CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 13, 452-461 (1979)

T Lymphocytes and Plasma Inhibitory Factor in ACTH-Dependent Cushing’s Patients

BATYA SHOHAT, AMI KLEIN, HAIUTA KAUFMANN, ILANA BLUM, AND ISRAEL CHOWERS

Clitkal Laborutory crnd Endoc~rinologiral Departmc~nt. Bvilinson Medical Center,

Petah-T&vu, Isrd

Received November 22. 1978

In this study the cell-mediated immune activity of lymphocytes obtained from seven patients with ACTH-dependent adrenal hyperplasia (Gushing’s disease) was investi- gated. The percentage of E rosette-forming peripheral blood mononuclear cells as well as the absolute T cell number was greatly decreased in the patient group (618 2 333 cells/ mm3) as compared to the controls (1222 2 456 T cells/mm3). The functional activity of T lymphocytes of patients with Cushing’s disease, as measured by a local graft versus host reaction (GVHR), was normal, resulting in a reaction of same magnitude as those pro- duced by 20 x 10” normal human lymphocytes. The whole heparinized plasma obtained from the seven patients abolished the ability of normal human lymphocytes to mount a normal local GVHR and to form spontaneous E rosettes. The inhibitory factor was not found in the two patients tested 1 year following left adrenalectomy and irradiation of the hypophysis. Fractionation of the plasma demonstrated that the inhibitory factor is found in the lipid fraction corresponding chromatographically to triglycerides. Control experi- ments performed with different concentrations of triolein demonstrated that the com- pound effects normal human lymphocytes.

INTRODUCTION

Gushing’s syndrome is characterized by a continuous overproduction of cortisol and low levels of lymphocytes. Britton et al. (1) recently reported a decrease in the number of hyporeactive T lymphocytes in a patient with Cushing’s syndrome due to adrenal adenoma. They attributed these manifestations to the high levels of cortisol.

In the present study, the number of T lymphocytes and their immunocompe- tence were studied in a series of seven patients with ACTH-dependent Cushing’s disease. The effect of the heparinized plasma on the immunocompetence of nor- mal human lymphocytes was also investigated.

MATERIAL AND METHODS

Human lymphocytes. Blood was obtained by venipuncture from seven patients with Cushing’s syndrome. In two patients blood was obtained both before and after adrenalectomy. The lymphocytes were separated from the heparinized blood by the Ficoll- Hypaque sedimentation technique, Minimal contamination with monocytes was obtained.

Patients. All seven patients presented with obesity osteoporosis and cushingoid faces. All had high levels of 17 ketosteroids and 17 hydroxysteroids, high urinary levels of free 11 hydroxycorticosteroids, and high levels of cortisol in plasma without diurnal variation and lymphopenia. The response of the suppressibility of the hypothalamic-pituitary adrenal axis by dexamethasone is shown in Table 3.

452 00!&1229/79/080452-10$01.00/O Copyrkht 0 1979 by Academic Press, Inc. All tights of reproduction in any form reserved.

T LYMPHOCYTES IN GUSHING’S PATIENTS 453

Six of the patients were treated by unilateral adrenalectomy and pituitary irradi- ation with 5000 R during 6 weeks. The seventh patient was treated with OpDDD and irradiation of the hypophysis.

Patients 1, 2, and 3 recovered fully following treatment but in patients 4 and 6 there was a recurrence of the disease 6 months after surgery. Patient No. 5, who was treated with OpDDD’ and irradiation of the hypophysis, showed a recurrence of her symptoms 3 months after treatment was started. Patient No. 7 showed no response to treatment but went into remission after treatment with OpDDD.

E rosettes. The number of T cells were determined by a modification of the sheep red blood cell (SRBC) rosette formation test described by Jondal et al. (2). A suspension containing 1 .O x lo6 lymphocytes in a volume of 0.25 ml was mixed with an equal volume of 1.0% sheep red blood cells in PBS containing 10.0% fetal calf serum previously absorbed with SRBC. The mixture obtained was then incu- bated at 37°C for 5 min, centrifuged at 200s for 5 min, and then incubated again for 1 or 24 hr at 4°C. The significance of the difference between the results obtained in each patient and those in the control group was assessed by means of student’s t test.

E-rosette inhibition test. Purified mononuclear cells obtained from normal vol- unteers at a concentration of 2 x 1OYO. 1 ml were incubated for 1 hr in 0.5 ml PBS and 0.1 ml plasma obtained from each patient with. Cushing’s syndrome. After in- cubation the cells were washed twice in PBS and resuspended at a concentration of 5. IO6 cells/ml. Their capacity to form E rosettes was then determined.

Graft-I’ersus-host reaction (GVHR). A O.l-ml sample of the lymphocyte sus- pension containing 20 x lo6 lymphocytes was injected intradermally into the closely shaven abdominal skin of immune-suppressed inbred Lewis rats. Five days later 0.4 ml of 1.0% Evans blue was injected iv into each animal. Five hours later the entire abdominal skin was excised and the blue stain was measured with calipers. A mean diameter of 3.0 mm assessed as a positive reaction, 1-2 mm as weakly positive, and less than 1 mm as a negative reaction (3-5).

Inhibition of GVHk. Normal human lymphocytes were incubated for 1 hr at 37°C with whole plasma obtained from all seven Cushing’s patients and with fractionated plasma from four of the patients. Following incubation the cells were washed twice in phosphate-buffered saline (pH 7.4) and then inoculated intrader- mally into immune-suppressed rats as described above. Control experiments were performed with normal human lymphocytes which were incubated in (a) whole plasma and (b) fractionated plasma obtained from normal subjects. In addition normal human lymphocytes were incubated with hyperlipemic plasma containing high levels of triglycerides and with triolein.

Fractionation of plasma. Plasma was fractionated into two main fractions, protein and lipids. The protein fraction of plasma consisted of sediments obtained using 20, 40, and 80% ammonium sulfate solutions as well as their remaining dissolved proteins. Dialysis was carried out against PBS. The lipid fraction was obtained by extraction of plasma with chloroform. The extract was dried and the residue dissolved in 0.2 ml of ethanol. To this residue solution [ 1 ,2-3H]cortisol was

’ Op DDD = ortho-paw-prime DDD = 1,l dichloro-2-(o-chlorophenyl)-2-p-chlorophenyl) ethane. MIF = Migration Inhibitory Factor.

454 SHOHAT ET AL.

added and placed on G (Merck) tic plates and run in a solution system of ether: petrol ether:acetic acid l‘o:‘sH): 1, in par&e1 with the following markers: choles- terol, triolein, atid ethyl myristate. Following chromatography the marker regions were sprayed with 50% sulfuric acid containing 5 mg% methyl orange and heated at 120°C for 20 min (6). The plates were divided according to the polarity of the markers to five zones: (1) origin; (2) zone corresponding to cholesterol; (3) zone corresponding to the region between cholesterol and triolein; (4) zone corre- sponding to triolein; and (5) zone corresponding to ethylmyristate.

RESULTS

Inoculation of 20 x lo6 normal human lymphocytes from 40 donors into immune-suppressed rats resulted in a skin reaction of 3.5 mm diameter, 40 x lo6 lymphocytes produced a larger reaction of 5.5 mm, and 60 x lo6 produced an even more pronounced reaction, 6.5 mm.

The functional activity of T cells in the patient group as measured by a local GVHR was found to be normal in all of the patients, i.e., a positive GVHR of 3-5.5 mm diameter being obtained (see Table 1).

In all seven patients with ACTH-dependent Cushing’s disease the percentage (47 + 13) and the absolute number (618 + 330/mm3) of T cells were low. A significant difference between the patients and the normal controls was found. In the patients tested both before and after successful operation there was a rise in these values (Table 1). The clinical data of four patients are summarized in Table

TABLE 1 T LYMPHOCYTES-NUMBER AND FUNCTION IN ACTH-DEPENDENT GUSHING’S PATIENTS

Patient Age sex Rosettes

(Es) T cell GVHR” Cortisol Triglycerides

(mm3 ? SD) (mm) (ia%) b%m

(1) Preop Postop

(2 weeks) (1 year)

(2) Postop (2 weeks) (1 year)

(3) Postop (3 months)

(4) Postop (unsuccessful)

(3 hop

(6) Preop Postop

(3 weeks) (3 months)

(7) Postop (unsuccessful)

Mean Control group

20 x 106 40 x 106 60 x 106

20 F 36

20 39 21 63

16 M 44 53

46 F 56

52 F 39

65 F 71

24 F 27

24 58 58

48 F 35

47 z 13 20-55 M+F 58 + 8

201 r 80

420 k 65 776 + 100

827 + 110 854 + 80

204 k 55

686 ? 51

603 f 70

453 + 75

13482200

428 2 120

618 r 333 1222 + 456

+ 3.5 26.2 200

+5 +4 12.4

+3 13.1 45 +3 9.6

+ 3.5 5.2 233

+ 3.5 24.0

+ 5.5 34.5 205

+3 33.7 60

+3

+ 4.5 26.3 75

120 + 3.5

3.5 5.5 6.5

a Normal local graft-versus-host reaction measuring 3-5 mm diameter.

TABL

E 2

CLIN

ICAL

DA

TA

IN

SEVE

N PA

TIEN

TS

WIT

H GU

SHIN

G’S

DISE

ASE

Urine

ex

cretio

n of

Pl

asm

a 1 I

-OH

CS

1 I-O

HC

S 24

hr

(/a%

) Pl

asm

a AC

TH

Prev

ious

Be

fore

Af

ter

Befo

re

Afte

r Be

fore

Af

ter

surg

ery

No.

N

ame

Sex

Age

surg

ery

surg

ery

surg

ery

surg

ery

surg

ery

surg

ery

and

X-ra

y

I B.

A.M

. F

20

985

169

26.2

12

.2

81

loo

7 ye

ars

2 G

.Y.

M

16

450

105

20.7

9.

3 11

0 10

0 1

year

3

F.K.

F

46

612

37

27.7

5.

2 25

0 30

3

mon

ths

4 M

.V.

F 52

30

.3

24

6 ye

ars

5 R

.G.

F 65

75

4 35

.5

60

No

surg

ery

6 B.

D.

F 24

14

00

150

33

12

125

100

1 m

onth

7

O.V

. F

48

884

325

26.3

20

.7

110

110

2 ye

ars

456 SHOHAT ET AL.

2. The response of the suppressibility of the hypothalamo-pituitary axis by dexa- methasone is shown in Table 3. It was noteworthy that the plasma obtained from three patients prior to operation and from two patients 2 weeks thereafter, as well as from two patients in whom operation had been unsuccessful, abolished the ability of normal human lymphocytes to mount a GVHR and to form E rosettes in normal percentages. In contrast the plasma obtained from one patient 3 months after operation and from two patients 1 year following successful adrenalectomy was not found to contain the inhibitory factor affecting the normal competence of lymphocytes to mount a GVHR. The plasma, however, still affected the capability of normal human lymphocytes to form E rosettes in normal percentages.

Fractionation of the plasma of these patients before or after unsuccessful ad- renalectomy demonstrated that the inhibitory factor was located on the tic plate on a region not corresponding to cortisol, as all the radioactivity of the added labeled cortisol remained in the origin. The inhibitory factor was located in a region chromatographically corresponding to triglycerides. The fractions corre- sponding to lipids more and less polar than triglycerides (including cortico- steroids) showed no inhibitory action and failed to affect the ability of normal lymphocytes to mount a GVHR or to form E rosettes (Tables 3-6). In addition, the fractions of patient’s plasma proteins obtained by ammonium sulfate sedimentation had no effect whatsoever on the normal human lymphocytes.

Incubation of normal human lymphocytes in vitro with cortisol in a concentra- tion up to 320 pg/ml did not affect their ability to mount a normal GVHR. On the other hand incubation of normal human lymphocytes with triolein at a concentra- tion of 1 mg/ml inhibited the normal human lymphocytes to mount a normal GVHR, while at a concentration of 12 mg triolein/ml a complete abolition of GVHR was found in spite of the fact that this concentration had no effect on the viability of the lymphocytes. Hyperlipemic plasma also affected the ability of normal lymphocytes to mount a GVHR.

DISCUSSION

The only report we have found in the scientific literature concerning T cells of Cushing’s syndrome (1) attributed the low number of T cells and their impaired immunocompetence, in this case due to adrenal adenoma, to the high level of cortisol present in the patient.

Our study of seven patients with Cushing’s disease, although demonstrating low levels of T cells, showed these cells to have normal function activity in the GVHR. Cantor et al. (7) have shown that at least two types of reactive T-lymphoid cells participate in the GVHR. In our opinion both subpopulations of T cells can be found in peripheral blood as well since in a previous study augmentation of the GVHR was obtained by preincubating normal human blood with a thymic hor- mone (8). Support of this opinion may be found in the recent studies by Kaplan et ul. (9) and Horowitz et al. (lo), who found precursor T cells in normal peripheral human blood. In addition, one of us has found that the T lymphocytes initiating a GVHR release a MIF (11). Suppressor T cells can also inhibit the GVHR (12). Therefore the capability of the lymphocytes from Cushing’s patients to induce a GVHR may be considered to represent the normal ability of at least two sub- populations of T cells to recognize a foreign antigen, with the probable release of

TABL

E 3

t; TH

E SU

PPRE

SSIB

ILITY

O

F TH

E HY

POTH

ALAM

O-PI

TUIT

ARY

ADRE

NAL

AXIS

BY

DE

XAM

ETHA

SONE

IN

SE

VEN

PATI

~NYS

SU

FFER

ING

3

FROM

CU

SHIN

G’S

DISE

ASE

z

24-h

r Ur

ine

17

KS-m

g g

24-h

r Ur

ine

170

HCS-

mg

3

Patie

nt

Durin

g 24

-hr

Urine

I I

-OHC

S-pg

Du

ring

E

No.

Cont

rol

2 m

g De

xa”

8 m

g De

xa

Cont

rol

2 m

g De

xa

8 m

g De

xa

Cont

rol

2 m

g De

xa

8 m

g De

xa

z

1 7.

2 3.

1 -

35.8

5.

6 -

450

87.3

2

19.7

8.

6 t2

-

30.2

8.

9 -

985

75

-

3 19

.4

5 50

.3

8 z

- -

1400

98

-

4 11

.3

11

7.8

29.3

19

.7

9 14

.5

731

350

226

0 5

10.7

5.

9 3

30

22

II 88

4 32

5 20

0 m

-

6 19

.8

5.4

27.4

12

.8

7 13

7.

5 8.

1 9

35

17.7

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.4

754

306

216

2

‘I De

xam

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sone

.

m

458 SHOHAT ET AL.

TABLE 4 EFFECTOFPATIENTS'PLASMAON NORMAL HUMAN LYMPHOCYTESASCOMPAREDTOTHE

EFFECTOF CORTISOL ANDTRIOLEIN

Patient

(1) Preop Postop

(2 weeks) (1 year)

(2) Postop (2 weeks) (1 year)

(3) Postop (3 months)

(4) Postop (unsuccessful) (6 years)

(5) Preop

(6) Preop Postop

(3 months)

(7) Post-unsuccessful operation

Cortisol 320 &ml

Normal plasma

Hyperlipemic plasma

Triolein 1 mg/ml

Triolein 2 mg

Triolein 12 mg

Age Sex

Normal lymphocytes + patient plasma

E rosettes 95 GVHR

20 F 24 -0

16 M 16 M

46

57 F

24 F

24 F

24

F

F

F

30 - 46 + 3.5

41 -0 36 + 4.5

19

Not done

47

6

37

30

65

68

f4

-0

-+2

-0

+ 3.5

-0

+ 3.5

+ 3.5

.25

.21

.l/

-

MIF. Furthermore, it may be assumed that no suppressor T cells are present in these Cushing’s patients since these cells have been demonstrated to be able to induce a normal GVHR.

Another main finding in our study was the finding of an inhibitory factor in the plasma of these patients. The inhibitory factor was shown to be neither a protein nor a lipoprotein or cortisol or cholesterol but probably a triglyceride-like com- pound. Regulation of lymphocyte stimulation by a series of low density lipopro- teins (LDL) was described by Curtiss and Edington (13). The LDL was found to be composed of protein and lipid while our inhibitory fraction obtained from plasma of Cushing’s patients did not contain any lipoprotein but mainly its lipid fraction. Furthermore, the LDL did not influence E-rosette formation while our plasma fraction did. An E-rosette inhibitory factor (RIF) isolated from sera of patients with hepatitis B virus infection is also serum lipoprotein. Four hours at least are required for reduction in rosette number by this fraction (14). Our factor

T LYMPHOCYTES IN GUSHING’S PATIENTS 459

TABLE 5 EFFECT OF PLASMA FRACTIONS OF GUSHING’S PATIENTS ON NORMAL HUMAN T LYMPHOCYTES

GVHR

Plasma Chloroform extract

Patient 1 Preoperation Postoperation

(1 year)

- ,! -

fb +

Patient 2 (2 weeks) (I year)

- -

+ +

Hyperlipemic plasma 0.25lO.25

0 No local graft-versus-host reaction, I’ + Local normal graft-versus-host reaction measuring from 3-5 mm diameter.

affects the E-rosette formation in less than 1 hr. Our factor was isolated in a completely different manner from that of RIF and it was proven to be an unpolar substance. Its polarity equals those of triglycerides (triolein). We cannot rule out the possibility that the factor is an unpolar steroid; however, it was demonstrated not to be cortisol.

Control experiments performed by us with hyperlipemic plasma containing high levels of triglycerides as well as triolein showed similar inhibitory effects on the ability of normal human lymphocytes to mount a local GVHR. These experiments demonstrate that triglycerides have inhibitory effects on the functional activity of human T lymphocytes.

It is noteworthy that Paradinas et al. (15) recently showed that the intravenous

TABLE 6 EFFECTS OF PLASMA FRACTIONS OF CUSHINGS’ PATIENTS ON NORMAL HUMAN T LYMPHOCYTES

Patient 4 Postop

GVHR ! Patient 6 Preop Postop

Normal volunteer

Plasma

GVHR

Whole % so, Chloroform extract fractions

plasma 80 40 20 I 2 3 4 5

- 0 + + + + + - - + + +

- + + + + +++ - - + + 0 + + + + + + + +

+ + + + + + + + +

” - No local graft-versus-host reaction. ’ + Local normal graft-versus-host reaction measuring from 3-5 mm diameter.

460 SHOHAT ET AL.

administration of cortisol to patients induced an increase in free fatty acids sub- sequent to an increase in triglyceride levels in the plasma. ACTH was also shown to promote an increase in free fatty acids after 4 hr which is followed by an increase in triglycerides.

Kigoshi el al. have also found in experimental studies in mice that administra- tion of cortisol induced a decrease in lymphoid cells in the thymus, spleen, and mesenteric nodes, and also found that the remaining cortisol-resistant cells con- tain significantly higher concentrations of free fatty acids (FFA), triglycerides (TG), and cholesterols esters (16). These findings may throw some light on what might happen in the Cushing’s patients, in whom the overproduction of cortisol probably initiates a chain of reactions including the increased production of TG- like compounds which may act directly as inhibitory substances upon the T cells.

Fauci ef al. have also shown that the lymphocytes found to be present in the circulation immediately after administration of cortisone are as responsive to mitogen as normal lymphocytes (20). In our study we found that the direct in vitro exposure of normal human lymphocytes to high nonphysiological levels of cortisol failed to inhibit their ability to mount a GVHR, a finding in accordance with well- documented reports that the T cells responsible for inducing the GVHR consti- tuted a cortisol-resistant population (18, 19).

The observations of a normal immune competence found in T cells of all Cush- ing’s patients despite their reduced number can be correlated to the findings of Fauci and Dale (20), Yu et nl. (21), Levine and Claman (22), and still more recently Fauci ( 17), who have all demonstrated that both short- and long-term administration of corticosteroids leads to a redistribution of the circulating lymphocytes with a profound circulating lymphocytopenia and accumulation of functionally mature T and B cells in the bone marrow.

In conclusion, the inhibitory factor found in the plasma of Cushing’s patients was demonstrated not to be cortisol but may be a factor produced by the over- production of cortisol which probably initiates a chain of reactions including the increased production of triglyceride-like compounds which may act directly as inhibitory substances upon the T cells.

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Narinetty. Ed.), Vol. 1, pp. 139- 155, Dekker, New York, 1967. 7. Cantor, H., and Asofsky, R., .I. Erp. h-lc~cl. 135, 764, 1972. 8. Douer. D., Shohat, B.. Pinkhas. J., and Trainin, N. Isr. ./. Med. Sci. 14. 870, 1978. 9. Kaplan, J., and Peterson, W. D. 1 C‘liri. Inrrnrrn~rl. Inz~trrtnoputAol. 9, 436. 1978.

10. Horowitz, S. D.. and Goldstein. A. L.. C‘lir~. /nr,r~urio/. Im~nrrnopurhr~l. 9, 408. 1978. II. Shohat, B., in preparation. 12. Gherson, R. K.. Tnrrrsplarzt. Ret,. 26, 171, 1975. 13. Curtis, L. K., and Edington, T. S., ./. Inzntroir~l. 116, 452, 1975. 14. Chisari, F. V., and Edington, T. S.. ./. E.r.rp. Med. 142, 1092, 197.5. 15. Paradinas. J., Prieto. C., Arrantz. J., Para, M. T.. and Velasco. R. J. Mrd. 7, 131, 1976. 16. Kigoshi. S.. and Akiama, M.. Experientia 32, 927. 1976.

T LYMPHOCYTES IN GUSHING’S PATIENTS 461

17. Fauci, A. S., Imnwnology, 28, 669, 1975. 18. Fernandes, G., Yunis, E., and Good, R. A., Clin. Immunol. Imtnunoparhol. 4, 304, 1975. 19. Cohen, J. J., Fischbach, M., and Claman, H. N., J. Immunol. 105, 1146, 1970. 20. Fauci, A. S., and Dale, D. C., J. C/in. Invrst. 53, 240, 1974. 21. Yu. D. T. Y., Clements, P. J., and Paulus, H. E., Peter. J. B., and Levy, J.. J. C/in. Invest. 53,

565, 1974. 22. Levine, M. A., and Ciaman, H. N., S&ww 167, 1515, 1970.