the effect of hydrocortisone on the kinetics of normal …€¦ · · 2017-04-13vascular...

Blood, Vol. 46, No. 2 (August), 1975 235

The Effect of Hydrocortisone on the Kinetics of

Normal Human Lymphocytes

By Anthony S. Fauci and David C. Dale

Lymphocyte kinetic studies employing

51chromium-labeled autologous lympho-

cytes were performed in nine normalvolunteers in order to determine the ef-fects of hydrocortisone administration onthe recirculating versus the nonrecirculat-ing intravascular lymphocyte pools. Fol-lowing infusion of labeled cells, therecirculating portion of the labeled cellsrapidly equilibrated with the total intra-

vascular lymphocyte pool and the vastlylarger total-body recirculating lymphocytepool, so that by 1 hr following infusion21.8% ± 3.2% of the labeled lympho-cytes were left in the circulation. Fourhundred milligrams of intravenous hydro-cortisone administered 24 hr after infusionof labeled cells caused a profound buttransient lymphocytopenia which was

maximal at 4 hr with return of lymphocytecounts to normal by 24 hr after injection.Concomitant with the lymphocytopeniathere was a dramatic increase in lym-phocyte specific activity (cpm per 106

lymphocytes), while the total lymphocyte-associated radioactivity remaining in thecirculation was unchanged, indicating thatcorticosteroid administration depleted theunlabeled recirculating cells. As the lym-phocyte counts returned to normal follow-ing hydrocortisone, the specific activityalso returned to normal. These studiesindicated that hydrocortisone administra-tion caused a transient lymphocytopeniaby a preferential depletion of the recircu-lating portion of the intravascular lympho-cyte pool.

C ORTICOSTEROID ADMINISTRATION in normal humans has been

demonstrated to cause a transient depletion of lymphocytes, predomi-

nantly of the thymus-derived (T) cell population)’2 It has been proposed that

this corticosteroid-induced lymphocytopenia results not from a destruction of

cells3 but from a redistribution of lymphocytes out of the circulation into other

body compartments,l’2 much like that which has been observed in certain ani-

mal species.�8 The phenomenon of the recirculation of lymphocytes has been

well established both in animals9� and in man.12 Since recirculating lympho-

cytes can freely migrate into and out of the circulation9� and since they are

composed predominantly of T lymphocytes,’#{176}’13 it is reasonable to assume that

the corticosteroid-induced depletion of predominantly T lymphocytes is due to

a redistribution of these intravascular recirculating lymphocytes. In order to

test this hypothesis, radioactive chromium (51Cr)-labeled lymphocyte kinetic

studies have been employed to determine the effects of acutely administered

hydrocortisone on the recirculating lymphocyte pool in normal human volun-

teers.

From the Laboratory of Clinical Investigation, National Institute of Allergy and Inftctious

Diseases, National Institutes of Health, Bethesda, Md. 24YJ14.

Submitted October 3, 1974; accepted March 5, 1975.

Address for reprint requests: Anthony S. Fauci, M. D., National Institutes of Health, Bldg. 10,

Room 1 JB-09, Bethesda, Md. 20014.

© 1975 by Grune & Stratton. Inc.

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236 FAIJCI AND DALE

MATERIALS AND METHODS

Subjects

Nine normal volunteers (six females, three males; ages 19-25 yr) were used in the study. In-

formed consent was obtained, and the subjects were taking no medications during the study. Five

subjects received a single injection of 400 mg of hydrocortisone sodium succinate (OHC) intra-

venously (i.v.)1 at the time designated below. Four subjects served as controls and did not re-

ceive OHC.

Separation and 5tCr-Iabeling of Leukocytes

At 8 a.m. on the first day of the study, 450 ml of citrate dextrose anticoagulated blood were with-

drawn from the antecubital vein via a 16-gauge needle and collected in a sealed sterile plastic bag

to which two satellite bags were connected (Fenwal Laboratories, Division of Travenol Labora-

tories, Morton Grove, Ill.). Separation and labeling of leukocytes were performed by a slight

modification of a previously described 14 One hundred milliliters of 6% dextran in 0.9%

saline (Abbott Laboratories, North Chicago, Ill.) were added to the bag of whole blood, and the

cells were sedimented at room temperature at 1 g for 45 mm. The leukocyte-rich supernate was

expressed into a satellite bag using a plasma extractor (Fenwal Laboratories), and platelets were

removed by differential centrifugation (150 g for 3 mm at 20’C). The white blood cells (WBC)

were incubated in 20 ml of plasma dextran with 500 �sCi of 51Cr (sodium radiochromate, E. R.

Squibb & Sons, Inc., Radiopharmaceutical Dept., New Brunswick, N.J.) for 30 mm at 37’C. Fifty

milligrams of ascorbic acid (Eli Lilly & Co., Indianapolis, Ind.) were then added to reduce the

remaining unbound chromate. The labeled cells were washed twice with and resuspended in 50 ml

ofautologous plasma-dextran and reinfused over a 1-3-mm period. The total time from withdrawal

of blood to reinfusion oflabeled cells was approxmately 24-3 hr. and the viability of the reinfused

labeled cells was always greater than 95% (trypan blue dye exclusion test). The employment of

these sealed plastic bags with attached satellite bags allowed the separation, labeling, washing,

and reinfusion of cells to be accomplished without breaking sterile conditions or exposing the

blood components to air. All cell types in the leukocyte-rich fraction obtained from dextran

sedimentation were labeled and reinfused. The lymphocyte-associated radioactivity was deter-

mined from Hypaque-Ficoll-separated cells from an aliquot of preinfusion cells and from blood

samples taken throughout the study. The reasons for this approach will be discussed further on.

Hypaque-Ficoll separation’5 of mononuclear cells was performed on an 8-ml aliquot of labeled

cells taken from the bag before reinfusion and from 20-mi samples of blood drawn at the desig-

nated time intervals after reinfusion. These Hypaque-Ficoli-separated mononuclear cells contained

approximately 85% lymphocytes, 15% monocytes, and less than 1% granulocytes. During the study,

several Hypaque-Ficoll-separated cell samples drawn 24 hr or more after the reinfusion of labeled

cells were plated on glass petri dishes to remove monocytes by glass adherence and so determine

precisely what per cent of the radioactivity of these separated cells was in the lymphocyte fraction.

It was found that greater than 95% of the radioactivity was associated with the lymphocytes. This

lack of monocyte-associated radioactivity in the cell suspensions is best explained by the fact

that most of the reinfused labeled monocytes had been cleared from the circulation by this time.

To rule out the possibility that any of the radioactivity of the Hypaque-Ficoll-separated cells was

associated with labeled platelets (since variable numbers of platelets are often seen in smears of

Hypaque-Ficoll-separated cell fractions), platelet counts and radioactivity determinations were

performed on the cell fractions before and after differential centrifugation to remove platelets. In

calculations throughout the study, we determined the lymphocyte-associated radioactivity as well as

the specific activity of lymphocytes (cpm per 106 lymphocytes) from these Hypaque-Ficoll-

separated cell samples. Cells were counted in a Coulter Counter (Model Fn, Coulter Electronics,

Inc.), and radioactivity was measured in an automatic gamma counter (Series 1185, Nuclear-

Chicago, Des Plaines, Ill.). The total lymphocyte-associated radioactivity contained in the circu-

lation at each individual time point was determined by converting the lymphocyte-associated

radioactivity in the 20-ml sample of blood to the total amount in the intravascular compartment,

assuming blood volume as 7% of body weight. �

Blood samples were drawn at 15 mm and 1, 2, 3, 6, 12, and 24 hr after the reinfusion of labeled

cells. The five subjects who received the single dose of 400 mg OHC intravenously got their dose




HYDROCORTISONE AND LYMPHOCYTES 237

immediately after the 24-hr sample. Blood samples were drawn 4 and 8 hr after OHC administra-

tion (28 and 32 hr after infusion of cells), and further samples were drawn daily for 6 days. Sub-

jects who did not receive OHC had blood samples drawn at the same time intervals. In addition to

the blood samples taken for determination of lymphocyte radioactivity, samples for WBC counts

and differentials were taken at each time interval.

There are several reasons why we chose to label and reinfuse dextran-sedimented WBC and per-form our more involved Hypaque-Ficoli separation of lymphocytes and determination of lympho-

cyte-associated radioactivity on the blood samples drawn after reinfusion of labeled cells. First,

it was learned from preliminary experiments that as minimal manipulation of cells as possible

prior to reinfusion lessened the chance of altering or damaging cells and appreciably increased

the proportion and time of survival after reinfusion. Second, Hypaque-Ficoll separation and

labeling of purified lymphocytes before reinfusion would entail manipulating cells outside the

sterile, sealed bags. This procedure would increase the small but nonetheless definite chance of

microbial contamination. Third, there is no real advantage in this particular study in removing

granulocytes before labeling the cells, since the half-life of granulocytes’7 is sufficiently short that

most of the labeled granulocytes would no longer be present by the time the OHC was administered

at 24 hr to determine the effect on lymphocyte kinetics. Fourth, removal of granulocytes and

monocytes by nylon or glass-bead column purification prior to labeling was avoided, since this

procedure has been shown to remove selectively certain lymphocyth populations)8”9

At the time each blood sample was drawn, body surface counts were measured over the spleen,sternal bone marrow, liver, and lung using a Tri-Carb scintillation spectrometer with a shielded

2-inch thallium-activated, sodium iodide crystal (Model 3002, Packard Instrument Co., Downers

Grove, Ill.).20

RESULTS

Since the initial 51Cr labeling of cells was done on the leukocyte-rich super-

nate from dextran-sedimented whole blood, it was necessary to rule out the

possibility that the radioactivity which was assayed in the samples drawn follow-

ing reinfusion of labeled cells was due to contaminating platelets, granulocytes,

or red blood cells (RBC). As mentioned above, determinations of lymphocyte-

associated radioactivity and specific activity of lymphocytes were done on

Hypaque-Ficoll-separated cells. These suspensions were macroscopically free

of RBC. This fact was confirmed by examination of cytocentrifuged smears

stained with Wright-Giemsa stain. It was found that only rare RBC per high-

power field were seen in these smears. However, in order to rule out totally the

possibility that these few contaminating RBC were significantly contributing to

the activity of the samples, Hypaque-Ficoll suspensions of specimens from four

subjects at different time periods in the study were examined for activity prior

to and following hypotonic lysis of RBC. It was found that this maneuver de-

creased the activity of the specimens by only 5.5% ± 1.2%. This small decrease

was due most likely to the minimal damage to the mononuclear cells themselves

by the hypotonic exposure as well as to the small amount of elution of label

consistently seen with washing alone.

As mentioned previously, granulocytes make up only about 1% of the

Hypaque-Ficoll mononuclear cell layer, and so any contribution by granulo-

cytes to the total activity of the cell suspension would be negligible. In addition,

the half-life of granulocytes is sufficiently short’7 that most of the labeled granu-

locytes would have been cleared from the circulation by 24 hr after reinfusion.

The most significant potential problem with contamination is with labeled

platelets. It is clear that Hypaque-Ficoll-separated mononuclear cells may con-

tain substantial numbers of platelets. Despite the fact that platelets were re-




238 FAUCI AND DALE

Table 1. Effect of Removal of Platelets on Lymphocyte

Specific Activity of Mononuclear Cell Suspensions

Centrifugations’

0 1 2

Suspension A

Platelets per ml 4 x 106 0.4 x 106 0

Lymphocyte SAt 23.1 23.3 21.0

Suspension B

Platelets per ml 6 x 106 1 x 106 0.4 x 106

Lymphocyte SA 29.6 28.4 26.2

counts and lymphocyte SA determinations were done on original

each of two differential centrifugations.

tSA, specific activity.

moved by differential centrifugation prior to 51Cr-labeling, it was not certain

that all platelets were removed, and thus some were labeled and reinfused with

the labeled WBC. In order to determine what contribution, if any, labeled plate-

lets made to the activity of the Hypaque-Ficoll suspensions which were assayed

for lymphocyte activity, the effect of removal of platelets from the suspensions

by differential centrifugation (150 g for 3 mm at 20#{176}C)upon the specific activity

of lymphocytes was determined in two subjects. Table 1 shows that removal of

all detectable platelets from suspension A and greater than 90% of the platelets

from suspension B resulted in only minimal decreases in the lymphocyte specific

activity. These data indicate that neither contaminating RBC, granulocytes, nor

platelets significantly influenced the activity of the mononuclear cell suspen-

sions.

Following reinfusion of cells, rapid equilibration of labeled lymphocytes with

the total intravascular as well as extravascular compartments occurred. This

equilibration was similar to that described in previous lymphocyte kinetic

studies.’4 By 1 hr following reinfusion of labeled cells, the mean per cent of total

injected lymphocyte-associated radioactivity remaining in the circulation for all

nine subjects was 2 1.8% ± 3.2%, with a range of 12.0%-36.2%. In order to de-

termine more accurately the kinetics of labeled versus unlabeled lymphocytes

remaining in the circulation after equilibrium had been reached, together with

the effect of OHC administration on these kinetics, the amount of lymphocyte-

associated radioactivity left in the circulation at any given time, as well as the

specific activity of lymphocytes, was expressed as the per cent of the 1-hr value.’6

Figure 1 illustrates the effect of a single dose of 400 mg OHC on lymphocyte

kinetics. Prior to the administration of OHC (at 24 hr), the total circulating

lymphocyte counts showed very little variability and were the same in the treated

and control groups. Likewise, the per cent of the 1-hr lymphocyte specific

activity, as well as the per cent of the 1 hr-lymphocyte-associated radioactivity

remaining in the circulation, were the same for the treated and control groups,

and both showed a gradual linear decay. Following administration of a single

400 mg dose of OHC intravenously, there was a dramatic decrease in total cir-

culating lymphocyte counts which was maximal at 4 hr. Counts returned to

normal by 24 hr following OHC administration. Thereafter, the lymphocyte

suspensions and after




Hours Days


0-c Controls; no OHC (n=4)

� 400 mg OHC given IV.

immediately following

� 3000 24 hr sample ln=51

�1000 IT-

O.

C)>.

4-

cr�5)

1<U

w -IU>.

-.�J--J

I-F.. �Z) �wUUO��IO.LUO.U

>.-J

Fig. 1. Effect of hydrocortisone adminis-tration on the kinetics of reinfused 51Cr- �labeled autologous lymphocytes. Time after � � Q

reinfusion of labeled cells is indicated on the ‘-

horizontal scale. Data on the vertical scale for �

the lymphocyte specific activity and the u.

lymphocyte-associated radioactivity remain- �ing in the circulation at a given time are I-

given as the per cent of th. i-hr value for �

each subsequent time point. Hydrocortisone �.

was administered immediately after the 24-hrsample was taken. The means (± SEM) forthe treated and control groups are shown. TIME

counts in both groups were the same for the remainder of the study (6 days). At

the same time as the decrease in lymphocyte counts in the OHC-treated group

was seen, there was a marked increase in lymphocyte specific activity which re-

turned to normal as the lymphocyte counts returned to normal and subsequently

resumed the gradual linear decay pattern identical to that of the untreated

group. However, during the period of corticosteroid-induced lymphocytopenia,

the amount of lymphocyte-associated radioactivity was unchanged and con-

tinued to follow a gradual linear decay pattern identical to that of the control

group. Hence, the depletion of circulating lymphocytes caused by corticosteroid

administration was predominantly from the unlabeled lymphocytes in the cir-

culation. This conclusion was determined by the fact that the amount of

lymphocyte-associated radioactivity in the circulation remained unchanged

while lymphocyte specific activity markedly increased during the decrease in

total circulating lymphocyte counts.

In addition to the fact that there was no increase in egress of labeled lympho-

cytes from the circulation following OHC administration, neither was there

any redistribution of labeled cells already present in the spleen, bone marrow,




240 FAUCI AND DALE

c-c Controls; no OHC ln=4l

‘--a 400 mg OHC given IV.

120 immediately following

24 hr measurement ln=5l

�200U ii�i I

� 100 Bone MarrowI-U<80

�40

�20

(I) (� LI I I

� 100 Liver

�20U

100 Lung -�Fig. 2. Effect of hydrocortisone administration

80 on the organ distribution of reinfused 51Cr-Iabeled

60 �-- - autologous lymphocytes detectable by surface body

40 �‘t counting. Labeled cells are reinfused at time 0 on thehorizontal scale. Data on the vertical scale for the or-20 gan activity are given as the per cent of the 1-hr

- . value for each subsequent time point. Hydrocortisone36 12 24 48 3 4 5 6 was administered immediately after the 24-hr time

Hours Days point. The means (± SEM) for the treated and con-

TIME trol groups are shown.

liver, and lung. Figure 2 illustrates that once the labeled cells entered these

tissues there was no change in the distribution of labeled cells in the treated

group following administration of OHC.

DISCUSSION

The phenomenon of the recirculation of lymphocytes has been clearly

demonstrated in certain animal species9” as well as in man.’2 Two distinct pop-

ulations of lymphocytes have been shown to exist in certain animal species

based on circulation properties.9” The recirculating portion of the intravascu-

lar lymphocyte poo1, comprised of approximately two-thirds of lymphocytes in

the circulation, belongs to a vastly larger total-body recirculating lymphocyte

pool.’#{176}These intravascular recirculating cells can freely migrate into and out of

the circulation. They are in constant equilibrium with the extravascular portion

of this total-body recirculating poo1 contained in the thoracic duct lymph and

certain areas of the spleen, lymph nodes, and bone marrow.��l’2L The major

proportion of these recirculating lymphocytes has been shown to be thymus

derived.’0”3 The smaller fraction of intravascular lymphocytes belongs to the





nonrecirculating pool which is not capable of freely migrating from the cir-

culation, and these cells probably live out their life span within the intravascu-

lar � In the present study, despite the fact that the reinfused cells

were minimally manipulated and were greater than 95% viable, there was a

rapid initial clearance of approximately 78% of labeled lymphocytes within the

first hour after reinfusion. Thereafter there was a gradual and steady decay of

lymphocyte-associated radioactivity remaining in the circulation. This observa-

tion is very similar to the findings in previous human lymphocyte kinetic

14�l6 The explanation has been put forth that the recirculating fraction of

the reinfused lymphocytes which is capable of freely migrating out of the circu-

lation, rapidly equilibrates with the vastly larger total-body recirculating pool.’4”6

The lymphocyte-associated radioactivity remaining within the intravascular

circulation is due then, for the most part, to the nonrecirculating fraction of the

reinfused labeled lymphocytes which cannot migrate out of the circulation. Since

the recirculating labeled lymphocytes have become so diluted in the much larger

total-body pool, one would expect them to contribute very little to the radio-

activity in the circulation as they continue to recirculate between the intra-

vascular and extravascular compartments.

The present study shows that the transient lymphocytopenia caused by a

single dose of400 mg ofOHC is due to a depletion of recirculating lymphocytes

from the intravascular compartment. Precisely at the point of OHC-induced

maximal lymphocytopenia, there was a dramatic increase in lymphocyte specific

activity, while the lymphocyte-associated radioactivity remaining in the circu-

lation was no different from that of the control group (Fig. 1). These data clearly

demonstrate that the lymphocytes which had been depleted from the circulation

by OHC administration were unlabeled recirculating cells, and the labeled non-

recirculating lymphocytes in the circulation were not noticeably affected. Of

interest is the fact that, as the total lymphocyte count returned to normal 24 hr

following OHC administration, the lymphocyte specific activity returned to and

followed the identical decay curve as that of the untreated control group, in-

dicating that the depleted cells had been replaced in the circulation by unlabeled,

recirculating lymphocytes which were capable of reentering the intravascular

compartment and restoring the total lymphocyte count to normal. This finding

is quite consistent with previous findings that administration of 400 mg of OHC

to normal subjects results in a preferentially greater depletion of the T-lymphocyte

population of circulating lymphocytes defined functionally and by surface

markers.’ This predominantly T-lymphocytopenia was maximal at 4 hr follow-

ing OHC, with a return to normal total numbers and proportions of lympho-

cyte populations by 24 hr. The present observation of return to the normal

linear regression of specific activity following transient depletion of cells to-

gether with previous evidence3’22 that suprapharmacologic doses of corti-

costeroids in vitro do not cause a lysis or death of normal human lymphocytes

argues strongly for a redistribution phenomenon as the explanation of the

corticosteroid-induced lymphocytopenia noted here. Studies in the mouse,�6

guinea pig,7 and rat8 have shown that corticosteroids cause a circulating lym-

phocytopenia by effecting a redistribution of T-lymphocytes out of the circula-

tion into other body compartments, particularly the bone marrow. In addition,




242 FAUCI AND DALE

this phenomenon of corticosteroid-induced redistribution of lymphocytes can

actually cause a lymphocytosis in special circumstances. Shaw et al.23 demon-

strated that corticosteroid administration caused a temporary lymphocytosis in

patients with chronic lymphocytic leukemia which was explained, at least in

part, by the redistribution of lymphocytes from peripheral organs such as the

spleen into the circulation.

Surface organ radioactivity counting demonstrated that, after equilibrium

had been reached following reinfusion, and after labeled cells had distributed to

different tissues, there was little redistribution of label in tissues following OHC

administration. This finding was not surprising, since the labeled recirculating

lymphocytes had already equilibrated and left the circulation by that time, and

we have seen (Fig. 1) that OHC administration caused no appreciable depletion

of labeled lymphocytes from the circulation. Surface body counting of organ

radioactivity is at best a very crude measurement. At the time of OHC ad-

ministration, equilibrium had already been reached, and the lymphocytes which

were depleted from the circulation were nonlabeled recirculating cells which

had replaced the recirculating lymphocytes during equilibration. It is unlikely

that any further shifts of labeled cells between organs at the time of OHC ad-

ministration would be appreciated by surface counting in experiments of this

sort because of the tremendous dilution factor in the total-body recirculating

lymphocyte pool and the necessarily small amount of radioactive label allow-

able in normal volunteer studies.

The present study demonstrates that in humans the lymphocytopenia induced

by acutely administered corticosteroids is due to a preferential depletion from

the circulation of recirculating lymphocytes. These cells under normal circum-

stances are capable of migrating into and out of the intravascular compartment

and are in equilibrium with the total-body recirculating lymphocyte pool. The

mechanisms whereby corticosteroid administration causes this selective re-

distribution of the intravascular portion of the recirculating lymphocyte pool

remain unknown. However, the fact that enzymatic alteration of molecular

configurations on the surface of lymphocytes profoundly affects their recirculat-

ing capacities24’2S suggests that the present alterations in circulation may be

directly associated with pharmacologically induced changes in the lymphocyte

surface.

ACKNOWLEDGMENT

The authors wish to acknowledge the expert technical assistance of Mrs. Rhoda Hubert.

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1975 46: 235-243

AS Fauci and DC Dale The effect of Hydrocortisone on the kinetics of normal human lymphocytes

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