- Email: [email protected]
Effects of polychlorinated biphenyls on T-lymphocytes using a mofified erythrocyte rosette inhibition test
Chemosphere, Vol.19, Nos.l-6, P r i n t e d in Great B r i t a i n
EFFECTS USING
pp 823-828,
1989
OF POLYCHLORINATED BIPHENYLS A MOFIFIED ERYTHROCYTE ROSETTE
B.G.J.HEINZOW;
0 0 4 5 - 6 5 3 5 / 8 9 $3.00 + P e r g a m o n Press plc
.OO
ON T-LYMPHOCYTES I N H I B I T I O N TEST
H.-R.TINNEBERG*
Instit. of E n v i r o n m e n t a l T o x i c o l o g y of S l e s v i g - H o l s t e i n , D-23oo KIEL, F l e c k e n s t r . 4 , FRG. *Dep. of G y n e c o l o g y and O b s t e t r i c s , U n i v e r s i t y T~bingen, D - 7 4 o o TUBINGEN, Schleichstr., FRG.
ABSTRACT Effects of Propranolol, Dihydroergotamine, PCB 28 ( 2 , 4 , 4 [ t r i c h l o r b i p h e n y l ) and PCB 153 ( 2 , 2 $ 4 , 4 $ 5 , 5 t h e x a c h l o r b i p h e n y l ) , P e n t a c h l o r p h e n o l and DDE on T-cells were i n v e s t i g a t e d using a m o d i f i e d E rosette i n h i b i t i o n assay. The environmental c o n t a m i n a n t s DDE, PCB 28 and 153 and PCP were i d e n t i f i e d as potent inhibitors of rosette formation, the drugs P r o p r a n o l o l and Dihydroe r g o t a m i n e and F l u n a r i z i n e had no effect. It is s p e c u l a t e d that the e n v i r o n m e n t a l c o n t a m i n a n t s i n t e r a c t with the sheep red blood cell receptor or specific m e m b r a n e functions of human Tlymphocytes.
KEYWORDS
PCB's;
environmental contaminants;
immunotoxicity;
T-lymphocytes;
rosette-
inhibition.
INTRODUCTION It has been shown that drugs, p e s t i c i d e s and other environmental contaminants affect the humoral and c e l l u l a r immune r e s p o n s e in various species and man (Tiefenbach and Lange,1980; D e s c o t e s , 1 9 8 6 ) . Damage to the immune sytem by x e n o b i o t i c s may f a c i l i t a t e i n f e c t i o u s d i s e a s e s and even n e o p l a s i a (Thigpen et ai.,1975; Luster et ai.,1987). It is therefore n e c e s s a r y to assess the l y m p h o c y t e f u n c t i o n f o l l o w i n g e x p o s u r e to drugs and e n v i r o n m e n t a l c o n t a m i n a n t s . The E rosette f o r m a t i o n with sheep red blood cells (SRBC) is one of the characteristics of h u m a n T - l y m p h o c y t e s . This r e s p o n s e can be i n h i b i t e d by drugs (Bach et ai.,1969; Dunne et ai.,1987; R y h ~ n e n ,1979) , and pesticides. However in vitro drug c o n c e n t r a t i o n s used in the past were much higher than usually e n c o u n t e r e d in vivo, w h i c h limits the i m p l i c a t i o n of these results. Using a m o d i f i e d E - r o s e t t e i n h i b i t i o n test o r i g i n a l l y d e v e l o p e d by Bach and Antoine (1968) we were able to evoke effects at very low c o n c e n t r a t i o n s (Heinzow et ai.,1988).
823
824
METHODS Details of the m o d i f i e d E r o s e t t e i n h i b i t i o n assay given by Tinneberg and c o w o r k e r s (1984,1985) are d e p i c t e d in fig. I. 350 ul of s e p a r a t e d human07 lymphocytes, suspended in Hank" s b a l a n c e d salt solution (HBSS) (I cells/ml), were c e n t r i f u g e d and the p e l l e t was p r e i n c u b a t e d for 30 min at 37 °C either with 125 ul serum of p r e g n a n t w o m e n (positive control) or male serum (negative control) or b u f f e r c o n t a i n i n g the d i l u t e d x e n o b i o t i c s . After washing, 50 ul of the i n c u b a t e d l y m p h o c y t e s were added to tubes c o n t a i n i n g 125 ul s e r i a l l y d i l u t e d m o n o c l o n a l m o u s e a n t i b o d y to h u m a n T cells (Dako T11, Dakopatts, Denmark) and 25 ul guinea pig c o m p l e m e n t and then~ incubated for 60 min. at 37 °C. 50 ul of w a s h e d sheep red blood cells (10 ~ cells/ml) were added and the number of r o s e t t e s per 100 l y m p h o c y t e s counted twice in Thoma chambers by f l u o r e s c e n c e m i c r o s c o p y . L y m p h o c y t e s with 3 or more adherent red cells were scored as rosettes. C o n t r o l s and each d i l u t i o n of the x e n o b i o t i c were tested in triplicate. The rosette i n h i b i t i o n titer (RIT) is the r e c i p r o c a l of that d i l u t i o n of the antibody (ng Dako T11/ml) w h i c h gave 25 % or more i n h i b i t i o n of rosette formation compared to the mean of an internal control w i t h o u t OKT 11 Previous e x p e r i m e n t s (Tinneberg et ai.,1984) i n d i c a t e that an RIT below I0 ng Dako T 1 1 / m l reflects s i g n i f i c a n t i n h i b i t i o n (p 0.01), values b e t w e e n 10 [ and 12 are c o n s i d e r e d m a r g i n a l (p 0.05) and above 12 not significant. The e x p e r i m e n t s were r e j e c t e d if either the p o s i t i v e or n e g a t i v e control did not score the e x p e c t e d values. Stock s o l u t i o n s c o n t a i n i n g P r o p r a n o l o l (ICI), Dihydroergotamine (Sandoz), F l u n a r i z i n e (Janssen), DDT (Riedel-de Haen) , 2 , 4 , 4 " - T r i - and 2,4,5- Hexachlorobiphenyl (PCB 28 and 153 ,Promochem) and P e n t a c h l o r p h e n o l (Riedel de Haen) were freshly d i l u t e d in HBSS using DMSO. Solvent without drugs or e n v i r o n m e n t a l c o n t a m i n a n t s showed no interference. Figure
I :
Schematic representation of the RIT I
l
tested chemical ~
3 X
RC or GPC (t:5)
washing
~
+ monoclonal anti Tt 1
centrifug~tion ~
resospension
(HBSS)
dilution series
adjust to
prelncubation
Incubation
SRBC
acridine orange ] ~
10"'7/ml
30 mln. 37 o C
90 rain. 37 ~ C
10"'8 /ml
+ Iluorescence
human lymphocytes
1
(260 x magn.)
RC or GPC (1:5)
washing
(HSSS)
,,
I,
+ monoclonal anti T11
dilutionseries
centrilugation ~
resuspenslon
825
RESULTS Mean rosette i n h i b i t i o n titers (ng Dako T11/ml) (± SD) versus different concentrations (mol/l) of xenobiotics are shown in figure 2-5. C neg indicates the negative control with male serum and C pos the positive control using p r e g n a n c y serum. Titers below 12 ng/ml indicate significant (p < 0.05) i n h i b i t i o n of E r o s e t t i n g . Rosette formation of human T l y m p h o c y t e s was impaired after preincubation with the e n v i r o n m e n t a l c o n t a m i n a n t s PCB 28 and 153, DDE and PCP. The tested drugs did n o t _ i n h i b i t E ^ r o s e t t i n g f o l l o w i n g preincubation at c o n c e n t r a t i o n s b e t w e e n I0 -j and 10 -~ mol/l. The minimal c o n c e n t r a t i o n s of the e n v i r o n m e n t a l c o n t a m i n a n t s eliciting a significant m o n o c l o n a l ..antibody. sparing effect ( rosette inhibition) in the test ranged from I0 -Iz to I0 -10 mol/l.
DISCUSSION Immunomodulation by environmental chemicals has been of increasing concern. Several studies have i n d i c a t e d that drugs, heavy metals, organop h o s p h a t e s and c h l o r i n a t e d h y d r o c a r b o n s have the p o t e n t i a l to affect immune responses and immune p a r a m e t e r s (Descotes,1986). The specific c e l l u l a r and molecular m e c h a n i s m s of these i n t e r a c t i o n s are still unclear. This has been p a r t i c u l a r l y h a m p e r e d by the fact that in vitro testing had to u t i l i z e much higher c o n c e n t r a t i o n s c o m p a r e d to the in vivo situation. In p r e v i o u s experiments using a standard E rosette assay, the i n h i b i t o r y effects of Lidocaine (Ryh~nen,1979) and P r o p r a n o l o l (Dunne et al., 1987) have been attributed to u n s p e c i f i c m e m b r a n e related m e c h a n i s m s . In our test Flunarizine and Propranolol two h y d r o p h o b i c drugs and DHE a secale a l k a l o i d with high binding a f f i n i t y to g l y c o p r o t e i n s (Nimmerfall and R o s e n t h a l e r , 1 9 8 0 ) did n o h interfere with T l y m p h o c y t e function at low c o n c e n t r a t i o n s c o m p a r a b l e to those e n c o u n t e r e d in therapy. Many environmental contaminants have been shown to target specific biological sites . PCB's bind to the l y m p h o c y t e A h - r e c e p t o r and induce biological response thereupon (Silkworth et ai.,1986). The E rosette inhibition test has been p a r t i c u l a r l y used as an in vitro m e t h o d for the c h a r a c t e r i s a t i o n and i d e n t i f i c a t i o n of an i m m u n o s u p p r e s s i v e protein r e l e a s e d in p r e g n a n c y , the early p r e g n a n c y factor (Morton et ai.,1976; Noonan e_~t ai.,1979). Less anti l y m p h o c y t e serum (ALS) or monoclonal antibody was req u i r e d when non-pregnancy l y m p h o c y t e s were p r e t r e a t e a d with pregnancy serum (Morton et ai.,1977). The present study shows i n t e r f e r e n c e with Erosette f o r m a t i o n a T l y m p h o c y t e function by e n v i r o n m e n t a l cont'aminants in a m a n n e r similar to the early p r e g n a n c y factor (EPF) . It is t e m p t i n g to s p e c u l a t e that the tested environmental contaminants elicit their i m m u n o s u p p r e s s i v e effects by a similar m e c h a n i s m . In previg~s i n v e s t i g a t i o n s (Lee et ai.,1979) DDT inhibited E r o s e t t i n g by 50% at 10 mol/l . No in v i t r o data on PCB 153 the major PCB c o n g e n e r in human body fat are available, but immune s u p p r e s s i o n has been reported in e x p e r i m e n t a l animals (Koller and T h i g p e n , 1 9 7 3 ; Thomas and H i n s d i l l 1978) and in victims of Y u - C h e n g (Chang et ai.,1982) f o l l o w i n g e x p o s u r e to PCB's. Our results may hold some e x p l a n a t i o n for the d i s c r e p a n c y b e t w e e n in vivo and in vitro data and may offer a p e r s p e c t i v e for further i n v e s t i g a t i o n of the underlying mechanisms of i m m u n o m o d u l a t i n g effects of environmental contaminants. The effects o b s e r v e d in the more s e n s i t i v e m o d i f i e d E rosette assay suggest a d i r e c t a c t i o n on l y m p h o c y t e s e.g i n t e r f e r e n c e with epitopes of the CD 2 - r e c e p t o r or specific m e m b r a n e function.
826
,Fi~. 2
30
I
E h20
Cneg
0 0 C~
I I I
C~
c10
"~
D2
I
.+.
T
"'-T----~"" 1---~ .... ,+
1 !Cpos
F-
.1_
I
I
I
I
I
controls -3
-5
-7
-9
-11
I
-13
log [ m o l / t ] Fi,g. 3
30-
E ~p-
Cneg
20
""
T--~[---i "'' $ DHE
! PCB-153
0 v 0 ¢q Cn
~10I-rr"
TCp0s 0
1 I
I
:ontrols -3
-5
I
I
I
-7 -9 log [tool/I]
I
-11
I
-13
I
-I.5
827
Fig.
30-
IE 20 -
4
ICneg
I--0 C~ r]
T
T PCP
~nlO
c"
I']---:
Cpos 0
!
I
controls -3
I
-5
I
I
-7 -9 log [mot/l]
I
I
-11
Fig.
30-
E ~20
-
-13
T
ICneg
5
T DDE
Fo
O E]
! /i
c10
~ /..,_I
CY
TCpos
0
I
I
controls -3
-5
/ \$--I I 1 i~j.
I
I
-7 -9 log [mol/I ]
I
-11
I
-13
I
-15
828
In vivo impairment of c e l l u l a r immune r e s p o n s e s in animals (Street Sharma,1975) and of the d e l a y e d type of h y p e r s e n s i t i v i t y r e a c t i o n in (Chang et ai.,1982) is c o n s i s t e n t with our findings.
and man
In c o n c l u s i o n it should be c o n s i d e r e d if the current testing tiers for the a s s e s s m e n t of the i m m u n o t o x i c p o t e n t i a l of x e n o b i o t i c s should be e x p a n d e d by the m o d i f i e d E rosette assay. As a b i o a s s a y using h u m a n l y m p h o c y t e s this is an e x t r e m e l y s e n s i t i v e test for the d e t e c t i o n of i n t e r f e r e n c e with T cell fun c t i o n at n a n o m o l a r and p o s s i b l y p i c o m o l a r c o n c e n t r a t i o n s .
REFERENCES Bach J.F., B. Antoine (1968).In vitro detection of immunosuppressive a c t i v i t y of a n t i - l y m p h o c y t e sera. N a t u r e 217 ,658-659. Bach J.F., M. Dardenne, C. F o u r n i e r (1969). In vitro e v a l u a t i o n of imraunos u p p r e s s i v e drugs. N a t u r e 222 ,998-999 Chang K.J., K.H. Hsieh, S.Y. Tang, T.C. Tung (1982). I m m u n o l o g i c e v a l u a t i o n of p a t i e n t s with p o l y c h l o r i n a t e d b i p h e n y l poisoning. J.Toxicol.Environ. Health 9, 217-223. D es c o t e s J. (1986). Immunotoxicology of drugs and chemicals Elsvier Amsterdam Dunne J.V., C.J. Peters, T.L.Moore, J.H. V a u g h a n (1976). Differential effects of p r o p r a n o l o l on l y m p h o c y t e rosette f o r m a t i o n and r e s p o n s e to plant mitogens. A r t h r i t i s R h e u m a t i s m 21, 767-773. H e i n z o w B.G.J., H.-R. Tinneberg, C.Boie (1988) Effects of some lipophilic xenobiotics on T-lymphocytes u s i ng a modified erythrocyte rosette i n h i b i t i o n test. Res. Commun. Chem. Pathol. Pharmacol. 6-1,277-280. Koller L.D., J.R. T h i g p e n (1973). B i p h e n y l - e x p o s e d rabbits. Am. J. Vet. Res. 34, 1605. Lee T.-P., R.Moscati, B°H. Dark (1979). Effects of p e s t i c i d e s on hulaan leukocyte functions. Res. Comm. Chem. Pathol. Pharmacol. 23, 597-605 (1979) Luster M.J., J.R. Blank, J.H. Dean (1987). M o l e c u l a r and c e l l u l a r basis of c h e m i c a l l y induced i m m u n o t o x i c i t y . Annu. Rev. Pharmacol. Toxicol. 27, 23-49 M o r t o n H., V. Hegh, G.J.A. Clunie (1976). Studies of the rosette inhibition test in p r e g n a n t mice. Proc. R. Soc. Biol. 193, 413-19 M o r t o n If., B.Rolfe, G.J.A. Clunie (1977). An early p r e g n a n c y factor d e t e c t e d in h u m a n serum by tHE rosette i n h i b i t i o n test. L a n c e t i, 394-397. N o o n a n F.P., W.J. Halliday, H. Morton, G.J.A. Clunie (1979). Early p r e g n a n c y factor is i m m u n o s u p p r e s s i v e . Nature 278, 649-651. R y h ~ n e n P. (1974). L i d o c a i n e i n h i b i t i o n of rosette formation. Med. Biol. 57, 196-198. S i l k w o r t h J.B., L. A n t r i m , G . S a c k (1986). Ah R e c e p t o r m e d i a t e d s u p p r e s s o n of the a n t i b o d y r e s p o n s e in mice is p r i m a r i l y d e p e n d e n t on the Ah phenotype of lymphoid tissue. Toxicol. Appl. Pharmacol. 86, 380-90. Street J.C., R.P. Sharma (1975). A l t e r a t i o n of induced c e l l u l a r and humoral immune responses by p e s t i c i d e s and c h e m i c a l s of environmental concern. Toxicol. Appl. Pharmacol. 32, 587-602. Thomas P.T., R.D. H i n s d i l l (1978). E f f e c t of p o l y c h l o r i n a t e d b i p h e n y l s on the immune r e s p o n s e s of rhesus m o n k e y s and mice. Toxicol. Appl. Pharmacol. 44, 41-51. T i e f e n b a c h B., P. Lange (1980). Studies on the action of d i m e t h o a t e on the immune system. Arch. Toxicol. Suppl. 4, 167-170. T i n n e b e r g H.R., R. Staves R., D.S. Lang (1985). In: Early p r e g n a n c y factors (Ellendorf F., E. Koch eds.). P e r i n a t o l o g y Press, 195-210. Tinneberg H.R., R. Staves, K. Semm ( 1 9 8 4 ) . I m p r o v e m e n t of the rosette i n h i b i t i o n assay. Am. J. Reprod. Immunol. 5, 151. (1984)
EFFECTS USING
pp 823-828,
1989
OF POLYCHLORINATED BIPHENYLS A MOFIFIED ERYTHROCYTE ROSETTE
B.G.J.HEINZOW;
0 0 4 5 - 6 5 3 5 / 8 9 $3.00 + P e r g a m o n Press plc
.OO
ON T-LYMPHOCYTES I N H I B I T I O N TEST
H.-R.TINNEBERG*
Instit. of E n v i r o n m e n t a l T o x i c o l o g y of S l e s v i g - H o l s t e i n , D-23oo KIEL, F l e c k e n s t r . 4 , FRG. *Dep. of G y n e c o l o g y and O b s t e t r i c s , U n i v e r s i t y T~bingen, D - 7 4 o o TUBINGEN, Schleichstr., FRG.
ABSTRACT Effects of Propranolol, Dihydroergotamine, PCB 28 ( 2 , 4 , 4 [ t r i c h l o r b i p h e n y l ) and PCB 153 ( 2 , 2 $ 4 , 4 $ 5 , 5 t h e x a c h l o r b i p h e n y l ) , P e n t a c h l o r p h e n o l and DDE on T-cells were i n v e s t i g a t e d using a m o d i f i e d E rosette i n h i b i t i o n assay. The environmental c o n t a m i n a n t s DDE, PCB 28 and 153 and PCP were i d e n t i f i e d as potent inhibitors of rosette formation, the drugs P r o p r a n o l o l and Dihydroe r g o t a m i n e and F l u n a r i z i n e had no effect. It is s p e c u l a t e d that the e n v i r o n m e n t a l c o n t a m i n a n t s i n t e r a c t with the sheep red blood cell receptor or specific m e m b r a n e functions of human Tlymphocytes.
KEYWORDS
PCB's;
environmental contaminants;
immunotoxicity;
T-lymphocytes;
rosette-
inhibition.
INTRODUCTION It has been shown that drugs, p e s t i c i d e s and other environmental contaminants affect the humoral and c e l l u l a r immune r e s p o n s e in various species and man (Tiefenbach and Lange,1980; D e s c o t e s , 1 9 8 6 ) . Damage to the immune sytem by x e n o b i o t i c s may f a c i l i t a t e i n f e c t i o u s d i s e a s e s and even n e o p l a s i a (Thigpen et ai.,1975; Luster et ai.,1987). It is therefore n e c e s s a r y to assess the l y m p h o c y t e f u n c t i o n f o l l o w i n g e x p o s u r e to drugs and e n v i r o n m e n t a l c o n t a m i n a n t s . The E rosette f o r m a t i o n with sheep red blood cells (SRBC) is one of the characteristics of h u m a n T - l y m p h o c y t e s . This r e s p o n s e can be i n h i b i t e d by drugs (Bach et ai.,1969; Dunne et ai.,1987; R y h ~ n e n ,1979) , and pesticides. However in vitro drug c o n c e n t r a t i o n s used in the past were much higher than usually e n c o u n t e r e d in vivo, w h i c h limits the i m p l i c a t i o n of these results. Using a m o d i f i e d E - r o s e t t e i n h i b i t i o n test o r i g i n a l l y d e v e l o p e d by Bach and Antoine (1968) we were able to evoke effects at very low c o n c e n t r a t i o n s (Heinzow et ai.,1988).
823
824
METHODS Details of the m o d i f i e d E r o s e t t e i n h i b i t i o n assay given by Tinneberg and c o w o r k e r s (1984,1985) are d e p i c t e d in fig. I. 350 ul of s e p a r a t e d human07 lymphocytes, suspended in Hank" s b a l a n c e d salt solution (HBSS) (I cells/ml), were c e n t r i f u g e d and the p e l l e t was p r e i n c u b a t e d for 30 min at 37 °C either with 125 ul serum of p r e g n a n t w o m e n (positive control) or male serum (negative control) or b u f f e r c o n t a i n i n g the d i l u t e d x e n o b i o t i c s . After washing, 50 ul of the i n c u b a t e d l y m p h o c y t e s were added to tubes c o n t a i n i n g 125 ul s e r i a l l y d i l u t e d m o n o c l o n a l m o u s e a n t i b o d y to h u m a n T cells (Dako T11, Dakopatts, Denmark) and 25 ul guinea pig c o m p l e m e n t and then~ incubated for 60 min. at 37 °C. 50 ul of w a s h e d sheep red blood cells (10 ~ cells/ml) were added and the number of r o s e t t e s per 100 l y m p h o c y t e s counted twice in Thoma chambers by f l u o r e s c e n c e m i c r o s c o p y . L y m p h o c y t e s with 3 or more adherent red cells were scored as rosettes. C o n t r o l s and each d i l u t i o n of the x e n o b i o t i c were tested in triplicate. The rosette i n h i b i t i o n titer (RIT) is the r e c i p r o c a l of that d i l u t i o n of the antibody (ng Dako T11/ml) w h i c h gave 25 % or more i n h i b i t i o n of rosette formation compared to the mean of an internal control w i t h o u t OKT 11 Previous e x p e r i m e n t s (Tinneberg et ai.,1984) i n d i c a t e that an RIT below I0 ng Dako T 1 1 / m l reflects s i g n i f i c a n t i n h i b i t i o n (p 0.01), values b e t w e e n 10 [ and 12 are c o n s i d e r e d m a r g i n a l (p 0.05) and above 12 not significant. The e x p e r i m e n t s were r e j e c t e d if either the p o s i t i v e or n e g a t i v e control did not score the e x p e c t e d values. Stock s o l u t i o n s c o n t a i n i n g P r o p r a n o l o l (ICI), Dihydroergotamine (Sandoz), F l u n a r i z i n e (Janssen), DDT (Riedel-de Haen) , 2 , 4 , 4 " - T r i - and 2,4,5- Hexachlorobiphenyl (PCB 28 and 153 ,Promochem) and P e n t a c h l o r p h e n o l (Riedel de Haen) were freshly d i l u t e d in HBSS using DMSO. Solvent without drugs or e n v i r o n m e n t a l c o n t a m i n a n t s showed no interference. Figure
I :
Schematic representation of the RIT I
l
tested chemical ~
3 X
RC or GPC (t:5)
washing
~
+ monoclonal anti Tt 1
centrifug~tion ~
resospension
(HBSS)
dilution series
adjust to
prelncubation
Incubation
SRBC
acridine orange ] ~
10"'7/ml
30 mln. 37 o C
90 rain. 37 ~ C
10"'8 /ml
+ Iluorescence
human lymphocytes
1
(260 x magn.)
RC or GPC (1:5)
washing
(HSSS)
,,
I,
+ monoclonal anti T11
dilutionseries
centrilugation ~
resuspenslon
825
RESULTS Mean rosette i n h i b i t i o n titers (ng Dako T11/ml) (± SD) versus different concentrations (mol/l) of xenobiotics are shown in figure 2-5. C neg indicates the negative control with male serum and C pos the positive control using p r e g n a n c y serum. Titers below 12 ng/ml indicate significant (p < 0.05) i n h i b i t i o n of E r o s e t t i n g . Rosette formation of human T l y m p h o c y t e s was impaired after preincubation with the e n v i r o n m e n t a l c o n t a m i n a n t s PCB 28 and 153, DDE and PCP. The tested drugs did n o t _ i n h i b i t E ^ r o s e t t i n g f o l l o w i n g preincubation at c o n c e n t r a t i o n s b e t w e e n I0 -j and 10 -~ mol/l. The minimal c o n c e n t r a t i o n s of the e n v i r o n m e n t a l c o n t a m i n a n t s eliciting a significant m o n o c l o n a l ..antibody. sparing effect ( rosette inhibition) in the test ranged from I0 -Iz to I0 -10 mol/l.
DISCUSSION Immunomodulation by environmental chemicals has been of increasing concern. Several studies have i n d i c a t e d that drugs, heavy metals, organop h o s p h a t e s and c h l o r i n a t e d h y d r o c a r b o n s have the p o t e n t i a l to affect immune responses and immune p a r a m e t e r s (Descotes,1986). The specific c e l l u l a r and molecular m e c h a n i s m s of these i n t e r a c t i o n s are still unclear. This has been p a r t i c u l a r l y h a m p e r e d by the fact that in vitro testing had to u t i l i z e much higher c o n c e n t r a t i o n s c o m p a r e d to the in vivo situation. In p r e v i o u s experiments using a standard E rosette assay, the i n h i b i t o r y effects of Lidocaine (Ryh~nen,1979) and P r o p r a n o l o l (Dunne et al., 1987) have been attributed to u n s p e c i f i c m e m b r a n e related m e c h a n i s m s . In our test Flunarizine and Propranolol two h y d r o p h o b i c drugs and DHE a secale a l k a l o i d with high binding a f f i n i t y to g l y c o p r o t e i n s (Nimmerfall and R o s e n t h a l e r , 1 9 8 0 ) did n o h interfere with T l y m p h o c y t e function at low c o n c e n t r a t i o n s c o m p a r a b l e to those e n c o u n t e r e d in therapy. Many environmental contaminants have been shown to target specific biological sites . PCB's bind to the l y m p h o c y t e A h - r e c e p t o r and induce biological response thereupon (Silkworth et ai.,1986). The E rosette inhibition test has been p a r t i c u l a r l y used as an in vitro m e t h o d for the c h a r a c t e r i s a t i o n and i d e n t i f i c a t i o n of an i m m u n o s u p p r e s s i v e protein r e l e a s e d in p r e g n a n c y , the early p r e g n a n c y factor (Morton et ai.,1976; Noonan e_~t ai.,1979). Less anti l y m p h o c y t e serum (ALS) or monoclonal antibody was req u i r e d when non-pregnancy l y m p h o c y t e s were p r e t r e a t e a d with pregnancy serum (Morton et ai.,1977). The present study shows i n t e r f e r e n c e with Erosette f o r m a t i o n a T l y m p h o c y t e function by e n v i r o n m e n t a l cont'aminants in a m a n n e r similar to the early p r e g n a n c y factor (EPF) . It is t e m p t i n g to s p e c u l a t e that the tested environmental contaminants elicit their i m m u n o s u p p r e s s i v e effects by a similar m e c h a n i s m . In previg~s i n v e s t i g a t i o n s (Lee et ai.,1979) DDT inhibited E r o s e t t i n g by 50% at 10 mol/l . No in v i t r o data on PCB 153 the major PCB c o n g e n e r in human body fat are available, but immune s u p p r e s s i o n has been reported in e x p e r i m e n t a l animals (Koller and T h i g p e n , 1 9 7 3 ; Thomas and H i n s d i l l 1978) and in victims of Y u - C h e n g (Chang et ai.,1982) f o l l o w i n g e x p o s u r e to PCB's. Our results may hold some e x p l a n a t i o n for the d i s c r e p a n c y b e t w e e n in vivo and in vitro data and may offer a p e r s p e c t i v e for further i n v e s t i g a t i o n of the underlying mechanisms of i m m u n o m o d u l a t i n g effects of environmental contaminants. The effects o b s e r v e d in the more s e n s i t i v e m o d i f i e d E rosette assay suggest a d i r e c t a c t i o n on l y m p h o c y t e s e.g i n t e r f e r e n c e with epitopes of the CD 2 - r e c e p t o r or specific m e m b r a n e function.
826
,Fi~. 2
30
I
E h20
Cneg
0 0 C~
I I I
C~
c10
"~
D2
I
.+.
T
"'-T----~"" 1---~ .... ,+
1 !Cpos
F-
.1_
I
I
I
I
I
controls -3
-5
-7
-9
-11
I
-13
log [ m o l / t ] Fi,g. 3
30-
E ~p-
Cneg
20
""
T--~[---i "'' $ DHE
! PCB-153
0 v 0 ¢q Cn
~10I-rr"
TCp0s 0
1 I
I
:ontrols -3
-5
I
I
I
-7 -9 log [tool/I]
I
-11
I
-13
I
-I.5
827
Fig.
30-
IE 20 -
4
ICneg
I--0 C~ r]
T
T PCP
~nlO
c"
I']---:
Cpos 0
!
I
controls -3
I
-5
I
I
-7 -9 log [mot/l]
I
I
-11
Fig.
30-
E ~20
-
-13
T
ICneg
5
T DDE
Fo
O E]
! /i
c10
~ /..,_I
CY
TCpos
0
I
I
controls -3
-5
/ \$--I I 1 i~j.
I
I
-7 -9 log [mol/I ]
I
-11
I
-13
I
-15
828
In vivo impairment of c e l l u l a r immune r e s p o n s e s in animals (Street Sharma,1975) and of the d e l a y e d type of h y p e r s e n s i t i v i t y r e a c t i o n in (Chang et ai.,1982) is c o n s i s t e n t with our findings.
and man
In c o n c l u s i o n it should be c o n s i d e r e d if the current testing tiers for the a s s e s s m e n t of the i m m u n o t o x i c p o t e n t i a l of x e n o b i o t i c s should be e x p a n d e d by the m o d i f i e d E rosette assay. As a b i o a s s a y using h u m a n l y m p h o c y t e s this is an e x t r e m e l y s e n s i t i v e test for the d e t e c t i o n of i n t e r f e r e n c e with T cell fun c t i o n at n a n o m o l a r and p o s s i b l y p i c o m o l a r c o n c e n t r a t i o n s .
REFERENCES Bach J.F., B. Antoine (1968).In vitro detection of immunosuppressive a c t i v i t y of a n t i - l y m p h o c y t e sera. N a t u r e 217 ,658-659. Bach J.F., M. Dardenne, C. F o u r n i e r (1969). In vitro e v a l u a t i o n of imraunos u p p r e s s i v e drugs. N a t u r e 222 ,998-999 Chang K.J., K.H. Hsieh, S.Y. Tang, T.C. Tung (1982). I m m u n o l o g i c e v a l u a t i o n of p a t i e n t s with p o l y c h l o r i n a t e d b i p h e n y l poisoning. J.Toxicol.Environ. Health 9, 217-223. D es c o t e s J. (1986). Immunotoxicology of drugs and chemicals Elsvier Amsterdam Dunne J.V., C.J. Peters, T.L.Moore, J.H. V a u g h a n (1976). Differential effects of p r o p r a n o l o l on l y m p h o c y t e rosette f o r m a t i o n and r e s p o n s e to plant mitogens. A r t h r i t i s R h e u m a t i s m 21, 767-773. H e i n z o w B.G.J., H.-R. Tinneberg, C.Boie (1988) Effects of some lipophilic xenobiotics on T-lymphocytes u s i ng a modified erythrocyte rosette i n h i b i t i o n test. Res. Commun. Chem. Pathol. Pharmacol. 6-1,277-280. Koller L.D., J.R. T h i g p e n (1973). B i p h e n y l - e x p o s e d rabbits. Am. J. Vet. Res. 34, 1605. Lee T.-P., R.Moscati, B°H. Dark (1979). Effects of p e s t i c i d e s on hulaan leukocyte functions. Res. Comm. Chem. Pathol. Pharmacol. 23, 597-605 (1979) Luster M.J., J.R. Blank, J.H. Dean (1987). M o l e c u l a r and c e l l u l a r basis of c h e m i c a l l y induced i m m u n o t o x i c i t y . Annu. Rev. Pharmacol. Toxicol. 27, 23-49 M o r t o n H., V. Hegh, G.J.A. Clunie (1976). Studies of the rosette inhibition test in p r e g n a n t mice. Proc. R. Soc. Biol. 193, 413-19 M o r t o n If., B.Rolfe, G.J.A. Clunie (1977). An early p r e g n a n c y factor d e t e c t e d in h u m a n serum by tHE rosette i n h i b i t i o n test. L a n c e t i, 394-397. N o o n a n F.P., W.J. Halliday, H. Morton, G.J.A. Clunie (1979). Early p r e g n a n c y factor is i m m u n o s u p p r e s s i v e . Nature 278, 649-651. R y h ~ n e n P. (1974). L i d o c a i n e i n h i b i t i o n of rosette formation. Med. Biol. 57, 196-198. S i l k w o r t h J.B., L. A n t r i m , G . S a c k (1986). Ah R e c e p t o r m e d i a t e d s u p p r e s s o n of the a n t i b o d y r e s p o n s e in mice is p r i m a r i l y d e p e n d e n t on the Ah phenotype of lymphoid tissue. Toxicol. Appl. Pharmacol. 86, 380-90. Street J.C., R.P. Sharma (1975). A l t e r a t i o n of induced c e l l u l a r and humoral immune responses by p e s t i c i d e s and c h e m i c a l s of environmental concern. Toxicol. Appl. Pharmacol. 32, 587-602. Thomas P.T., R.D. H i n s d i l l (1978). E f f e c t of p o l y c h l o r i n a t e d b i p h e n y l s on the immune r e s p o n s e s of rhesus m o n k e y s and mice. Toxicol. Appl. Pharmacol. 44, 41-51. T i e f e n b a c h B., P. Lange (1980). Studies on the action of d i m e t h o a t e on the immune system. Arch. Toxicol. Suppl. 4, 167-170. T i n n e b e r g H.R., R. Staves R., D.S. Lang (1985). In: Early p r e g n a n c y factors (Ellendorf F., E. Koch eds.). P e r i n a t o l o g y Press, 195-210. Tinneberg H.R., R. Staves, K. Semm ( 1 9 8 4 ) . I m p r o v e m e n t of the rosette i n h i b i t i o n assay. Am. J. Reprod. Immunol. 5, 151. (1984)