Dose-related depression of pha-induced stimulation of human lymphocytes by hydrocortisone

Int. J. Immunopharmac., Vol. 3, pp. 21-29

0192-0561/81 / 0101-0021 $02.00/0

,c, Pergamon Press Ltd. 1981. Printed in Greal Britain.

DOSE-RELATED DEPRESSION OF PHA-INDUCED STIMULATION OF HUMAN LYMPHOCYTES BY HYDROCORTISONE A. J. ROBERTSON,J. H. GIBBS,R. C. POTTS,R. A. BROWN,*MARGARETC. K. BROWNING~"and J. SWANSON BECK~ Departments of Pathology, Mathematics* and Biochemical Medicine,l University of Dundee, Ninewells Hospital and Medical School, Dundee, U.K. (Received 8 April 1980 and in final form 6 June 1980) Abstract--Hydrocortisone inhibits PHA-induced lymphocyte stimulation by reducing the number of cells entering Gt-phase and by slowing the volume growth rate during the first 24 h of culture: the kinetics of cellular recruitment is unmodified. This inhibition has a linear log-dose response curve and there is marked intersubject variation in normal adults. Pre-incubation of the cells with hydrocortisone greatly potentiates its inhibitory effect: when added after the start of PHA stimulation, hydrocortisone becomes progressivelyless effective until it has lost most of its activity by 5 h, even though the cells do not show any microscopic evidence of having entered the Gl-phase at that time. The experiments have elucidated the mode of action of hydrocortisone inhibition on the early stages of PHA-induced lymphocyte stimulation and shown an unexpectedly large intersubject variation in responsiveness that might have therapeutic significance. The methods that have been employed could be adapted for clinical monitoring of the immunosuppressive effect of glucocorticoids in individual patients and for screening synthetic steroids for glucocorticoid activity.

Glucocorticoids are potent immunosuppressors. Although the general mechanisms by which glucocorticoids exert their intracellular action are now fairly well understood (Baxter & Rousseau, 1979) relatively little is known about differences in reactivity between the various classes of lymphoid cells. For example, glucocorticoid receptors are said to be present in equivalent amounts and to have the same affinity and binding specificity in T-cells and non-T-cells (Lippman & Barr, 1977), but most suppressor T-cells are relatively sensitive whilst helper T-cells and B-cells are relatively resistant (Fauci, 1978). Moreover, from recent studies on peripheral blood lymphocytes from normal people, it has been claimed that there is considerable intersubject variation in the number of glucocorticoid receptors per cell (Gleispach, Schumik, Zollner & Esterbauer, 1979). We have recently introduced new methods for measuring mitogen-induced stimulation based on analysis of changes in cell volume distribution by a stochastic model (Brown, McWalter, Slidders, Gibbs & Beck, 1979; Gibbs, Brown, Robertson, Potts & Beck, 1979b). These methods give information about the pre-S-phase kinetics of the first cell cycle after stimulation and they have proved to be precise and highly reliable in our routine clinical laboratory for

measuring the inherent growth potential of lymphocytes from normal subjects and from patients with a wide variety of diseases. We have now studied the effects of physiological and pharmacological concentrations of hydrocortisone on the pre-S-phase kinetics of PHA-stimulated human peripheral blood mononuclear cells (>90°/0 lymphocytes) and shown that there is substantial intersubject variation in responsiveness of cells to inhibition of growth by hydrocortisone. EXPERIMENTAL PROCEDURES Lymphocyte cultures Twenty-one healthy adults (14 men and 7 women, aged 18-70 years) were studied. Some of the subjects (numbers 2, 4, 7 and 21) were healthy menlbet~ of Ilae laboratory staff. The remaiader werg'patiCnts attending a dyspepsia outpatient cli~lic in whom no active disease was detected: only one patient (subject number 10) was on medication, this being diazepam, but the tablets were being taken only intermittently. None of the subjects were smokers and, although several were social drinkers, none gave a history of drinking alcohol in excessive amounts: none of the subjects had consumed alcohol within the 24 h prior to the removal of the blood sample. Two patients

~/Correspondence to: Professor J. Swanson Beck, Department of Pathology, Ninewelis Hospital and Medical School, Dundee DDI 9SY. 21

22

A. J. ROBERTSONel al.

had significant previous medical history: subject orifice, and 0.5 ml manometer, settings used being number 9 had suffered a myocardial infarct 21 yr 'attenuation - 1' and 'aperture - 32'. The output of previously and subject number 17 had undergone a this machine was accumulated in a multi-channel vagotomy for a duodenal ulcer 4 yr previously: this analyser (Model C1000, Coulter Electronics Ltd.), latter patient had been asymptomatic and free from with settings 'base channel threshold - 1 5 ' and ulceration, as determined by duodenoscopy, during 'window width - 8 0 ' . The counter produces volumethe year previous to the removal of the blood sample. related pulses that are accumulated in 100 contiguous No attempt was made to determine the stage of the size-ranges in the channelyzer until the mode channel menstrual cycle in the female subjects: none were reaches a pre-set value of 400. With these settings, taking any form of contraceptive pill. Peripheral the first channel starts at 147.6 ~m 3, the channel venous blood samples were collected in tubes con- width is 7.9 ~m 3 and the last channel ends at 927.0 taining lithium heparin anticoagulant (Searle Diag- /~m3. nostic, High Wycombe, England) from the subjects The size distribution profiles obtained in this when they were in a rested state, between the hours manner were analyzed using an iterative stochastic of 10.00 am and 1.00 pm. model (Brown et al., 1979; Gibbs et al., 1979b) by The mononuclear cells were separated from the which the percentage 6o) of cells responding to P H A blood samples by Ficoll-Paque (Pharmacia Fine stimulation is calculated. We have chosen to describe Chemicals, Uppsala, Sweden) density gradient centri- the volume growth in terms of the simple function fugation (BOyum, 1968) and washed three times in (a + bv), where a is a constant (the basal growth rate) our standard tissue culture fluid--TC199 (Gibco- and b is another constant (the incremental growth Bio-Cult, Paisley, Scotland) buffered at pH 7.3 with rate) which is multiplied by the current volume (v) of HEPES and supplemented with L-glutamine (200 the responding cell. It is important to realise that for raM), penicillin (200 I.U./ml) and streptomycin (100 most cells, b will be the major descriptor of overall ~g/ml). Suspension of these cells were examined by growth rate since it is weighted by the current volume the method of Potts, Gibbs, Robertson, Brown & of the cell, whereas a does not vary and so it is Beck (in press) to determine the relative numbers of unimportant except with very small cells (Gibbs, lymphocytes and of other types of leucocytes: all Brown, Robertson & Beck, 1979a). In our evaluation preparations used in these experiments showed less of this method (Gibbs et al., 1979b) it was shown that than 1007ocontamination with cells of other types and intra-experimental variation within repetitive tests on more than 95070 viability. single large blood samples was relatively small: the The cells were cultured in Cooke microtiter plates percentage of responding cells and their incremental (Sterilin Ltd., Teddington, England) for 22-24 h; growth rates had coefficients of variation of 5.8°70 the precise duration of culture was noted when each and 4.6% respectively. sample was measured. Each round-bottomed well The influence of hydrocortisone on the manner of contained 2 x l0 s cells in 100 /~l TC199 and 20 /~l recruitment of lymphocytes after P H A stimulation autologous serum: either 25 /al of the solution of was studied by measuring the elapsed-time growth hydrocortisone or solvent (TC 199 with 207opropylene after 9 h culture and at 3-hourly intervals until 24 h glycol) alone was added to each well, followed by 25 with the method used previously to study the mode /~l of the optimum dilution of P H A (Wellcome of recruitment of cells into Gl-phase in unmodified Reagents Ltd., Beckenham, England) (10.0 ~l P H A stimulation (Brown, Gibbs, Robertson, Potts H A l 5 / m l TC199). The plates were sealed with & Beck, 1980). adhesive tape (Flow Laboratories, Irvine, Scotland) Statistical analysis o f results before incubation at 37°C. The data were analysed using the Generalised Measurement o f number o f responding cells and Linear Interactive Modelling Package produced by growth rates the Royal Statistical Society (Baker & Nelder, 1978). The methods used for assessing the degree of lymphocyte stimulation have been described in detail RESULTS elsewhere (Gibbs et al., 1979b). Briefly, after the appropriate period of culture, the cells in the wells Hydrocortisone effect on lymphocyte growth after were disaggregated by gentle pipette mixing and main recruitment completed dispersed by repeated inversion in 20 ml Isoton II Results from a typical experiment on the effect of (Coulter Electronics Ltd., Harpenden, England). hydrocortisone on PHA-induced stimulation of The suspension was counted in an electronic particle lymphocytes from a normal adult human subject are counter (Model Fn, Coulter Electronics) with 100 ~m shown in Fig. 1. When the size distribution profile of

Subject Variation in Response of Human Lymphocytes to Hydrocortisone

in the well while the remainder are unchanged in volume. In the presence of hydrocortisone, the P H A stimulation effect was clearly reduced: mathematical analysis showed that this partial suppression was a consequence o f reduction in both the percentage of growing cells and in their averaged growth rate during the elapsed period in culture, Table 1 surnmarises the analysis of variance of the whole series o f 217 observations on the percentage of cells responding to P H A stimulation at varying doses

0.0a I I

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23

Z

v

95

20

40 60 Channel number

80

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Fig. 1. Volume distributions of human peripheral blood lymphocytes measured after culture for 24 h at 37°C with an electronic particle counter in 100 contiguous size ranges ("channels"). The solid line (X) is the profile of ceils cultured without mitogen. The heavier interrupted line (Y) shows the appearance of the cultures stimulated with optimal dose of PHA; the lighter line (Z) shows the partial suppression of the lymphocyte stimulation reaction by addition of hydrocortisone to the culture medium. The mathematical model can be used to calculate from the shape of these profiles the percentage (p) of cells growing in each stimulated culture and two constants (a) and (b) describing their rate of volume growth. the cells o f the unstimulated culture was compared with that of P H A stimulated cells, the peak was seen to be shifted to the right and the distribution had developed a more pronounced tall: these changes are a consequence of growth o f a proportion o f the cells

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20

Residual

Fig. 2. Probability-scale plot of residuals from analysis of variance of observations of percentages of growing cells in PHA-stimulated lymphocyte cultures in the presence of various hydrocortisone concentrations. The generally linear distribution indicates that these residuals are distributed normally.

Table 1. Statistical analysis of percentage of cells undergoing volume (G]-phase) growth in lymphocytes from 21 normal subjects stimulated by PHA in the presence of various doses of hydrocortisone

Source of variation

H JI I I ~ l l m

Mean square

11410

2246,3

Variance ratio

282

55.5

Different slopes for all subjects

101,5

2.51

Residuals

40.46

--

Significance level

P<0.001

P<0.00I

Pc-0,001 --

24

A. J. ROBERTSONet al.

Table 2.

Variation in log-dose related depression by hydrocortisone of numbers of cells recruited into the first G r p h a s e in PHA-induced stimulation of lymphocytes from 21 normal human subjects

Regression line of log-dose response Slope

Subject no.

l 2 3 4 5 6 7 8 9 l0 11 12 13 14 15

16 17 18 19 20 21

Value

SE

-6.517 - 5,933 -4.770 -4,660 -4.334 -4,207 - 4.187 -3.860 -3,670 -3.612 -3.173 -3.090 - 3.019 2.944 2.645 -2.590 -2.323 -2.007 -0.865 -0.707 -0.681

1.011 0.876 0.771 0.876 1.011 0.771 0.876 0.771 1.417 1,184 1,011 0.878 1.011 1.011 1.011 0.771 1.011 1.417 1.011 1,011 1,009

950/0 Confidence limits Lower Upper

-8,499 - 7.649 -6.281 -6.376 -6.316 -5.718 - 5.903 -5.371 -6.447 -5.933 -5.155 -4.810 - 5.001 - 4,926 - 4.627 -4.101 -4.305 -4.784 -2.846 -2.689 -2,658

Y-intercept (per cent cells growing in presence of 1/~g hydrocortisone/ ml)

Per cent of cells growing in absence of hydrocortisone

Age (yr)

Sex

49.37 43.60 51.05 56.02 64.62 71.11 24.97 62.50 34.53 61.79 60.89 60.51 49.06 70.14 75.80 65.59 74.85 54.66 48.52 87.12 84.89

84.6 73.0 83.6 82.0 86.3 93.6 49.0 83.4 48.0 81.9 73.5 75.2 84.7 84.8 88.8 78.8 86.2 85.6 63.6 87.5 88.6

47 20 45 21 50 50 25 47 70 40 18 59 25 25 21 32 40 30 25 40 30

M M M M M M M M F M F F F M M F M M F F M

-4,535 - 4.217 -3.259 -2.944 -2.352 -2.696 - 2.471 -2.349 -0.893 - 1.291 -1.191 - 1.370 - 1.037 - 0.962 - 0.663 - 1,079 -0.341 -0.770 - 1.117 - 1.274 - 1.297

Table 3. Statistical analysis of incremental growth rate during G r p h a s e of lymphocytes from 21 normal subjects responding to P H A stimulation in the presence of various doses of hydrocortisone

Source of variation

Mean square

Variance ratio

Significance level

Log-dose response relationship

7.60

79.64

P < 0.001

Different intercepts for all subjects

6.87

72.26

P<0.001

Different slopes for all subjects

0.339

3.56

P<0.001

Residuals

0.095

--

--

Subject Variation in Response of Human Lymphocytes to Hydrocortisone of hydrocortisone from the 21 subjects. All three variance ratios are highly significant and we may therefore conclude that there is very strong evidence of (a) a log-dose relationship for the responses of each subject over the range of doses investigated and (b) intersubject variability with respect to both slope and intercept (percentage of cells growing when hydrocortisone concentration was 1 /~g/ml). The ordered residuals from this analysis are plotted on a normal probability scale in Fig. 2: apart from six possible outliers, the general pattern is strongly linear. A further analysis omitting the observations corresponding to the outliers gave the same conclusions. The linearity of the plot confirms the standard assumptions necessary for the validity of the analysis of variance. A graph of the ordered slope estimates plotted on a normal probability scale (Fig. 3) demonstrates by its closeness to a straight line the approximate normality of the distribution of the slopes for all 21 subjects.

25

ficant and we may therefore conclude that there is strong evidence of (a) a log-dose relationship for incremental growth variation over the range of doses investigated and (b) intersubject variation for slopes and intercepts (incremental growth rates when the hydrocortisone concentration was 1 /ag/ml). The residuals were distributed normally.

Hydrocortisone effects on the kinetics of recruitment o f cells into G j-phase Within the concentration range of 0.03 to 14.7/ag hydrocortisone/ml, the percentage of responding cells decreased with increasing dose of glucocorticoid but reached a plateau level at the same time as the control cultures. There was no evidence of a difference in the duration of the lag period before the onset of volume growth, when the glucocorticoid treated cultures were compared with the controls (Fig. 4). Thus it would appear that although the percentage of cells recruited is dose dependent, the time course of recruitment into G~-phase is not modified by hydrocortisone. 100

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Fig. 3. Probability-scale plot of slopes of log-dose response estimates for depressive effect of hydrocortisone on percentage of responding cells in PHA-induced lymphocyte stimulation. The strongly linear distribution indicates that this measurement is distributed normally. The values of the slopes for changes in percentage of responding cells, their standard errors and a 95*/o confidence interval for each slope are shown in Table 2. These slopes did not show any relation to the age of the subject: however, the range of variation was greater in men than in women (/)<0.05). Table 3 summarises the analysis of variance of the 217 observations of incremental growth rate from the 21 subjects. All three variance ratios are highly signi-

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Time (h) Fig. 4. Calculated numbers of growing cells in cultures of lymphocytes from a normal subject in the presence and absence of hydrocortisone sampled at intervals during the first day (+ no hydrocortisone added; * 0.25 /~g hydrocortisone/ml). The similarity in the development of the response suggests that the time-course of recruitment of cells is not modified by the addition of hydrocortisone to the culture fluid.

Effect o f differing temporal relationship between hydrocortisone exposure and PHA stimulation Addition of hydrocortisone to lymphocyte cultures 6 h before PHA stimulation resulted in a much greater (three- to five-fold) inhibition in numbers of

A.J. ROBERTSONet al.

26

+

80

+ +

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60 ,n

= t-

40

O

20

*

-6

-4

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HC pre-incubation

•

,

2

4

HC post-incubation

Interval between addition of Hydrocortisone and PHA (h)

Fig. 5. The effect of the time at which hydrocortisone is added to cultures on its suppressive action on PHA-stimulation of lymphocytes from a normal subject (+ no hydrocortisone added; * 0.23 /ag hydrocortisone/ml). The ceils are much more sensitive when pre-incubated with the hormone and become progressively more resistant if hydrocortisone is added after the onset of PHA stimulation, so that by 5 h, the cells are almost completely resistant to its action. responding cells (and a comparable reduction in incremental growth rate) than synchronous addition of the same concentration of hydrocortisone (Fig. 5). By contrast, addition of hydrocortisone at hourly intervals after P H A stimulation progressively reduced the inhibitory effect, so that by 5 h, stimulation with PHA was virtually unaffected. DISCUSSION

Subject variation in depression of lymphocyte stimulation by hydrocortisone Addition of hydrocortisone to cultures of normal human lymphocytes, stimulated with optimal doses of PHA, causes a reduction in the number of cells that are stimulated and slows the growth rate of the responding cells in the G~-phase of the first stimulated cell cycle: however, the kinetics of recruitment are not modified. Comparable changes are seen in the absence of hormone when the cells are stimulated by suboptimal concentrations of PHA (Gibbs, Potts, Brown, Robertson & Beck, unpublished results), but the mode of action of hydrocortisone must be different since there is no evidence that the glucocorticoid can bind the mitogen to reduce its availability in the same way that certain normal and

pathological sera can 'neutralise' PHA (Amlot & Unger, 1976; Sohnle & Collins-Lech, 1979). It is therefore probable that hydrocortisone alters the potential responsiveness of the cells by raising their stimulation threshold. Naturally-occurring serum inhibitors, such as those found in lepromatous leprosy are capable of diminishing the number of responding cells, but do so without modifying the growth rate or recruitment kinetics of the cells in the resistant subpopulation (Sherif, Robertson, Potts, Gibbs, Brown & Beck, unpublished experiments) and hence also differ in the mode of action from hydrocortisone. It is clear from our experiments that hydrocortisone inhibits PHA action at concentrations corresponding to the non-protein bound levels found in the plasma in physiological and pharmacological stages. Ilfield, Krakauer & Blaese (1977) have previously shown that the participation of human lymphocytes in the mixed lymphocyte reaction is also inhibited by comparable concentrations of hydrocortisone but they did not study the effect on the cell cycle kinetics. The inhibitory action had a log-dose response curve in both groups of experiments and so it is comparable to most hormone-receptor and drugacceptor dependent effects. The extent of inter-

Subject Variation in Response of Human Lymphocytes to Hydrocortisone subject variation in apparently normal people (tenfold range) was greater than we had anticipated when the experiments were started. It is not possible to explain these results on the basis of comparable variation in the plasma protein binding of the added glucocorticoid since (a) human sera do not show any great variation in their content of transcortin-binding globulin (Doe, Fernandez & Seal, 1964); (b) the plasma concentrations of transcortin-binding globulin are low relative to the pharmacological concentrations of hydrocortisone studied in our experiments, consequently variation in transcortin-binding globulin will have little effect on the slope of the logdose response curves; and (c) although plasma transcortin-binding capacity shows diurnal variation (Angeli, Frajria, De Paoli, Fonzo & Ceresa, 1978), this factor is largely irrelevant to our experiments since the blood samples were removed from rested subjects at a standard time of day to avoid great fluctuations in endogenous glucocorticoid levels. Furthermore it is very unlikely that the considerable intersubject variability in dose-responsiveness is a direct consequence of heterogeneity of the relative sizes of the individual lymphocyte subpopulations, each with different responsiveness since the lymphocyte subpopulations show variation in numbers and relative proportions of a much smaller order, but this possibility cannot be dismissed since dose-response tests have not yet been performed on purified subpopulations of lymphocytes. A number of mechanisms have been adduced to explain the variations in cellular sensitivity to glucocorticoids observed in comparative studies between species, organs and various lymphoid cell lines (Harris & Baxter, 1979): these include: (a) temporary modifications in receptor binding characters by interaction of other metabolites; (b) induction of changes in numbers of receptors by recent metabolic experience; (c) genetic variation in number and characteristics of receptors; and (d) inherent or acquired variation in the capacity of cells to perform metabolic activity triggered by glucocorticoid binding. We have attempted in our experiments to minimise such influences by selecting healthy people who were not taking drugs or alcohol regularly and who did not smoke. Consequently our findings should be representative of the responses of the circulating lymphocytes in normal people. We have found a ten-fold variation in the slopes of the logdose response curves: clearly, changes in the relative numbers of cells in the various subpopulations, whether a consequence of diurnal variation (Carter, Barr, Levin, Byers, Ponce, Fudenberg & German, 1975; Fan, Yu, Clements, Opelz, Goldberg & Bluestone, 1977; Tavadia, Fleming, Hume & Simpson,

27

1975; Eskola, Frey, Molfiar & Soppi, 1976; Abo & Kumagai, 1978), or cyclic menstrual variation (Raptopoulou & Goulis, 1977), or from disease (Robbins & Gershwin, 1978) or from drug effect on the cells [e.g. depression by aspirin, Panush & Anthony (1976) or potentiation by procainamide, Bluestein, Zvaifler, Weisman & Shapiro (1979)1, might further exaggerate the potential variability in glucocorticoid responsiveness. If glucocorticoids act in a similar manner in vivo, then there should be considerable intersubject variation in the plasma levels necessary to induce an immunosuppressive effect.

Mode of depression of lymphocyte stimulation by hydrocortisone It has been established with very potent synthetic glucocorticoids that pre-exposure diminishes the sensitivity of lymphocytes to P H A stimulation (Nowell, 1961; Elves, Gough & Israels, 1964; Darzynkiewicz & Pienkowski, 1969). We have now shown that a natural hormone at physiological concentrations has the same effect. Smith, Crabtree, Kennedy & Munck (1977) have shown that stimulation with a mitogen (Con A) also results in an increased number of glucocorticoid receptors, but with P H A the increased number of receptors did not reach significance for 20h (Lippman, 1979): thus the rate of increase in receptor numbers was much slower than that demonstrated after glucocorticoid treatment (Neifeld, Lippman & Tormey, 1977). From these experiments we would infer that the number of glucocorticoid receptors would remain unchanged during the first 5 h after P H A exposure, during which we detected rapidly progressive falling-off of sensitivity to hydrocortisone: consequently this steroid must have a potent influence on at least one of the complicated metabolic changes that take place during the early hours after stimulation (Ling & Kay, 1975; Decker & Marchalonis, 1978) and before there is clear cytological evidence that the cell has left the G~-phase: the nature of this biochemical mechanism remains unexplained. Weber, Skoog, Mattsson & Lindahl-Kiessling, (1974) have postulated that there are at least four steps in the early cellular response after mitogen stimulation and that the first three steps are potentially reversible: our work suggests that hydrocortisone acts by preventing some of the cells passing through Step II and also by retarding the progress of other cells through the succeeding steps in G~-phase. This hypothesis is supported by the results of Sloman & Bell (1980) who have shown that there is cyclic variation in the sensitivity of longer-term cultures of porcine lymphocytes to depression of bulked

28

A. J. ROBERTSONel al.

D N A synthetic rate by potent synthetic glucocorticoids: even though these experiments did not include cell cycle kinetic studies, the time relationships that were found infer that the cells are most sensitive in the Gl-phase. By contrast, Ilfield et al. (1977) had previously shown that the mixed lymphocyte reaction was not suppressed by the presence of hydrocortisone during the first 6 h of culture: we cannot explain this discrepancy from the results of our experiments and we must conclude that cells in the mixed lymphocyte reaction experiments respond to hydrocortisone in a different manner from most other systems where cell growth is arrested by glucocorticoids in the Gi-phase of the cycle (Frankfurt, 1968; Epifanova, 1971; Adolphe & Lechat, 1974; Wright, Appleton & Morley, 1974). Potential applications o f v o l u m e spectroscopy technique in i m m u n o p h a r m a c o l o g y

The doses of glucocorticoids used therapeutically to induce immunosuppression frequently give rise to serious side-effects, e.g. development of Cushingoid facies, weight gain, hypertension, carbohydrate intolerance and suppression of the hypothalamicpituitary-adrenal axis. Current practice assumes that all patients require a similar dose of glucocorticoids, but clinical experience and the results of our experiments imply that there is considerable individual

variation in responsiveness. Unfortunately no tests are available for routine clinical monitoring of the other effects of glucocorticoids, such as modification of the lymphocyte circulation kinetics or of antiinflammatory action. The tests that we have described in this paper might be appropriate for determination of minimum doses required to give adequate immunosuppression prior to starting treatment and also at intervals throughout treatment, so that the dosage of glucocorticoid could be tailored to the requirements of the individual patient with consequent increase in efficacy and reduction in the morbidity associated with this type of treatment. Clearly, the methods we have devised could be readily adapted to study the pharmacological action on human lymphocytes of the various synthetic steroids that have been shown to have glucocorticoid activity in classical endocrine or receptor binding assays. The new technique could provide complementary information on the relative potencies of the immunosuppressive action of the components and give some indication on the extent of intersubject variability of human peripheral blood lymphocytes. Acknowledgements--This work was partly supported by Grant K/MRS/50/C207 from the SHHD Biomedical Research Committee. The data terminal was donated by Tenovus Tayside. We are grateful to Mr R. S. Fawkes for the preparation of the diagrams.

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Subject Variation in Response of Human Lymphocytes to Hydrocortisone

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