Immunology Letters, 3 (1981) 267-271 Elsevier/North-Holland Biomedical Press

D E C R E A S E D A N T I B O D Y - D E P E N D E N T AND S P O N T A N E O U S C E L L - M E D I A T E D

C Y T O T O X I C I T Y IN P A T I E N T S WITH CHRONIC R E N A L F A I L U R E

Istvdn L~,NG*, Istv~in TARABA and Gyula PETR,~NYI Second Department of Medicine, Semmelweis University Medical School, H-1088 Budapest VIII, Szentkirdlyi utca 46, Hungary

(Received 18 May 1981) (Accepted 22 May 1981)

I. Summary 2. Materials and methods

Antibody-dependent and spontaneous lympho- cytotoxicity of blood mononuclear cells from 15 patients with chronic renal failure was studied in allogeneic K-562 and xenogeneic chicken erythrocyte test systems. Both antibody-dependent and spontane- ous cytotoxic activities were significantly decreased in uremic patients as compared to the corresponding mean values of healthy control persons and non- uremic patients with the same underlying diseases. The addition of autologous serum further reduced the impaired cytotoxic capacity of uremic lympho- cytes. Uremic sera also inhibited the antibody-depen- dent and spontaneous lymphocytotoxicity of healthy individuals.

2. Introduction

Defects in humoral [1,2] and cellular [3,4] immu- nity are well known in chronic renal failure. Impair- ment of resistance to infections of these patients is also well documented [5 ]. Since antibody-dependent cell-mediated cytotoxicity (ADCC) may play a role in the immunological defence mechanisms against infections [6] and in kidney graft rejection [7,8], we have studied the ADCC activity of 15 patients with chronic renal insufficiency. Simultaneously we deter- mined their spontaneous cytotoxic (NK cell) activity.

* Present address (to which correspondence should be directed): Dept. of Immunology, Arthur Stanley House, Middlesex Hospital Medical School, 40-50 Tottenham Street, London WlP 9PG, U.K.

3.1. Patients Fifteen patients (5 males and 10 females), whose

ages ranged from 19 to 59 years (mean 32.7 + 3.0 years), were studied. They were on long-term hospital- centred maintenance haemodialysis for a period from 60 to 290 days with a median of 190 days on haemo- dialysis. All patients had chronic renal failure with creatinine clearances less than 3.0 ml/min]1.73 m 2. Glomerulonephritis (GN) was the underlying disease in 6, hypertension (HT) in 4 and pyelonephdtis (PN) in 5 patients. No patient received any drugs influenc- ing immune function. Thirty, age- and sex-matched healthy individuals served as controls. Addition con- trol groups of non-uremic patients with chronic GN, chronic PN and HT were also included. Blood was obtained before a haemodialysis session, when blood urea nitrogen level was, in every case, greater than 21.0 mmol/1. Lymphocytes were separated by the method of Boyum [9]. Phagocytic cells were removed by carbonyl iron treatment. ADCC activity was simultaneously determined in a xenogeneic chicken red blood cell (CRBC) test system and in an aUogeneic assay using rabbit anti-K-562-coated K-562 target cells. NK cell activity was assessed on K-562 target cells alone. Effector:target cell ratios were 50:1 and 25:1 in the K-562 assays and 20:1 and 10:1 in the CRBC system. The incubation time was 4 h. Details of the cytotoxicity assays have been previously described [10].

In some experiments the effect of uremic sera on cytotoxicity was also tested. Uremic sera were heat inactivated (56°C 60 min) and absorbed with target

0165-2478/81/0000-0000/$2.75 © Elsevier/North-HoUand Biomedical Press 267

t~

O',

OO

Tab

le 1

A

nti

bo

dy

-dep

end

ent a

nd

sp

on

tan

eou

s ce

ll-m

edia

ted

cyto

tox

icit

y o

f p

atie

nts

wit

h c

hro

nic

ren

al f

ailu

re

Pat

ient

A

ge

Sex

(y

ears

) U

nd

er-

BU

N a

Ser

um

C

RB

C-A

DC

C %

K

-562

-AD

CC

%

lyin

g (m

mo

l/l)

cr

eati

nin

e di

seas

e (#

mo

l/l)

20

:1

10:1

5

0:1

25

:1

NK

%

50

:1

25:1

1 45

F

2 19

F

3 21

F

4 30

M

5

35

F 6

26

F 7

27

F 8

59

F

9 28

F

10

21

F 11

31

M

12

24

F

13

32

M

14

44

M

15

48

M

PN

27

.3

639

34.5

19

.3

27.2

14

.7

11.7

2.

5 G

N

28.7

80

3 37

.5

24.6

29

.3

16.1

14

.1

4.1

GN

21

.8

850

39.2

18

.4

31.2

17

.3

17.1

9.

3 G

N

28.1

97

3 16

.8

9.2

12.7

5.

9 2.

8 0.

2 H

T

38.2

10

70

28.4

16

.1

25.9

13

.7

10.9

4.

0 P

N

37.2

79

5 39

.2

24.2

35

.8

19.6

18

.1

10.2

H

T

41

.8

1259

34

.5

17.1

28

.1

14.0

12

.5

7.5

PN

32

.2

1168

37

.1

20.0

27

.8

18.4

18

.5

9.9

HT

6

6.0

14

85

30.0

17

.6

24.5

13

.9

15.1

6.

4 G

N

46

.8

1070

22

.7

13.9

26

.9

15.6

18

.2

9.6

PN

35

.1

1187

29

.7

16.6

23

.8

13.7

14

.6

9.5

PN

42

.1

946

26.9

5.

9 20

.2

8.6

10.3

3.

8 G

N

37.5

12

91

32.6

17

.8

25.1

14

.3

12.0

8.

3 H

T

30.4

16

62

21.1

8.

3 17

.6

6.3

6.9

2.6

GN

42

.1

1114

23

.8

13.0

19

.8

9.5

9.6

3.5

1-1

5

5 M

6G

N,5

PN

, (m

ean

± S

J3.)

32

.7 ±

3.0

10

F

4H

T

37.0

± 2

.7

mea

n ±

S.E

. o

f h

ealt

hy

co

ntr

ol

gro

up

(n

= 3

0)

mea

n ±

S.E

. o

f n

on

-ure

mic

GN

co

ntr

ol

gro

up

(n

= 1

5)

mea

n ±

S.E

. o

f n

on

-ure

mic

PN

co

ntr

ol

gro

up

(n

= 1

0)

mea

n ±

S.E

. o

f n

on

-ure

mic

HT

co

ntr

ol

gro

up

(n

= 2

0)

1087

± 7

0 30

.3 ±

1.8

"*

16.1

± 1

.4"

25.1

± 1

.5"*

13

.4 ±

1.1

"

52

.8±

2.9

3

0.4

±2

.5

39

.7±

2.6

2

2.8

±2

.0

57

.1±

3.3

3

1.6

±2

.8

43

.8±

3.1

2

5.9

±3

.0

49

.8±

34

2

7.8

±3

.0

36

.2±

34

1

9.9

±2

.7

50

.3±

3.2

2

9.4

±2

.7

41

.6±

2.7

2

4.3

±2

.4

12

.8±

1.1

"

28

A±

2.1

3

0.6

±2

.4

26

.1±

3.1

3

1.1

±2

.7

6.1

±0

.8"

16

.1±

1.8

1

5.3

±1

.6

14

.8±

2.0

1

6.7

±2

.0

*P <

0.0

1 as

co

mp

ared

to

the

corr

esp

on

din

g v

alue

of

the

hea

lth

y c

on

tro

l g

rou

p.

**P

< 0

.05

as c

om

par

ed t

o th

e co

rres

po

nd

ing

val

ue o

f th

e h

ealt

hy

co

ntr

ol

gro

up

. aB

lood

ure

a ni

trog

en.

cells prior to addition to the test systems. The final serum dilution was 1:5.

Statistical significance was evaluated by the Student's t-test.

4. Results

ADCC and NK cell activities of the 15 patients with chronic renal failure are shown in Table 1. In all the 3 test systems each individual patient's lympho- cytes exerted markedly lower cytotoxic activity than the corresponding mean values of the healthy control group. The mean values of the non-uremic patient groups did not differ significantly from those of the healthy control group.

The addition of autologous sera further reduced the originally impaired cytotoxic activity of each patient's lymphocytes (Table 2). Autologous sera from healthy individuals and non-uremic control patients did not significantly decrease cytotoxicity (data not shown). Furthermore, all uremic sera tested

on a panel of 10 healthy control lymphocytes markedly inhibited cytotoxicity in all 3 test systems (Table 3). Sera from healthy individuals and non-uremic control patients did not significantly reduce the cytotoxic activity o f normal lymphocytes suggesting that they did not contain immune complexes (data not shown).

5. Discussion

Impairment of resistance to infection in patients with chronic renal failure is well known [5]. Depres- sion of cell-mediated immunity has been observed as diminished or absent delayed skin reactivity [4,11]; reduction in time of aUograft rejection [3,12]; low response in mixed lymphocyte culture [4,13], spon- taneous [14] and lectin-induced blastogenesis [4,15]; lymphopenia [1,16] with decrease in both T- and B-cell subpopulations [ 17]; decreased macrophage phagocytic activity [ 18]; thymosin deficiency [ 19] and low leukocyte migration capacity [20]. In fact all these defects could contribute to the impairment

Table 2 Effect of autologous serum on the antibody-dependent and spontaneous cell-mediated eytotoxicity

of patients with chronic renal failure

Patient CRBC-ADCC % (20:1) K-562-ADCC % (50:1) NK % (50:1)

(a) (b) (a) (b) (a) (b)

1 34.5 11.0 27.2 8.2 11.7 4.2 2 37.5 11.8 29.3 11.4 14.1 6.6 3 39.2 23.5 31.2 16.9 17.1 9.9 4 16.8 6.7 12.7 4.2 2.8 0.9 5 28.4 9.7 25.9 9.3 10.9 4.8 6 39.2 26.3 35.8 22.9 18.1 11.0 7 34.5 10 .4 28.1 9.6 12.5 5.0 8 37.1 18.6 27.8 15.9 18.5 10.9 9 30.3 14.5 24.5 11.0 15.1 6.3

10 22.7 11.4 26.9 15.1 18.2 11.3 11 29.7 14.0 23.8 7.4 14.6 6.0 12 26.9 7.5 20.2 8.1 10.3 3.8 13 32.6 12.4 25.1 7.0 12.0 4.1 14 21.1 6.1 17.6 4.2 6.9 2.1 15 23.8 11.2 19.8 11.9 9.6 5.4

1-15 mean • S.E. 30.3 -+ 1.8 13.0 ± 1.5" 25.1 ± 1.5 10.9 +- 1.3" 12.8 ± 1.1 6.2 ± 0.8*

(a) Without autologous serum. (b) With autologous serum (final dilution 1:5). *P < 0.01 as compared to the corresponding value without autologous serum.

269

Table 3 Effect of uremic sera on the antibody-dependent and spontaneous cell-mediated

cytotoxicity of healthy control lymphocytes

Uremic serum Mean ± S.E. of 10 healthy control lymphocytes

CRBC-ADCC % K-562-ADCC % NK % (20:1) (50:1) (50:1)

54.9 +- 3.0 37.6 ± 2.8 27.5 ± 2.2

1 16.5 ± 3.1" 13.9 ± 3.1"* 11.0 ± 2.0** 2 24.7 ± 3.2** 12.0 ± 2.2* 13.8 ± 2.4*** 3 30.7 ± 2.7*** 18.8 ± 3.0*** 17.1 ± 1.9"** 4 20.3 ± 2.5* 11.3 ± 2.3* 6.9 ± 2.1" 5 22.0 -+ 2.6* 15.0 ± 2.6** 12.7 ± 1.8"** 6 32.9 -+ 2.7*** 22.9 ± 2.4*** 15.1 ± 2.1"** 7 18.7 ± 2.2* 14.3 ± 3.3** 8.5 ± 2.3* 8 25.3 ± 2.1"* 22.6 ± 2.5*** 14.9 ± 1.9"** 9 30.2 ± 2.8*** 17.7 ± 2.4** 10.5 ± 2.4**

10 25.6 ± 3.2** 21.8 ± 2.0*** 15.1 -+ 2.0*** 11 27.5 ± 2.5** 13.9 ± 2.9** 12.9 ± 1.9"** 12 13.7 ± 3.0* 15.8 ± 2.2** 10.4 ± 2.3** 13 17.6 ± 2.4* 7.5 ± 2.6* 9.6 ± 2.4** 14 16.5 ± 2.9* 9.4 ± 2.0* 11.3 ± 2.5** 15 30.4 ± 2.6*** 20.7 ± 2.7*** 16.5 ± 2.1"**

*P < 0.001 as compared to the corresponding mean value without serum. **P < 0:01 as compared to the corresponding mean value without serum.

***P < 0.05 as compared to the corresponding mean value without serum.

of resistance to infection in these patients. Though antibody-dependent cell-mediated cytotoxicity may play a role in the defense mechanism against certain infections [6] and in kidney graft rejection [7,8,21, 22], very little is known about the cytotoxic capacity of the lymphocytes from patients with chronic renal

failure [23,24]. In this study, uremic patients investigated showed

a decreased antibody-dependent and spontaneous cytotoxic capacity of their peripheral blood mono- nuclear cells on both allogeneic and xenogeneic target cells as compared to the corresponding mean values of healthy individuals and non-uremic patient groups with the same underlying diseases. This impairment was seen even when autologous uremic serum was n o t

present in the test system indicating that the well known inhibitory effect of uremic plasma or serum on lymphocyte effector function [4,25,26,27] can- not alone be responsible for this effect.

A reduction in the percentage of possible effector cells is unlikely to cause the defect in cytotoxic activ-

270

ity since the proportion of non-T-non-B- (null) lymphocytes is even higher in uremic patients as a consequence of the decrease in percentages of T- and B-cell subpopulations [17]. It seems unlikely that the uremic patients' effector cells were blocked by immune complexes since we could not demonstrate circulating immune complexes in our patients' sera. In addition, immunosuppressive-cytostatic drug effects could also be excluded, since no patient received this kind of therapy preceding the assays of cytotoxicity.

On the other hand, the addition of autologous serum further reduced the originally impaired ADCC and NK cell activity of uremic lymphocytes and uremic sera strongly inhibited that of healthy lympho- cytes. The suppressive effect of uremic serum or plasma on lymphocyte effector function has previ- ously been shown by inhibition of in vitro responses of lymphocytes to PHA [4], specific antigens [4] and aUoantigens [13,26]. Depression of the response of normal lymphocytes to PHA by serum or plasma from uremic patients was observed by Silk [25] and

by Holdsworth [27]. F rom these studies it can be suggested, that a dialyzable inhibitor is present in uremic plasma and we are currently investigating whether or not the 'ADCC-NK inhibi tor ' o f uremic serum is also dialyzable.

Summarising our findings we conclude that : (1) lymphocytes from uremic patients show an impaired ant ibody-dependent and spontaneous cytotoxic activity in vitro, which can be further reduced by the addit ion of autologous serum; and (2) sera from uremic patients also inhibit the ant ibody-dependent and spontaneous cytotoxic activity of heal thy lympho- cytes. Further studies are needed to elucidate the mechanism and biological significance of this inhibi- t ion.

References

[1] Wilson, W. E. C., Kirkpatrick, C. H. and Talmage, D. W. (1965) Ann. Intern. Med. 62, 1-14.

[2] Byron, P. R., MaUick, N. P. and Taylor, G. (1972) Int. Congr. Nephrology, Mexico, October 1972, Abstr. No. 993, p. 175.

[3] Dammin, G. J., Couch, N. P. and Murray, J. E. (1957) Ann. N.Y. Acad. Sci. 64,967-976.

[4] Huber, H., Pastner, D., Dittrich, P. and Braunsteiner, H. (1969) Clin. Exp. Immunol. 5, 75-82.

[5 ] Montgomerie, J. Z., Kalmonson, G. M. and Guze, L. B. (1968) Medicine 47, 1,.

[6] Jondal, M., Svedmyr, E., Klein, E. and Klein, G. (1976) in: Comparative Leukaemia Research, 1975 Bibl. Haemat. (Clemmesen, J. and Yohn, D. S. ed.) pp. 265-269, Karger, Basel.

[7] Ting, A. and Terasaki, P. I. (1975) Lancet 1,304-306.

[8] Stiller, C. R., Sinclair, N. R. STV., Abrahams, S., Ulan, R. A., Fung, M. and Wallace, A. C. (1975) Lancet 1, 953-954.

[9] B~yum, A. (1968) Stand. J. Clin. Lab. Invest. 21, Suppl. 97, 1-109.

[10] L~ng, I., Benczfir, M., Varga, M., Gergely, P. and Petr~nyi, Gy. (1977) Acta Med. Acad. Sci. Hung. 34,

1 2 3 - 1 2 7 . [11] Touraine, J. L., Touraine, F.', Revillard, J. P. Brochier,

J. and Traeger, J. (1975) Nephron 14,195-208. [12] Smiddy,F. G., BurweU,R. G. and Parsons,F. M. (1960)

Br. J. Surg. 48,328-333. [13] Elves, M. W., Israels, M. C. G. and Collinge, M. (1965)

Proc. Europ. Dialysis Transplant. Ass. 2,165-172. [14] Korz, R., Naber, A. and Essers, U. (1975) Kiln. Wsehr.

53, 21-27. [15] Reis, H. E., Heimsoth, V. H., Hirche, H., Eitelhuber, U.

(1974) Kiln. Wsehr. 52,437-443. [16] Jensson, O. (1958) Br. J. Haemat. 4,422-427. [17] Hoy, W. E., Cestero, R. V. M. and Freeman, R. B.

(1978) Nephron 20,182-188. [18] Ringoir, S., Van Looy, L., Van de Heyning, P. and

Leroux-Roels, C. (1975) Clin. Nephrol. 4,234-237. [19] Harris, J. and Sengar, D, (1975) Transplantation 20,

176-178. [20] Hanicki, Z., Cichocki, T., Komorowska, Z., Sulowicz,

W. and Smolenski, O. (1979) Nephron, 23,273-275. [21] D'Apice, A. J. P. and Morris, P. J. (1974) Transplanta-

tion 18, 20-26. [22] Jeannet, M. (1975) Lancet 1,573-574. [23] Thomas, J. M., Thomas, F. T., Kaplan, A. M. and Lee,

H. M. (1976) Transplantation 22, 94-100. [24] Dupont, E., Opelz, G., Gustafsson, L. A., Mikulski, M.

and Terasaki, P. I. (1977) Transplantation 23,165-168. [25] Silk, M. R. (1967) Invest. Urol. 5,195-199. [26] Kasakura, S. and Loewenstein, L. (1967) Transplanta-

tion 5,283-289. [27] Holdsworth, S. R., Fitzgerald, M. G., Hosking, C. S.

and Atkins, R. C. (1978) Clin. Exp. Immunol. 33, 95-101.

271