Otro Circadiano | Lymphocyte | Flow Cytometry

  1171 RITISH MEDICAL JOURNAL VOLUmE286 9 APRIL 1983 Diurnal variation of lymphocyte subsets identified by monoclonal antibodies JAMESV BERTOUCH, PETER J ROBERTS-THOMSON, JOHNBRADLEY Abstract Monoclonal antibodies specific for lymphocyte subsets were used to examine circulating lymphocytes obtained at frequent intervals from healthy subjects.   diurnal rhythm was found in the total numbers oflymphocytes, T cells, inducer/helper cells, suppressor/cytotoxic cells, Iapositive cells, and B cells. The lowest levels of all subsets were seen at 0900 hours and thehighest levels at2100. In some subjects the ratio ofhelper to suppressor cells variedconsiderably during the sample period, though theratio was relatively constantforthe group as awhole. Introduction Diurnal variation in the numbers of circulating leucocytes in man is well recognised. Exposure tolight and subsequent effect on the hypothalamic-pituitary adrenal axis is considered to be the probable mechanism,though exercise, food, and emotion may also be implicated. Studies ofdiurnal variations of T and B lymphocytes have mainly utilised rosetting techniques, mito- genic responses, and identification of surface immunoglobulinsandFc receptors.1-4 The discoveryof monoclonal antibodies specificfor lymphocyte subsets prompted anexamination for the presenceof diurnal rhythms in these subsets. We have used a panel of monoclonal antibodies to identify lymphocyte popula- tionsin blood taken at three hourly intervals from a group of healthy laboratory workers. Subjects and methods We studied nine healthy male volunteers from laboratory staff. Their mean age was 35 years(range 29-49), and nonewas taking any form of medication. Blood was collected from 0600 to 2100 hours at three hourly intervals. White cell counts wereperformed with a Coulter counter and differential counts measured in blood smears stained with Giemsa. All differential counts were performedby the same observer. Serum cortisol concentrations were measuredbyradioimmunoassay in a single batch for each subject (normalrange 140-690 nmol l; 5 0-25-0 isgI100 ml). Mononuclear cells were separated from each sample ofheparinised blood on   Ficoll-Hypaque gradient.All separations and subsequent preparative steps were performed by the same person. The cells were washed three times in Dulbecco sphosphate buffered saline and the final concentrationadjusted to 10 x 106 cells/l.   50 4l sample of this suspensionwas incubated for 20minuteswith an appropriate volume of each of   panel of monoclonal antibodies. These included FMC 16 positivecontrol (P32-microglobulin; I Beckman, unpublished observa- tions) x63 negativecontrol(supernatant from lgG 1 mouse myeloma cell line), FMC 1 (B cells),5 FMC 4 (Ia positive cells), T 28 (T cells), Department of Clinical Immunology, Flinders Medical Centre, Adelaide 5042, South Australia JAMES V BERTOUCH, FRACP, researchfellow PETER J ROBERTS-THOMSON, FRACP, staff specialist JOHN BRADLEY, FRCP, professorof clinical immunology Correspondence to: Professor John Bradley, Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, South Australia 5042. OKT 4 (inducer/helper), and OKT 8 (suppressor/cytotoxic; Ortho PharmaceuticalCorporation, NJ, USA). After washing, a second20 minute incubationwith fluorescein isothiocyanate labelled goat antimouseantibodywasperformed. After two washeswithDulbecco sphosphate buffered saline the cells were resuspended in this solution and the percentage of positively fluorescent lymphocytesmeasuredon a BectonDickinson FACS IV flowcytometer. The same observer measured all percentages using bothforward and 90degree light scatter to delineate lymphocytes from other cells. After analysis of the initial samples from each subject the flow cytometer was programmed to usethe same cursor positions (measure- ment parameters) for all further samples from that subject. Thiswasdone to eliminate any observer bias. The absolute numbers of each subset were calculated by multiplying thepercentage of positively fluorescent cells by the total number of lymphocytes obtained from the blood smear. The results were analysedusing Friedman s two way analysis of variance by ranks. Mean and standard errors for each sample time were calculated from logarithmic data and the results plotted on semilogarithmic graph paper. Results There was a diurnal rhythm inthe total lymphocyte count and all subsets, with   minimum count at 0900 hours and a maximum count at 1800-2100 (figure). Analysisof these results showed a highly significant degree ofvariance (p < 0-005). The helper :suppressor cell ratio for the whole groupwas remarkably constant at roughly 2:1 throughout the test period. Nevertheless, large individualvariations occurred-for example, in subjects 1 and 3 (table). Serum cortisol concentrations peaked at 0600 hours and then fell throughout the day to 2100 hours  figure . 10 5. Cel count xl 015- f ->_r___   _TTotal ---- i\ \ lymphocytes T28 1~~~~~~~~~~~~ OKT4 Cort sol OKT8 FNC   FMC 4   -~~~~~~~~~~~~~~ c50c , 100   10 Serum cortisol (nmol /I 060C O9X 120W1500 1800 210C T me (hours) Mean  ± SEM numbers of peripheral lymphocytes and corresponding subsets at each sample time (solid lines) and mean  ±SEM serum cortisol concentration for each sample time (dashed line). Samples taken from0600 to 2100 hours. Conversion: SI to traditional units-Cortisol:   nmol/l0 04 ,ug/100 ml.  1172 BRITISH MEDICAL JOURNAL VOLUME 286 9 APRIL 1983 Helper:suppressor cell ratios inall subjects duirin7g test period Subject No Time (hours) 1 2 3 4 5 6 7 89 0600 1 1 284923 17 36 181521 0900 2-7 1 7 3 21.91.925   1-2 2 0 1200 2 1 20 55 2 1 15 26 1614 26 1500 2320 5422 18 32 1912 23 1800 222 24-92-4 1-6 281-6 1 4 2.1 Discussion Our findings confirm the known diurnal rhythm of total lymphocyte numbers and illustrate the similar rhythm of several lymphocyte subsets as identified by monoclonal antibodies. We examined only men, as the effect on subsets of the menstrual cycle and other factors such as oral contraceptives is not known. When absolute cell numbers were examined the largest diurnal variations occurred in total T cell and helper cell populations. Suppressor cells showed less variation, and the least change was seen in B cells and Ia positive cells. These last two populations have a closecorrelation, which is further evidence for the conceptof circulating Ia positive cells usually belonging to the B cellclass. The ratio ofhelper to suppressor cells wasremark- ably constant for the whole group (figure). The mean figures, however, had large standard errors, and hence variations occurred in every subject. The table shows the ratiosfor all subjects, and in two (subjects 1 and 3) the ratio doubled during the test period. These two subjects were re-examinedand similar pronounced changes in ratios again seen. Possibly some normal subjects may always show this degreeof change in ratios, and further studies are required to examine this. Hence ratios calculated from samples obtained at different times of the day must be compared with c ution The diurnal rhythm is presumably secondary to compart- mentalisationof lymphocytes in various organs such as lymph nodes, spleen, andbonemarrow. Endogenous glucocorticoids are probably at least partially responsible forthis.24 Kinetic studies of radiolabelled lymphocytes show redistribution of the recirculating population outof thevascular compartment into peripheral lymphoid organs after administration of gluco- corticoids. -9 In our study the relation between serum cortisol concentrations and cell counts was not directly inverse, as the mean peak of cortisol values was at 0600,while cell counts were lowestthree hours later.   postulated delay in the cortisol redistribution through body compartments beforeexerting an effect may account for this. Evidence for this proposal may be found from other studies where the maximum depressive effect of oral prednisolone was notseen for several hours after the dose.7 10 In conclusion we found a highly significant diurnal rhythm forseveral lymphocyte subsets. There was no disproportionate change in any particular subpopulation. The rhythm may be due to the effect of endogenous cortisol. Variations in the total lymphocyte count reflect similar changes in different sub- populations. JVB is a grateful recipient of a National Health and MedicalResearch Councilpostgraduate scholarship. We thank J Webster for flow cytometry measurements and R McEvoy fordifferential white cell counts. Cortisol measurements wereperformed by the department of clinical biochemistry, and the department of haematology prepared the blood films. T 28 was a gift from PCL Beverley, ICRF, Human Tumour Immunology Group, UniversityCollege London. The typescript was prepared by MarleneMolnar. References Bartter FC, Delea CS.   map of bloodand urinary changes related to circadian variations in adrenal cortical function in normal subjects. Ann NY Acad Sci 1962;98:969-75. 2 Tavadia HB, Fleming KA, Hume PD, Simpson HW Circadianrhythmi- city of human plasma cortisol and PH induced lymphocyte transforma- tion. Clin Exp Immunol 1975;22:190-3. 3 Abo T, Kumagai K. Studiesof surface immunoglobulins on human B lymphocytes. III. Physiological variations of SIg cells in peripheral blood. Clin Exp Immunol 1978;33:441-52. Abo T, Kawate T, Itoh K, Kumagai K. Studies on the bioperiodicity ofthe immune response. I. Circadian rhythms of human T, B and K cell traffic in the peripheral blood. J Immunol 1981 ;126:1360-3. 5 Brooks DA, Beckman I, Bradley J, et al. Human lymphocyte markers defined by antibodiesderived from somatic cell hybrids. II.   hybridoma secreting antibody against a marker specific for human B lymphocytes. Clin Exp Itimmunol 1980;39:477-85. O Beckman IGR, Bradley J, Brooks DA, et al. Human lymphocyte markers defined by antibodiesderived from somatic cell hybrids. I.   hybridoma secreting antibody againstan antigen expressed by human B and null lymphocytes. Clin Exp Immunol 1980;40:593-601. 7 Fauci AS, Dale DC. Alternate day prednisolonetherapyand human lymphocyte subpopulations. J Clin Invest 1975;55 :22-32. 8 Fauci AS. Mechanism of corticosteroid action on lymphocyte subpopula- tions. I. Redistributionof circulating T and B lymphocytes to the bone marrow. Imnnmunology 1975 ;28 :669-80. 9 Fauci AS. Glucocorticoid effects on circulating human mononuclear cells. J Reticuloendothel Soc1979; suppl:727-38.  ' Yu DT, Clements PJ, Paulus HE, et al. Human lymphocyte subpopula- tions. Effect of corticosteroids. ClGn Invest 1975;53:565-71. (Accepted 19 Februiary 1983) ONE HUNDRED YEARS  GO Sir JamesHanbury, late Principal Medical Officer of the Army under Lord Wolseley in Egypt, was under examination before Lord Morley s Committee on Friday,the 19th instant, and again in thecourseof the present week. Several matters will probably be cleared up when theevidencegiven by this medical officer, who, of course, holding the high position which he did, was Lord Wolseley s chief medical adviser in sanitary matters, is published. Among other points, we may hope to get anexplanationregarding the issue of spirit-rationsto the troops in Egypt. The distribution of spirits to thetroops was quite contrary to the views ofthe commander-in-chief, if we may take as accurate thereport of Lord Wolseley s recent speech to the temperance societies at Blackburn, which appeared in the Times of the 19th instant. According to thereport referred to, Lord Wolseley told the deputations that he himselffirmlybelieved that, if we couldonly havean army which not only wore Her Majesty s colours, but also theblue ribbon, it would be the finest army eversent into the field to represent this country; yet, during therecent campaign in Egypt, the doctors told him it was very necessary that the men should have grog; and he was obliged, owing to great pressure put upon him, to allow it occasionally. The recordedexperience of some ofthe ablest medical officers has shown that alcoholic drinksneither give help as regardsbodily exertion, nor protect the constitutionagainst the invasionof disease, and, with respect to hot climates,that they aggravate rather than lessenthe effects of heat; moreover,Lord Wolseley was able to quote, with regard to Egypt itself, that, during Sir Ralph Abercrombie s expedition to that country in theyear 1800, the good conduct and healthofthetroops landed were attributed to the fact that no liquor was issued to them-testimony which was confirmed by Sir James McGrigor in his Medical Sketchesof the Expedition to Egypt. Under all thesecircumstances, it seems strange that the issue of grog to the men should have been pressed upon the commander-in-chief ofthe army recently engaged in active operations in Egyptby the medical officers. As, however, the particular conditions under which the advice was given have not as yet been published, it seemsonly right to abstain for thepresent from comments on theobservations made by Lord Wolseley on the subject to the deputations from the Blackburn temperance societies. It is greatly to be hoped that the opportunity of forming an impartial judgment on the subject will be afforded when thereport of Lord Morley s Committee, and the evidence onwhich thereport will bebased, havebeen presented to Parliament. The matter is one of more thanpassing interest.(Briti.sh AMedical yournal 1883 ;i: 169.)