-6
208
126

(nmol/l)

0948

121

p<0-01

Conversion: SI to traditional units-Cortisol: 1 nmol/I 36-3 ng/100 ml.

and cells forming rosettes with sheep erythrocytes8 has been reported previously. Our use of monoclonal antibodies in
16Peak time

14-

Cell number
x 108,1

12
Amplitude

10
0

0800 1100

1400 1700 2000 2300 0200 0500 0800 Time of day

FIG 1-Circadian variation of Leu-3a + cells (helper subset of T cells). (Each point represents mean value in 10 subjects.) Mesor 11 66; peak time 2232; circadian variation (2 X amplitude/mesor)= 590' .

conjunction with flow cytometry permitted further subclassification of the lymphocyte population and greater precision in enumeration. It would have been impossible to analyse this number of samples and to count the cells in each sample by light or fluorescent microscopy. The development9 and characterisation"o -12 of the HNK-1 monoclonal antibody enabled us to identify cells with NK and K cell function by surface labelling, without the necessity for time consuming functional assays. The variation of numbers of circulating B and T cells points to sizable shifts of cells into and out of the circulation. The traffic of cells through other lymphoid tissue, including bone marrow, may allow for contact between antigen primed regulatory T cells and B cells. The lack of variation of HNK-1 + cells correlates with the fact that only 0700 of nucleated bone marrow cells have this phenotype.' It also correlates with the finding that in lymphoid tissues HNK-1 + cells are predominantly located within germinal centres,'2 which are not regarded as part of the lymphocyte recirculation pathway." Tht striking inverse relation of the numbers of circulating T and B cells and plasma cortisol concentration, in conjunction with the reported T lymphopenia in patients with Cushing's syndrome'4 and transient lymphopenia after single doses of exogenous corticosteroid,"5 16 suggest a causal interrelation. The results have certain clinical implications. For example, would the efficiency of antilymphocyte chemotherapy be improved if administration was timed to produce peak plasma

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T cell subsets for immunologic monitoring and treatment in recipients of renal allografts. N EnglJ' Med 1981;305:308-14. ,a Ritchie AWS, Gray RA, Micklem HS. Right angle light scatter: a necessary parameter in flow cytofluorimetric analysis of human peripheral blood mononuclear cells. J Immunol Methods (in press). 4Nelson W, Tong YL, Halberg F. Methods for cosinorrhythmometry. Chronobiologia 1979 ;6 :305-23. Pownall R, Knapp MS. Immune responses have rhythms: are they important? Immunology Today 1980 October: vii-x. 6 Bartter FC, Delfa CS, Halberg F. A map of blood and urinary changes related to circadian variations in adrenal cortical function in normal subjects. Ann NY Acad Sci 1962;98:969-83. 7Abo T, Kumagi K. Surface studies of immunoglobulins on human B lymphocytes. III. Physiological variations of SIg+ cells in peripheral blood. Clin Exp Immunol 1978;33:441-52. 8 Abo T, Kawate T, Itoh K, et al. Characterization of rhythms and cell components in circadian variations of human and mouse lymphocytes. Chronobiologia 1979;6:71-2. Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J' Immunol 1981;127: 1024-9. Abo T, Cooper MD, Balch CM. Characterization of HNK-1+ (Leu-7) human lymphocytes. I. Two distinct phenotypes of human NK cells with different cytotoxic capability. J Immunol 1982;129:1752-7. Abo T, Balch CM. Characterization of HNK-1 + (Leu-7) human lymphocytes. II. Distinguishing phenotypic and functional properties of natural killer cells from activated NK-like cells. J Immunol 1982;129:1758-61. 12 Ritchie AWS, James K, Micklem HS. The distribution and possible significance of cells identified in human lymphoid tissue by the monoclonal antibody HNK-1. Clin Exp Immunol 1983;51 :439-47. 13 Ford WL. Lymphocyte migration and immune responses. Prog Allergy 1975;19: 1-59. 4 Shohat B, Klein A, Kaufmann H, et al. T lymphocytes and plasma inhibitory factor in ACTH-dependent Cushings patients. Journal of Clinical Immunology and Immunopathology 1979;13:452-61. 1 Fauci AS, Dale DC. The effect of in vivo hydrocortisone on subpopulations of human lymphocytes. J Clin Invest 1974;53:240-6. 16 Yu DTY, Clements PJ, Paulus HE, et al. Human lymphocyte subpopulations: effect of corticosteroids. J Clin Invest 1974;53:565-71. 1 Ratte J, Halberg F, Kuhl JFW, et al. Circadian variation in the rejection of rat kidney allografts. Surgery 1973;73:102-8. 18 Knapp MS, Cove-Smith JR, Dugdale R, et al. Possible effect of time on renal allograft rejection. Br MedJ7 1979;i:75-7.

concentrations when the maximum number of cells are in the circulation? Does rejection occur earlier if allografts are transplanted at night, when the number of T cells in the recipients' circulation are at their peak ? With respect to the last question, circadian variation in the rejection of rat renal allografts has been described,' 7 and the longest surviving grafts were those inserted when the numbers of circulating lymphocytes were at their trough. Sound data relating time of operation to onset of rejection, or graft survival, in man are lacking, although circadian variation in episodes of renal allograft rejection has been suggested.' 8 We have shown that in any attempt at immunological monitoring account must be taken of circadian variation of lymphocyte subpopulations as a source of variance in results. Conversely, we found the ratio of helper to suppressor cells to be fairly stable in our healthy subjects.
This study was supported by a grant from the Melville Trust. We thank Drs T Abo, C M Balch, and M D Cooper for the gift of the HNK-1 antibody; Professor G D Chisholm for discussion and encouragement; and Mr R A Gray for help with the fluorescence activated cell sorter analysis. ADDENDUM-SiX of the subjects were studied over a further 24 hour period, during which they were deprived of sleep. There were no significant differences in any of the rhythmic variations over this period when compared with the first set of data for these six subjects.

References
Reinherz EL, Schlossman SF. Regulation of the immune responseinducer and suppressor T lymphocyte subsets in human beings. N Eng 1 7 Med 1980;303:370-3. 2 Herzenberg LA, Herzenberg LA. Analysis and separation using the fluorescence activated cell sorter (FACS). In: Weir DM, ed. Handbook of experirmental immunology. 3rd ed. London: Blackwell Scientific, 1978:22.1-22.21. Cosimi AB, Colvin RB, Burton RC, et al. Use of monoclonal antibodies to

(Accepted 24 March 1983)

Renal transplantation in children with occult neurogenic bladders drained by intermittent self catheterisation
0 H STANLEY, T L CHAMBERS, B D PENTLOW
Abstract Children with neurogenic bladders have usually not been considered for renal transplantation because of the potential complications caused by chronic infections of the urinary tract. Two girls with occult neurogenic bladders who were practising intermittent self catheterisation were given renal transplants. Both took prophylactic antibacterial agents after transplantation, and both experienced infections at some stage, but none of these infections produced a deterioration in renal function. In one patient creatinine clearance fell and signs of
obstruction appeared, but this was because the patient had been catheterising herself only two or three times a week. When she resumed catheterisation four times a day creatinine clearance rose and the urographic and renographic appearances returned to normal. These results suggest that, with adequate catheterisation and urinary chemoprophylaxis, infection is not a particular problem in children with bladder dysfunction who undergo renal transplantation.

Regional Department of Paediatric Renal Dialysis and Transplantation, Southmead Hospital, Bristol BS10 5NB O H STANLEY, MRCP, paediatric registrar T L CHAMBERS, FRCP, consultant paediatrician B D PENTLOW, FRCS, consultant transplant surgeon Correspondence to: Dr T L Chambers.

Introduction Intermittent clean self catheterisation is an accepted form of managing neurogenic bladders in childhood which does not produce major problems arising from sepsis.1 It might be supposed that the immunosuppression associated with renal transplantation would lead to problems with infection. We describe two children with occult neurogenic bladders who received renal transplants while practising intermittent self catheterisation after micturition.