JOURNAL OF SURGICAL RESEARCH 16, 172-182 (1974) Effect of Exogenous and Endogenous Glucocorticosteroids on the In Vitro Stimulation of Lymphocytes from Sedated and Awake-Restrained Healthy Baboons* AFTAB AHMED, PH.D., CLIFFORD M. HERMAN, M.D., RICHARD C. KNUDSEN, PH.D., JONAS SODE, M.D.? DOUGLAS M. STRONG, PH.D., AND KENNETH W. SELL, M.D., PH.D. EXTENSIVE EXPERIENCE WITH THE USE of pharmacologic doses of syn- thetic glucocorticosteroids for treatment of overwhelming systemic sepsishas accumu- lated in recent years. This has produced a large body of observations on the wide- spread hemodynamic [21, 271 and meta- bolic [19, 26, 321 effects of these agents. However, while their well-known immu- nosuppressive properties have been used to advantage in the prevention of trans- planted organ rejection, the immunologic From Clincial Medical Sciences Department, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland 20014. Supported by the Bureau of Medicine and Surgery Work Unit MR041.02.01.0009, MR041.02.01.0014A2GX, and CICC 2-08-322. * The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles outlined in the Animal Welfare Act (PL 89-544 as amended) and followed the guidelines prescribed in DHEW Publication No. (NIH) 72-23, formerly PHS Publication No. 1024, Guide for Laboratory Animal Facilities and Care. The authors acknowledge the technical as- sistance of Mrs. Carol Knebel and Miss Kathy Gerstenberg. Additional laboratory support was given by Navy Hospital Corpsmen Uy, Baldwin, Populus, Chisholm, Thompson, Mayfield, Chester, Eichman, Merecki, and by Mr. J. Magee. 7 Endocrinology Branch, Medical Service, Naval Hospital, Bethesda, MD 20014. Submitted for publication April 26, 1973. consequences of very large doses given to septic patients have not been evaluated. The effect on the circulating lymphocytes, in particular, and the potential therapeutic benefit or detriment of thus altering com- ponents of the immune response is essen- tially unknown. Recent research in cellular immunology has pointed out the heterogeneity of the lymphoid cell compartment within a given species [7, 13,141. The two major subpopu- lations are the thymus derived “T-cells” and the bursa of Fabricius equivalent “B-cells.” The T-cells seem to be directly concerned with cell-mediated immunity, and the B-cells with the production of hu- moral antibody. Evidence is also accumu- lating that cooperation between T and B lymphocytes is needed for the production of the immune response to certain antigens [5]. The situation in primates and man is less clear, but studies of some selective human immunologic deficiency states sug- gest a similar compartmentalization of lymphocytes into thymus-derived T-cells and bursa equivalent bone marrow-derived B-cells. Nowell, in 1960, discovered that phyto- hemagglutinin (PHA), a kidney bean ex- tract, could induce blast-like transforma- tion in lymphoid cells [24]. Since then, mitogenic agents have been used extensive- ly to study the immune capacity of humans and experimental animals. Blomgren and Svedmyr [2], using experimentally induced Copyright @ 1974 by Academic Press, Ine. All rights of reproduction in an,v form reserved. 172
Transcript
JOURNAL OF SURGICAL RESEARCH 16, 172-182 (1974)
Effect of Exogenous and Endogenous Glucocorticosteroids
on the In Vitro Stimulation of Lymphocytes from Sedated
and Awake-Restrained Healthy Baboons*
AFTAB AHMED, PH.D., CLIFFORD M. HERMAN, M.D.,
RICHARD C. KNUDSEN, PH.D., JONAS SODE, M.D.?
DOUGLAS M. STRONG, PH.D., AND KENNETH W. SELL, M.D., PH.D.
EXTENSIVE EXPERIENCE WITH THE USE of pharmacologic doses of syn- thetic glucocorticosteroids for treatment of overwhelming systemic sepsis has accumu- lated in recent years. This has produced a large body of observations on the wide- spread hemodynamic [21, 271 and meta- bolic [19, 26, 321 effects of these agents. However, while their well-known immu- nosuppressive properties have been used to advantage in the prevention of trans- planted organ rejection, the immunologic
From Clincial Medical Sciences Department, Naval Medical Research Institute, National Naval Medical Center, Bethesda, Maryland 20014.
Supported by the Bureau of Medicine and Surgery Work Unit MR041.02.01.0009, MR041.02.01.0014A2GX, and CICC 2-08-322.
* The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large.
The experiments reported herein were conducted according to the principles outlined in the Animal Welfare Act (PL 89-544 as amended) and followed the guidelines prescribed in DHEW Publication No. (NIH) 72-23, formerly PHS Publication No. 1024, Guide for Laboratory Animal Facilities and Care.
The authors acknowledge the technical as- sistance of Mrs. Carol Knebel and Miss Kathy Gerstenberg. Additional laboratory support was given by Navy Hospital Corpsmen Uy, Baldwin, Populus, Chisholm, Thompson, Mayfield, Chester, Eichman, Merecki, and by Mr. J. Magee.
7 Endocrinology Branch, Medical Service, Naval Hospital, Bethesda, MD 20014.
Submitted for publication April 26, 1973.
consequences of very large doses given to septic patients have not been evaluated. The effect on the circulating lymphocytes, in particular, and the potential therapeutic benefit or detriment of thus altering com- ponents of the immune response is essen- tially unknown.
Recent research in cellular immunology has pointed out the heterogeneity of the lymphoid cell compartment within a given species [7, 13,141. The two major subpopu- lations are the thymus derived “T-cells” and the bursa of Fabricius equivalent “B-cells.” The T-cells seem to be directly concerned with cell-mediated immunity, and the B-cells with the production of hu- moral antibody. Evidence is also accumu- lating that cooperation between T and B lymphocytes is needed for the production of the immune response to certain antigens [5]. The situation in primates and man is less clear, but studies of some selective human immunologic deficiency states sug- gest a similar compartmentalization of lymphocytes into thymus-derived T-cells and bursa equivalent bone marrow-derived B-cells.
Nowell, in 1960, discovered that phyto- hemagglutinin (PHA), a kidney bean ex- tract, could induce blast-like transforma- tion in lymphoid cells [24]. Since then, mitogenic agents have been used extensive- ly to study the immune capacity of humans and experimental animals. Blomgren and Svedmyr [2], using experimentally induced
Copyright @ 1974 by Academic Press, Ine. All rights of reproduction in an,v form reserved.
172
AHMED ET AL.: EXOGENOUS AND ENDOGENOUS GLUCOCORTICOYTEROIDS 173
immunologically impaired mice, found that PHA responsiveness is a property of T-cells. Stockman et al. [30] found that pokeweed mitogen (PWM) stimulated thy- mus-independent or B-cells. Janossy and Greaves [18] since then have shown that PWM stimulates both T and B cells but largely B cells and, therefore, has been used as an indicator of B-cell function. Stobo [29] found that there were subpopulations of thymocytes in mice, one small popu- lation which reacted to PHA and concana- valin-A (Con-A), and the other large frac- tion which reacted to Con-A alone. He showed that both PHA and Con-A bound equally well to murine thymocytes but that they specifically activated only those cells which differentiated within the thymus. Thymocytes and thymus-derived cells do not respond equally well to all mitogens, the relative degree of responsiveness being related to various stages of differentiation within the thymocyte and thymus-derived cell line. Andersson et al. [l] reported that spleen cells from thymectomized and lethally irradiated mice, reconstituted with anti-0 serum-treated bone marrow or spleen cells from congenitally athymic (nude) mice did not respond to Con-A but did re- spond to lipopolysaccharide of E. coli endo- toxin (LPS) . These data suggest that PHA and Con-A stimulate T-cells, PWM stimu- lates largely B-cells, and LPS stimulates only B-cells. The relative specificity of these various mitogenic responses if ex- trapolated to primates, therefore, permits the characterization of the different subsets within the lymphoid cells in the peripheral blood and makes it possible to study the kinetics and the effects of various pharma- cological agents on the immune response of the organism in vivo.
Naturally occurring adrenal cortical steroids and their synthetic analogs have been widely used in the treatment of a va- riety of immunologic, inflammatory and neoplastic diseases 141. Several studies on the effect of glucocorticosteroids on the cellular metabolism of rat and mouse thy-
mus cells have been published. Thymus cells from rats treated with steroids in viva have been shown to have a low capacity for nucleic acid synthesis and impaired RNA polymerase activity [ 11, 221. Steroids did not inhibit basal RNA synthesis in human cells but did inhibit PHA-stimu- lated RNA synthesis [25].
Relatively few data are available on the kinetics of lymphoid cell subpopulations after the administration of glucocorticoste- roids in primates. As the initial step in ex- ploring these issues, we have carried out an experiment to determine the effect of a sin- gle pharmacologic dose of methylpred- nisolone sodium succinate (Solu-Medrol, Upjohn, Kalamazoo, MI) on the size and composition of the circulating lymphocyte subpopulations in healthy, awake-re- strained baboons (Papio doguera) . Because the baboon must be sedated to allow any handling into or out of the cage, the rela- tionships between the stress of sedation, the stress of the awake-restrained state, and the endogenous cortisol response to these stresses was also evaluated. The investiga- tions again confirm that the subhuman primate can be used as an experimental model for the study of the effect of various drugs on immunologic and other metabolic functions [ 8-101.
MATERIALS AND METHODS
Animals. Fourteen adult male baboons (Papio doguera), weighing between 20 and 30 kg, were divided into two groups. Seven baboons acted as controls and received no exogenous corticosteroids. The other seven received a single intravenous dose of 30 mg/kg of Solu-Medrol.
Blood sampling. On seven separate days, a pair of baboons, each day after an over- night fast of lo-12 hr, were injected at 6:30 AM with 1 (1-phenylcyclohexyl) piperidinc hydrochloride (Sernylan) 1.0 mg/kg intra- muscularly for insertion of venous and uri- nary catheters. The animals were placed in a primate restraining chair and allowed to awaken. Blood samples were collected in
174 JOURNAL OF SURGICAL RESEARCH, VOL. 16, NO. 2, FEBRUARY 1974
sterile syringes containing preservative-free sodium heparin (Medical Chemical Corp., Chicago, IL) 8-10 units/ml of blood. The first sample was drawn at 7 AM and the sec- ond one 3 hr later. Immediately after the second sample, one baboon received a single injection of 30 mg/kg of Solu-Medrol intra- venously. A total of seven samples of lo-20 ml of heparinized blood was collect,ed from each baboon at hourly intervals. Ringers lactate solution was infused at the rate of 100 cc/hr throughout the experiment.
In vitro tests. WBC, differential, and lymphocyte counts were performed on each sample using standard laboratory tech- niques. Lymphocyte viability was deter- mined using l/S dilution of 0.4% trypan blue in Hanks’ Balanced Salt Solution (Grand Island Biological Co. (GIBCO) , NY).
In vitro lymphocyte cultures were car- ried out according to the procedure of Strong et al. [31]. Essentially, heparinized blood samples were layered over a Ficol- Hypaque gradient (sp gr 1.08) and then centrifuged at 450g for 45 min. The lymph- ocytes separated as a distinct band which was aspirated. Equal volumes of sterile RPMI-1640 medium (GIBCO, NY) were added to the lymphocyte band, and the resulting suspension was centrifuged at 150g for 15 min at 4%. The supernatant fluid containing Ficol Hypaque and plate- lets was discarded, and the cell button was resuspended in RPMI-1640 medium. The cell suspension was washed once more in a similar manner to insure removal of the density-grandient medium. The cell button was finally resuspended in RPMI-1640 media containing 100 units/ml penicillin, 100 pg/rnl streptomycin, 200 mM n-gluta- mine, and 10% heat-inactivated (56%, 30 min) fetal calf serum. Lymphocyte cell counts were determined by the use of an Autocytometer II (Fisher Scientific Co.). Cells were diluted to the appropriate con- centration. Use was made of a Hamilton syringe and repeating dispenser unit; 100 ~1 of the cell suspension were added to
each well of a microtiter plate (Falcon Plastics, No. 3040 Microtest II, Los An- geles, CA) and 100 ~1 of either media control or a specific mitogen in the ap- propriate concentration were added in quadruplicate cultures. The plate was fitted with a loose-fitting sterile lid (Linbro No. 55, Bellco Glass Co., Vineland, NJ). The cultures were then incubated in a 5% CO, humidified atmosphere at 37°C for 66 hr. Eighteen hours prior to termination of the lymphocyte cultures, 1 PCi of methyl-3H- thymidine (3H-TdR) (sp act 1.9 Ci/mmole, Schwartz-Mann, Orangeburg, NJ) in 20 ~1 of media was added to each well. The lymphocyte cultures were harvested using the multiple automated sample harvester described previously [ 161. Each sample was counted for 2 min and the counts per minute of incorporation of 3H-TdR were determined. Mean, standard error, and percent standard error were de- termined using the Wang 700-C advanced programming calculator.
Serum cortisol levels. Blood samples were drawn at appropriate time intervals and al- lowed to clot. Serum was then used for quantitation of cortisol levels using thin- layer chromatography and radioimmunoas- say. In the chromatographic system employed, methylprednisolone sodium suc- cinate (Solu-Medrol) separated completely from cortisol, and Solu-Medrol concentra- tions of 10 mg/lOO ml did not alter the analytical results obtained on commercial cortisol quality control serum (Hyland) .
Urinary cyclic adenosine monophosphate (cyclic AMP) levels. Urinary cyclic AMP levels were determined in 2- or 3-hr urine pools throughout the study. The first urine pool was between 7 AM and 10 AM, the sec- ond between 10 AM and 12 noon, the third between 12 noon and 2 PM, and the final pool between 2 PM and 4 PM. Cyclic AMP was measured by radioimmunoassay utiliz- ing the Schwarz-Mann (Schwarz-Mann, Orangeburg, NJ) cyclic AMP radioim- munoassay kit.
Mitogen assay. A sample of 100 ml of
AHMED ET AL.: Exogenous AND ENDOGENOUS GL~~~C~RT~CO~TERO~D~ 175
Table 1. PHA and Con-A Responses of Lymphocytes from Control and Solu-Medrol-Treated Baboonsa (Uptake oj 3H-thymidine, counts per minute + Standard Error)
7 AM 3204 + 172 3416 + 173 38364 zk 3960 47454 + 4473 71086 + 3905 73410 k 6830 10 AM 1818 ILc 208 lB99 f 57 190.52 f 1673 26772 f 3814 27324 k 2038 32291 f 3238 11 AM 1875 f 207 784 + 104 17329 z!x 2051 8800 27 1757 25877 i- 2236 2224 k 434
12 Noon 1922 i 19.5 852 * 88 18839 f 2888 9821 k 1518 30498 2~ 5214 2083 k 446 1 Flu 1991 f 190 838 I 69 18925 f 2081 10737 * 2227 23900 f 3064 1824 k 492 2 I’hZ 1940 + 222 846 + 52 20860 i 2570 11756 + 1889 24237 + 3308 1544 -1. 361
3 I’M 2108 * 281 822 * 72 20619 + 2424 10516 k 1536 25098 k 1767 1725 + 637 4 PM IQ16 i 171 886 + 42 20784 _+ 1769 12383 I2365 23752 k 2752 1521 f 303
n Data are the cumulative average (mean + standard error) of triplicate cultures run on seven control and seven Solu-hIedrol-treated baboons performed on seven diffefent days.
‘I All animals were injected with 1 mg/kg Sernylan intramuscularly before the 7 AM sample and the treated baboons were injected with Solu-Medrol 30 mg/kg intravenously just after the 10 AM sample. All samples were drawn in preservative-free heparin.
preservative-free heparinized blood was taken from an untreated baboon. Lympho- cytes were obtained by density gradient as described above. Lymphocytes were resus- pended in six fractions such that 100 ~1 of each fraction contained 1 X 105, 2 X 105, 3 X lo”, 4 X 105, and 5 X lo5 or 6 X loj lymphoid cells. Mitogen dose and concen- tration curves were performed with each cell concentration using .0125, 0.05, 0.1, 0.15, 0.2, and 0.570 final concentrations of PHA-P (Difco Labs, Detroit, MI) and 0.125, 0.5, 1.5, 2.0, and 3.0% final concen- trations of PWM (Difco Labs, Detroit, MI). Con-A (Calbiochem, San Diego, CA, Lot 210073) was used in concentrations of 0.1, 0.5, 1.0, 2.0, and 4.0, 5.0, 10.0 pg/cul- ture, and LPS 0111-B4 (Difco Labs, De- troit, MI) in concentrations of .5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 pg per lympho- cyte culture. Lymphocytes from baboons demonstrated low levels of stimulation to several lots of LPS tested. However, one lot of LPS produced significantly higher stimulation. This lot was used throughout the experiments.
RESULTS
Baseline experiments were carried out, in
an effort to determine the optimum culture conditions for baboon lymphocytes. The highest stimulation index
cpm of lymphocyte cultures with mitogen
cpm of lymphocyte cultures with no mitogen
was obtained using 4 X lo5 lymphoid cells/ culture. This cell concentration was, there- fore, used throughout the study. A mitogen dose-response curve was performed with various concentrations of PHA-P, Con-A, PWM, and LPS using 4 X lo5 cells/culture. Figure 1 A, B, C, and D shows peak activi- ties of each mitogen, respectively, when eul- tures were incubated for a total of 66 hours.
PHA-P induced the highest uptake of 3H-TdR at a final concentration of 0.1% (Fig. lA), Con-A at 4 pg/culture (Fig. 1B) , PWM at 1% final concentration (Fig. lC), and LPS at 16.0 pg/culture (Fig. 1D).
Tables 1 and 2 show the unstimulated and mitogen-stimulated lymphocyte re- sponses from untreated and Solu-Medrol- treated baboons. The unstimulated lym- phocyte response is included in both tables to facilitate a comparison of data. Solu- Medrol was injected into the baboons im- mediately after the 10 AM sample in “treated” animals. Therefore, t’he 7 AM and 10 AM samples in “untreated” and “treated” baboons were obtained under comparable conditions and before Solu-Medrol admin- istration to the “treated” group.
At 7 AM, unstimulated lymphocytes in- corporated approximately 3200 cpm 3H-
176 JOURNAL OF SURGICAL RESEARCH, VOL. 16, X0. 2, FEBRUARY 1974
Fig.
A),
IA ioo PHA-P
so
“* 80
: 70
% 60 is 6 50
p 40 if 5 30
s 20
PWM
t p/C”LTuRE I pgICULTURE
CONTROL CONTROL
1. Phytohemagglutinin-P (PHA-P), pokeweed mitogen (PWM), concanavalin-A and lipopolysaccharide (LPS) responses of lymphocytes from normal baboons.
(Con-
TdR. The incorporation of 3H-TdR by PHA-P, 72,000 cpm for Con-A, 24,000 cpm lymphocytes in response to mitogens was for PWM, and 57,000 cpm for LPS. Sam- increased to approximately 43,000 cpm for ples taken at subsequent time intervals for
Table 2. PWM and LPS Responses of Lymphocytes from Control and Solu-Medrol-Treated Baboonsa (Uptake of 3H-thymidine, counts per minute + Standard Error)
Sample Timeb
Control PWM (1%)
Untreated Treated Untreated Treated
LPS (160 pglculture)
Untreated Treated
7 AM
10 AM
11 AM
12 Noon 1 PM
2 PM
3 PM
4 PM
3204 zk 172 1818 A 208 187ii f 207 1922 * 195
1991 + 190
1940 f 222
2108 5 281
1916 + 171
3416 + 173 1599 i 57
784 + 104 852 IL 88 838 f 69 846 + 52 822 f 72 886 f 42
22513 * 1680 16164 * 1406 16304 + 1513 18881 f 2040 17221 f 1569
56155 + 4846 46946 + 4790 22022 f 2126 29160 f 2542
43363 + .5451 49210 f 7032 52121 k 8116
53217 + 6439
B Data are the cumulative average (mean f SE) of triplicate cultures run on seven control and seven Solu-Medrol-treated baboons performed on seven different days.
b All animals were injected with 1 mg/kg Sernylan intramuscularly before the 7 AM sample and the treated baboons were injected with Solu-Medrol 30 mg/kg intravenously just after the 10 AM sample. All samples were drawn in preservative-free heparin.
AHMED ET AL.: EXOGEh-OUR AND ENDOGEKOUS GLUCOCORTICORTEROIDS 177
all baboons showed reduced uptakes of 3H-TdR.
Table 3 shows the counts per minute of 3H-TdR incorporated for all samples from 10 AM on as a percentage of the 7 AM values (relative response). Two distinct effects on the incorporation of 3H-TdR by the baboon lymphocytes are seen. The first effect on the lymphocyte response occurred at 10 AM, several hours after the baboons had awakened in the primate restraining chairs. At this point, the incorporation of 3H-TdR was reduced in all animals. Incor- poration of 3H-TdR by unstimulated lym- phocytes was reduced to 56 and 46’$%, PHA-P stimulation to 49 and 5670, Con-A stimulation to 38 and 43%, PWM stimulation to 71 and 86%, and LPS stimu- lation to 82 and 83% of the 7 AM value, respectively.
Immediately after t,he 10 AM sample, Solu-Medrol was administered to one set of animals (Yreated group”). One hour after the injection of Solu-Medrol (11 AM
sample), a second effect was observed. Solu-Medrol treatment reduced the incor- poration of 3H-TdR by unstimulated lym- phocytes to 22.9% of the 7 AM value, whereas in the untreated control animals, t,he 3H-TdR incorporation by lymphocytes remained essentially unchanged (58% of 7 AM value). Solu-Medrol treatment also reduced the mitogenic responses. Stimula- tion by PHA-P was reduced to IS%,, by Con-A to 370, by PWM to 69%, and by LPS to 39% of the 7 AM values. Lympho- cytes from untreated baboons showed little change from the 10 AM values.
3H-TdR incorporation in subsequent samples from the Solu-Medrol-treated baboons (12 noon to 4 PM) showed only slight changes from the 11 AM values, with the exception of the LPS series where “H-TdR incorporation increased from 39.2% of baseline in the 11 AM sample to 94% in the 4 PM sample. The results are summarized in Fig. 2.
Table 5 shows the serum cortisol levels in the untreated and the Solu-Mcdrol- trcatcd baboons. In the untreated animals,
mean serum cortisol levels rose throughout the study from 40 pug/100 ml at 7 AM to
53 pg/lOO ml at 4 PM. In the Solu-Medrol- treated baboons, plasma cortisol values showed a greater variability among the in- dividual animals, but mean values were sig- nificantly lower than in the untreated group at 2, 3, and 4 PM (P < 0.05).
The leukocyte profile for control and ste- roid-treated baboons is shown in Table 4. The most pronounced change for both groups was observed between 7 AM and 10 AM. By 10 AM, the total WBC count dou- bled for the treated and tripled for the un- treated group, the percentage of PMN’s in- creased from approximately 50 to 82, the percentage of lymphocytes decreased from 43 and 45 to 15, and the total lymphocytes were reduced by 3570 and 30%.
Mean urinary cyclic AMP values (Table 5) showed no significant change throughout the study in the untreated animals. In the Solu-Medrol-treated baboons, on the other hand, mean urinary cyclic AMP rose from a pretreatment value of 4.45 * 0.41 pm/l g urinary creatine to a peak of 7.73 + 1.23 pg/lg urinary creatinine and remained ele- vated throughout the study (PI< 0.05).
DISCUSSION
If we assume that the normal serum cor- tisol level of the subhuman primate is simi- lar to that observed in man, then the data in Table 5 indicate that t.he mean cortisol in both groups of baboons at 7 AM is at least twice normal and already reflects a high degree of hypothalamic-pituitary- adrenocortical stimulation which probably resulted from the stress of the squeeze cage treatment for Sernylan injection before the 7 AM sample was obtained. The subsequent additional rise in plasma cortisol observed in the untreated baboons reflects the additional experimental stress as the animals awaken from tranquiliza- tion. Similar observations have been re- ported previously from our laboratory [S] .
In man, elevations in the total leukocyte count with an increase in PMN leukocytes,
Tabl
e 3,
Re
lative
Re
spon
ses*
of
Ly
mph
ocyte
s fro
m
Cont
rol
and
Solu-
Me&
al-Tr
eate
d Ba
boon
s Us
ing
Mito
genic
Ag
ents
Unst
imul
ated
lym
phoc
yte
resp
onse
PH
A-P(
o.l%
)
Mito
gen-
stim
ulate
d lym
phoc
yte
resp
onse
Con-
A (4
fig
) PW
M
(1%
)
--
LPS
(160
.u
g)
Sam
ple
time
cont
rol
Untre
ated
Tr
eate
d Un
treat
ed
Trea
ted
Untre
ated
Tr
eate
d Un
treat
ed
Trea
ted
Untre
ated
Tr
eate
d
10AM
56
.7
46.8
49
.6
11
AM
58.5
22
.9
45.1
12
No
on
59.9
24
.9
49.1
1
PM
62.1
24
.5
47.6
2
PM
60.5
24
.7
54.3
3
PM
65.7
24
.0
53.7
4
PM
59.8
25
.9
54.1
8 Re
spon
se
expr
esse
d as
per
cent
of
th
e 7
AM
resp
onse
.
Resp
onse
=
7 AM
Re
spon
se
(cpm
)
Expe
rimen
tal
(cpm
) x
100.
56.2
38
.4
43.9
71
.8
86.7
82
.2
83.6
18
.5
36.4
3.
0 72
.4
69.0
69
.5
39.2
20
.7
42.9
2.
8 80
.3
68.9
80
.4
51.9
22
.6
33.6
2.
4 76
.4
74.0
98
.6
77.2
24
.7
34.2
2.
1 74
.9
77.8
11
0.3
87.6
22
.1
35.3
2.
3 77
.2
80.5
10
7.4
92.8
26
.1
33.4
2.
0 81
.5
86.3
10
4.9
94.7
Tabl
e 4.
To
tal
WBC
, To
tal
Lym
phoc
yte,
%
Segs
, TO
Lym
phoc
yte
Coun
ts of
Co
ntro
l an
d So
lu-M
edro
l-Tre
ated
Ba
boon
sa
Sam
ple
timeb
WBC
X
lo3
Tota
l lym
phoc
ytes
Untre
ated
Tr
eate
d Un
treat
ed
Trea
ted
y&
SEGS
T0
Ly
mph
ocyte
s
Untre
ated
Tr
eate
d Un
treat
ed
Trea
ted
7AM
10
AM
11
AM
12
No
on
1 PM
2
PM
3 PM
4
PM
7.3
+ 0.
8 8.
4 Ik
1.7
3398
?c
678
39
96
_+ 1
136
22.7
f
2.0
18.5
i
1.0
2215
*
579
2862
t
547
22.8
f
2.6
17.5
j,
1.0
2881
k
424
3101
+
432
20.4
f
1.0
22.2
5
1.2
2440
+
405
1691
+
22.5
21
.4
zk
1.5
19.5
+
2.1
1696
*
319
1311
*
341
19.0
+
2.2
20.5
-t
3.3
1673
_+
361
11
39
i 31
8 20
.2
l!z 1
.0
18.6
+
1.5
2189
+
291
1047
rk
17s
19.2
+
1.8
18.9
+
1.6
1914
k
171
845
* 13
3
50.7
+
4.7
53.0
_+
5.4
45
.3
f 4.
5 43
.7
+ 5.
3 81
.9
f 2.
6 82
.4
k 3.
1 15
.0
* 2.
2 15
.6
+ 2.
6 85
.7
+ 1.
6 81
.3
j, 2.
3 12
.6
+ 1.
1 17
.1
f 1.
9 85
.7
f 2.
2 90
.1
+ 1.
6 12
.3
k 8.
3 7.
7 *
1.0
89.6
rt
1.9
92.0
k
1.9
8.3
* 1.
7 6.
9 f
1.5
91.0
Yc
l..?
91
.9
i 2.
1 8.
1 i
1.3
6.1
i 1.
5 88
.1
i: 1.
3 92
.6
+ 2.
0 10
.7
i: 1.
0 t5
.7
f 0.
5 87
.1
* 1.
5 92
.7
+ 1.
5 10
.0
+ 0.
6 4.
6 z!z
0.7
8 Da
ta
are
the
cum
ulat
ive
aver
age
(mea
n f
stand
ard
erro
r) of
tri
plica
te
cultu
res
run
on
seve
n co
ntro
l an
d se
ven
Solu-
Med
rol-tr
eate
d ba
boon
s pe
rform
ed
on
seve
n dif
fere
nt
days
. b
All
anim
als
were
in
ject
ed
with
1
mg/
kg
Sern
ylan
intra
mus
cula
rly
befo
re
the
7 AM
sa
mpl
e an
d th
e tre
ated
ba
boon
s we
re
inje
cted
wi
th
Solu-
Med
rol
30
mg/
kg
intra
veno
usly
just
af
ter
the
10
AM
sam
ple.
Al
l sa
mpl
es
were
dr
awn
in
pres
erva
tive-
free
hepa
rin.
AHMEI) ET AI,. : EXOGENOUS ASD EiYDOGENOUS GI~UCOCOHTICOYTEl~OIDS 179
60
SAMPLE TIME
, &-,.,.,-t 7 IO II I2 I 2 34
SAMPLE TIME
Fig. 2. Summary of the effect of exogenous (broken line) and endogenous (solid line) corticosteroids on the control, PHA-P (Oil%), PWM (l%), Con-A (4 pglculture), and LPS (16 pg/culture) response of baboon lymphocytes,
Table 5. Serum Cortisol and Urinary Cyclic-AMP Levels of Control and Solu-Medrol-Treated Baboons’
Samples’ Time
Serum Cortisol (rr/lOO ml) f SEh Urinary Cyclic AMP (&M/l g creatinine)
Untreated Treated Untreated Treated
7 AM 40.00 i 2.42 34.00 _+ 2.69f 3.70 * 0.75 4.43 rt 0.41
10 AM 44.14 i 2.87 38.42 jz 2.01 4.46 f 1.07 7.73 * 1.23’ 11 AM 43.83 i 1.56 38.14 * 3.22
a Data are the cumulative average (mean i standard error) of determinations run on seven control and seven Solu-Medrol-treated baboons performed on seven different days.
b Serum cortisol levels determined by chromatography and radioimmunoassay excluding exogenous Solu-Medrol.
d All animals were injected with 1 mg/kg Sernylan intramuscularly before the 7 AM sample and the treated baboons were injected with 30 mg/kg solu-medrol intravenously just after the 10 AM sample.
e = Level of significance <0.05. f = Level of significance <O.Ol. g = Level of significance <0.02.
1so JOURNAL OF SURGICAL RESEARCH, VOL. 16, NO. 2, FEBRUARY 1974
as well as absolute and relative lympho- cytopenia, are characteristically associated with cortisol excess, and it is likely that similar changes occur also in the subhuman primate. After a single injection of adreno- corticotropin, the effect is maximal at 4 hr in man [ 171.
The pronounced change in the leukocyte profile (Table 4) between 7 AM and 10 AM
with elevation of total leukocytes and seg- mented neutrophils, as well as a reduction in the percentage and the total number of lymphocytes, is, therefore, consistent with a stress-induced rise in endogenous plasma cortisol around .7 AM in our experimental animals. Along with the altered leukocyte profile, there was a marked reduction in the 3H-TdR incorporation by unstimulated lymphocytes (Table 1). These results indi- cate that a significant percentage of periph- eral lymphocytes has either been destroyed or removed from the peripheral blood through sequestration in Iymphoid tissues. This latter explanation would appear more likely since in vitro studies have shown that human peripheral blood lmphocytes suffered little damage when cultured with cortisol in contrast to rat lymphocytes which were severely damaged [28]. Little is known of the sequestering of lympho- cytes from peripheral blood into various compartments of the body. However, it has been reported that enzymes [15] and pertussis vaccine [20] can effect a redis- tribution of lymphoid cells. Recent studies in mice have shown that cortisol treatment increased the T-cell population in the bone marrow and that at least some of these T-cells came from the spleen [6].
In our studies, the lymphocytes which persisted in the peripheral blood must be cells which normally have a reduced ability to incorporate 3H-thymidine or lympho- cytes which have been so altered by the high endogenous cortisol levels that their ability to incorporate 3H-TdR has been impaired. These lymphocytes also showed a reduced ability to respond to mitogens. By 10 AM, all four mitogens showed a re-
duced ability to stimulate incorporation of 3H-TdR. PHA-P and particularly Con-A showed the most marked reductions in mitogenic activity. This would suggest that the lymphocytes remaining in the periph- eral blood are either largely B-cells (stimu- lated by PWM and LPS), or that the lym- phocytes most affected by high endogenous cortisol Ievels are T-cells (PHA-P stimu- lated or Con-A stimulated), particularly that subpopulation of T-cells which is sen- sitive to Con-A.
A second effect on the peripheral lympho- cytes, similar in nature to the first one, was observed after the intravenous injection of Solu-Medrol which was given immediately after the 10 AM sample. This effect was most pronounced 1 hr later (11 AM sample). By 11 AM, the unstimulated incorporation of 3H-TdR by lymphocytes from the treated baboons was reduced to 50% of the 10 AM value. An examination of the leuko- cyte profile (Table 4) showed that tota lymphocytes actually increased slightly during this period of time. The change in 3H-TdR incorporation after Solu-Medrol was, therefore, probably not due to destruction or sequestration of peripheral lymphocytes, but more likely reflected a pharmacological effect of the steroid on the lymphocytes; namely, a reduction in their ability to incorporate 3H-thymidine. These lymphocytes were also altered in their abil- ity to respond to the mitogens PHA-P, and most particularly Con-A whose ability to incorporate 3H-TdR was reduced from 43.9% to 3.0%. It would, therefore, appear that Solu-Medrol altered the reactivity of T-lymphocytes to mitogen stimulation, particularly the reactivity of that subpopu- lation of T-cells which is sensitive to Con-A.
The reduced thymidine uptake by un- stimulated and mitogen-stimulated lym- phocytes could be due to the inhibition of DNA and RNA synthesis. In vitro studies show that RNA synthesis [25] and DNA synthesis [33] are inhibited by steroids in the general range of l-100 rg per ml.
AHMED ET AL.: EXOGENOUS AND ESDO~ENOUS GLU~~C~RTI~~~~TEROIDS 181
Nowell [23] found that the PHA-in- duced blast transformation of human lym- phocytes was inhibited by prednisolone if
the steroid was added before or just after PHA. However, if the cells were exposed to PHA for more t.han 10 min, later addi- tion of prednisolone was ineffective [12).
It should be noted that baboon lympho- cytes were washed several times in tissue culture media and incubated in tissue cul-
ture media containing fetal calf serum. Thus, the in viva effect of Solu-Medrol was not abolished by in vitro washing and incu- bation procedures. In vitro studies have also shown that washing human leukocytes incubated for 3 hr with cortisol succinate (100 pg per ml) did not alter the inhibition of PI-IA-estimulated RNA synthesis [25].
After the 11 AM samples, the PHA-P and Con-A responses showed little change. However, the PWM and LPS responses in- creased, LPS increasing from 39.6% at 11 AM to 94.7% by 4 PM. This would suggest that the Solu-Medrol-induced altered lym- phocyte reactivity to PWM and LPS is re- versible as the tissue concentration of Solu-Medrol decreases, but that the altered lymphocyte reactivity to PHA-P and Con-A is not. Weston et al. 1341 report that cortisol inhibition in cell-mediated immune responses is located between the lympho- kine and the monocyte-macrophage. The effect of such cells on mitogen-stimulated lymphocytes in vitro should be studied to further evaluate the effect of cortisol.
Mean urinary cyclic AMP, normalized for urinary creatinine in order to minimize the effects of changes in glomerular filtra- tion rate, was significantly higher after the administration of Solu-Medrol than in the untreated control animals. The tissue sources responsible for this rise in urinary cyclic AMP are not known, but the obser- vation is intriguing in light of the many reports that cyclic AMP in some way par- ticipates in the immune responses of the or- ganism. The situation is complex since finite elevations of this nucleotide in the immunocytes can enhance the activity of
these cells, whereas excessive signals for adenyl cyclase activity may be inhibitory
to cellular events [3]. In summary, it appears that both
endogenous and exogenous glucocorticoste- roids alter the composition of the circulat- ing lymphocyte population. The en- dogenous cortisol response to the stress of squeeze-cage manipulation and t’he subsc- qucnt awakening in a restraining chair sup- pressed the relat’ive and total lymphocyte counts. There was an associated suppres- sion of 3H-TdR incorporation by unstimu- lated as well as by T-cell mitogen (PHA-P, Con-A), and B-cell mitogen (PWM, LPS) -stimulated lymphocytes.
The subsequent injection of methylpred-
nisolone further inhibited 3H-TdR incor- poration by unstimulated and stimulated lymphocytes. However, this inhibition by the exogenous steroid affected the T-cell subpopulation more markedly than it did the B-cells. The relative roles of T-cells and B-cells in responding to acute sepsis are unknown. This relative T-cell suppres- sion by a pharmacologic dose of methyl- prednisolone must be explored to determine if it occurs in the presence of systemic sep- sis and, if so, what the therapeutic conse- quences might be.
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