Journal of Immunological Methods, 24 (1978 ) 257--267
257
© Elsevier/North-Holland Biomedical Press
ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY AND NATURAL CYTOTOXICITY: EFFECT OF PRE-TREATMENT OF HUMAN LYMPHOCYTES WITH DEIONISED WATER AND AMMONIUM CHLORIDE
O. EREMIN, J. ASHBY and D. PLUMB Division of Immunology, Department of Pathology, University of Cambridge. Cam bridge, ~LK.
(Received 15 May 1978, accepted 5 June 1978)
Deionised water is an efficient red blood cell lytic agent with no demonstrable deleterious effects on ADCC and natural cytotoxicity (blood and tonsil). Treatment with isotonic ammonium chloride, however, produces a significant reduction of both ADCC and natural cytotoxicity. This reduction is less pronounced if the treatment is carried out at 4°C and is temporary, recovery occurring over a 24 h period of incubation at 37°C.
INTRODUCTION Red b l o o d cells, c o n t a m i n a t i n g b o t h h u m a n and animal l e u k o c y t e preparations, have been r e m o v e d b y lysis with various lytic p r e p a r a t i o n s including deionised w a t e r { T h o m s o n et at., 1 9 6 6 ) , h y p o t o n i c saline (Fallon et at., 1 9 6 2 ; J a n o w s k y et at., 1 9 6 4 ) and a m m o n i u m c h l o r i d e (Agostini and Ideo, 1 9 6 5 , Boyle, 1 9 6 8 ; R o o s and Loos, 1970). A l t h o u g h l y m p h o c y t e d a m a g e and d e a t h o c c u r following such t r e a t m e n t s ( T h o m s o n et al., 1 9 6 6 ) , a f t e r removal o f the cell ghosts and debris the r e s u l t a n t l y m p h o i d cell suspensions c o n t a i n viable, motile cells ( J a n o w s k y et al., 1 9 6 4 ) , with a well preserved intracellular a r c h i t e c t u r e (Agostini and Ideo, 1 9 6 5 ) . L y m p h o c y t e s p r e p a r e d in such a m a n n e r have been used in various in vitro assays, such as m i x e d l y m p h o c y t e - c u l t u r e i n t e r a c t i o n s ( H o d e s and T e r r y , 1 9 7 4 ) , a n t i b o d y ~ d e p e n d e n t cellular c y t o t o x i c i t y (ADCC) (De Bracco et al., 1 9 7 6 ) , natural c y t o t o x i c i t y (Kiessling et al., 1 9 7 5 ) and in rosetting r e a c t i o n s ( E r e m i n et at., 1 9 7 7 b ) . Evidence has a p p e a r e d , h o w e v e r , suggesting an alteration o f ADCC following a m m o n i u m c h l o r i d e t r e a t m e n t ( K o v i t h a v o n g s et at., 1 9 7 4 ; K o d e r a and Bean, 1 9 7 5 ; Yust et al., 1976). T h e aim o f this p a p e r is to evaluate and c o m p a r e the e f f e c t on ADCC and natural c y t o t o x i c i t y o f pre-treating l y m p h o c y t e suspensions ( b l o o d and tonsil) with deionised w a t e r and with a m m o n i u m chloride. T h e evidence p r e s e n t e d shows t h a t a m m o n i u m c h l o r i d e t r e a t m e n t significantly reduces
258 both ADCC and natural c y t o t o x i c i t y whilst deionised water ha~ no demonstrable deleterious effect. MATERIALS AND METHODS
Standard lymphocyte preparation L y m p h o c y t e s were prepared as previously outlined (Eremin et al., 1976). Blood l y m p h o c y t e s were isolated from heparinised venous blood on a FicollH y p a q u e gradient (B¢yum, 1968), washed and made up in tissue culture medium RPMI 1640 (TCM), containing 100 IU penicillin/ml, 100 ~g streptomycin/ml, 0.7 g sodium bicarbonate/l, 25 mM Hepes buffer/ml and 10% heat-inactivated foetal calf serum. Tonsillar l y m p h o c y t e s were isolated from fresh operative specimens, washed and made up in TCM. Carbonyl iron was used to remove phagocytic cells. Treated lymphocyte preparation Deionised water. A b o u t 0.3 ml of deionised water (10 drops) was added to the l y m p h o c y t e pellet (3 X 107 cells), mixed and allowed to stand for 20 sec at 20°C {room temperature). A similar volume of twice concent rat ed phosphate-buffered saline was then added, followed by an excess volume of TCM. The red cell ghosts were removed by centrifuging at 100 X g for 3 min, twice. Isotonic ammonium chloride (KHCO3 0.01 M, NH4C1 0.15 M, 0.1 mM EDTA -- pH 7.4} *. 7 ml of a m m o n i u m chloride was added to the lymphocyte pellet (3 X 10 ~ cells), mixed and allowed to stand for 7 min. This was then neutralized with an excess volume of TCM and centrifuged to remove the red cell ghosts. This procedure was used as it had previously been found to give the most efficient (virtually total) removal of contaminating red blood c e l l s - - c o m p a r a b l e with deionised water. To minimize l y m p h o c y t e cell loss, with o ut diminishing the lytic capacity for red blood cells, heatinactivated foetal calf serum (5%) was added to the a m m o n i u m chloride solution just prior to use. The lytic process was carried out at 20°C and 4°C. Lymphocyte markers L y m p h o c y t e rosetting assays were carried out as previously described (Eremin et al., 1976}. For all rosetting tests the l y m p h o c y t e s were made up to 2 X 10~/ml in TCM. Sheep red cell rosettes were used as markers for T l y m p h o c y t e s . Fc receptor-bearing l y m p h o c y t e s were detected by opsonic adherence o f ox red blood cells, sensitized with a subagglutinating dose of rabbit IgG anti-ox red blood cell antiserum. L y m p h o c y t e s with receptors for the third c o m p o n e n t o f c o m p l e m e n t were detected by rosette formation
* NHaCI, ammonium chloride; KHC03, potassium hydrogen carbonate; EDTA, ethylenediaminetetraacetic acid.
259 with ox red blood cells sensitized with rabbit IgM anti-ox red blood cell antiserum and mouse complement.
Cytotoxicity assays The details of the Slchromium (Cr) release cytotoxicity assays, used to measure ADCC and natural c y t o t o x i c i t y , have been published elsewhere (Eremin et al., 1977a, 1978a). CLA-4, a lymphoblastoid cell not lysed by phagocytic cells, was used as the target cell and l y m p h o c y t e c y t o t o x i c i t y was expressed in terms of S~Cr released. Killer (K) cell activity, responsible for ADCC, was measured 3--4 h after commencing the assay, whilst the natural killer (NK) cell, responsible for natural cytotoxicity was measured after an 18--24 h incubation period. Varying effector cell to target cell ratios were used: 50/1 and 25/1 in the case of blood, and 200/1 and 100/1 in the case of tonsil. Statistical analysis Percentage isotope released was calculated and assays were statistically analysed by analysis of variance on the logarithm of the percentage isotope released, using the Genstat computer package (Nelder, 1975). The significance of the results, at the 5% level, was determined using Duncan's multiple range test. RESULTS
L y m p h o c y t e preparation Following the standard preparation of blood and tonsil lymphoid cell suspensions, 98% of the cells were viable (phase contrast), containing 3% or less of monocytes and polymorphs. Pre-treatment of these lymphoid cell suspensions with deionised water and with a m m o n i u m chloride resulted in varying but comparable amounts of l y m p h o c y t e loss, usually less than 20%, the residual l y m p h o c y t e population being 98% viable (phase contrast). In the absence of foetal calf serum a much larger percentage of l y m p h o c y t e loss occurred with a m m o n i u m chloride treatment. L y m p h o c y t e markers No selective depletion of l y m p h o c y t e subpopulations occurred after treating the l y m p h o c y t e suspensions with a m m o n i u m chloride and with deionised w a t e r - - t h e T l y m p h o c y t e , Fc receptor and C3 receptor-bearing l y m p h o c y t e percentages being unaltered (see Table 1). A n tibody-dependen t cellular cy to toxicity (ADCC) As we have shown previously that K cell activity is minimal or absent in tonsil (Eremin et al., 1977a), data dealing with ADCC in blood only are presented here. It can be seen from Fig. 1 that there was a virtual abolition of ADCC after treatment with a m m o n i u m chloride at 20°C, but no altera-
TABLE 1 LACK O F E F F E C T O F T H E T R E A T M E N T S SUBPOPULATIONS Lymphocyte
Lymphocyte treatment
ON T H E D I F F E R E N T
LYMPHOCYTE
L y m p h o c y t e s u b p o p u l a t i o n s (%)
source
'F lymphocytes
Fc receptorbearing lymphocytes
CB receptorbearing lymphocytes
Blood
Standard preparation Deionised water I s o t o n i c a m m o n i u m chloride
66 68 62
31 29 28
2O 22 22
Tonsil
Standard preparation Deionised w a t e r Isotonic ammonium chloride
33 32 29
33 32 30
75 74 77
90 ~
go
80
80
70
70
~s
.
60 X
SO
5O
////#/
t.O
30
3O ~ ~ 0
20
20
f J
10
}:
o
10
~a
[Al m
!
!
25:1
50:1
0
[BI I
!
25:1
50:1
Lymphocyte- target celt ratio
Fig. 1. E f f e c t o f t r e a t m e n t w i t h a m m o n i u m c h l o r i d e a n d deionised w a t e r o n a n t i b o d y d e p e n d e n t cellular c y t o t o x i c i t y o f h u m a n b l o o d l y m p h o c y t e s against SlCr-labelled CLA-4 target cells c o a t e d w i t h h u m a n a n t i - H L A IgG. I n c u b a t i o n t i m e o f 3 h. U n t r e a t e d l y m p h o c y t e s (o c)); u n t r e a t e d l y m p h o c y t e s , b u t k e p t at 4°C for 45 rain (o o); l y m p h o c y t e s t r e a t e d w i t h i s o t o n i c a m m o n i u m c h l o r i d e at 20°C (:!-- -- ~ : . ) and at 4~C (x . . . . . x); l y m p h o c y t e s t r e a t e d w i t h deionised w a t e r (/,- . . . . . ;.). Statistically signific a n t r e d u c t i o n o f ADCC ( P < 0 . 0 5 ) w i t h a m m o n i u m c h l o r i d e t r e a t e d l y m p h o c y t e s at 20°C (A), (B) a n d at 4°C (B).
261 tion o f A D C C after t r e a t m e n t with deionised w a t e r at 20°C. If h o w e v e r , the e f f e c t o r l y m p h o c y t e s were p r e - t r e a t e d with a m m o n i u m chloride at 4°C, the r e d u c t i o n o f A D C C , a l t h o u g h significant, was n o t so m a r k e d . Fig. 4 shows t h a t after a 24 h i n c u b a t i o n period at 37°C, the l y m p h o c y t e s pre-treated with a m m o n i u m chloride at 4°C had fully recovered their K cell activity, whilst t h o s e pre-treated at 20°C, a l t h o u g h s h o w i n g p r o m i n e n t K cell activity, had n o t y e t fully recovered. This r e c o v e r y period was p r o l o n g e d f u r t h e r if the a m m o n i u m chloride treated l y m p h o c y t e s (4°C o r 20°C) were i n c u b a t e d at r o o m t e m p e r a t u r e (data n o t shown}.
90 ~
90
80
80
?0
70 6O
./.4/:INSJ
5O ~l
;i
i
I
"°
f
/
v /,//
/]
/ j
Q
/ n/
3O
30 20
20
'/
10
10
[AI I
!
0
25:1
[B] 50:1
0
!
!
25:1
50:1
Lymphocyte-target cell ratio
Fig. 2. Effect of treatment with ammonium chloride and deionised water on natural cytotoxicity of human blood lymphocytes against SlCr-labelled CLA-4 target cells. Incubation time of 18--24 h. Untreated lymphocytes (c~ ©); untreated lymphocytes, but kept at 4°C for 45 rains (v, ~); lymphocytes treated with isotonic ammonium chloride at 20°C ( L 1 - - - - ~ t : ] ) and at 4°C (x-- -- --x); lymphocytes treated with deionised water (/, . . . . . . A). Statistically significant reduction of natural cytotoxicity (P < 0.05) with ammonium chloride treated lymphocytes at 20°C (B), (A), except where marked NS -- 25/1(A).
262
Natural cytotoxicity Natural c y t o t o x i c i t y is s h o w n b y l y m p h o c y t e p r e p a r a t i o n s from b o t h b l o o d and tonsil ( E r e m i n et al., 1978a). T h e b l o o d NK cell appears to be similar t o the b l o o d K c e l l - - a n o n - t h y m u s - d e p e n d e n t , Ig-bearing l y m p h o c y t e with Fc r e c e p t o r s , b u t lacking C3 r e c e p t o r s (Eremin et al., 1977b, 1 9 7 8 b ) . T h e tonsil NK cell, on the o t h e r hand, appears to be a non-Igbearing T l y m p h o c y t e , lacking r e c e p t o r s for b o t h Fc and C3 (Eremin et al., 1 9 7 8 b ) . T h e e f f e c t o f the various t r e a t m e n t s will t h e r e f o r e be c o n s i d e r e d separately f o r b l o o d and tonsil l y m p h o c y t e preparations. Blood. F r o m Fig. 2 it can be seen t h a t natural c y t o t o x i c i t y is partially impaired after treating the b l o o d l y m p h o c y t e s with a m m o n i u m chloride at 20°C, b u t appears t o be u n a f f e c t e d if this t r e a t m e n t is carried o u t at 4°C.
60'
60
50
50 _
.-#%
i /'
257
© Elsevier/North-Holland Biomedical Press
ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY AND NATURAL CYTOTOXICITY: EFFECT OF PRE-TREATMENT OF HUMAN LYMPHOCYTES WITH DEIONISED WATER AND AMMONIUM CHLORIDE
O. EREMIN, J. ASHBY and D. PLUMB Division of Immunology, Department of Pathology, University of Cambridge. Cam bridge, ~LK.
(Received 15 May 1978, accepted 5 June 1978)
Deionised water is an efficient red blood cell lytic agent with no demonstrable deleterious effects on ADCC and natural cytotoxicity (blood and tonsil). Treatment with isotonic ammonium chloride, however, produces a significant reduction of both ADCC and natural cytotoxicity. This reduction is less pronounced if the treatment is carried out at 4°C and is temporary, recovery occurring over a 24 h period of incubation at 37°C.
INTRODUCTION Red b l o o d cells, c o n t a m i n a t i n g b o t h h u m a n and animal l e u k o c y t e preparations, have been r e m o v e d b y lysis with various lytic p r e p a r a t i o n s including deionised w a t e r { T h o m s o n et at., 1 9 6 6 ) , h y p o t o n i c saline (Fallon et at., 1 9 6 2 ; J a n o w s k y et at., 1 9 6 4 ) and a m m o n i u m c h l o r i d e (Agostini and Ideo, 1 9 6 5 , Boyle, 1 9 6 8 ; R o o s and Loos, 1970). A l t h o u g h l y m p h o c y t e d a m a g e and d e a t h o c c u r following such t r e a t m e n t s ( T h o m s o n et al., 1 9 6 6 ) , a f t e r removal o f the cell ghosts and debris the r e s u l t a n t l y m p h o i d cell suspensions c o n t a i n viable, motile cells ( J a n o w s k y et al., 1 9 6 4 ) , with a well preserved intracellular a r c h i t e c t u r e (Agostini and Ideo, 1 9 6 5 ) . L y m p h o c y t e s p r e p a r e d in such a m a n n e r have been used in various in vitro assays, such as m i x e d l y m p h o c y t e - c u l t u r e i n t e r a c t i o n s ( H o d e s and T e r r y , 1 9 7 4 ) , a n t i b o d y ~ d e p e n d e n t cellular c y t o t o x i c i t y (ADCC) (De Bracco et al., 1 9 7 6 ) , natural c y t o t o x i c i t y (Kiessling et al., 1 9 7 5 ) and in rosetting r e a c t i o n s ( E r e m i n et at., 1 9 7 7 b ) . Evidence has a p p e a r e d , h o w e v e r , suggesting an alteration o f ADCC following a m m o n i u m c h l o r i d e t r e a t m e n t ( K o v i t h a v o n g s et at., 1 9 7 4 ; K o d e r a and Bean, 1 9 7 5 ; Yust et al., 1976). T h e aim o f this p a p e r is to evaluate and c o m p a r e the e f f e c t on ADCC and natural c y t o t o x i c i t y o f pre-treating l y m p h o c y t e suspensions ( b l o o d and tonsil) with deionised w a t e r and with a m m o n i u m chloride. T h e evidence p r e s e n t e d shows t h a t a m m o n i u m c h l o r i d e t r e a t m e n t significantly reduces
258 both ADCC and natural c y t o t o x i c i t y whilst deionised water ha~ no demonstrable deleterious effect. MATERIALS AND METHODS
Standard lymphocyte preparation L y m p h o c y t e s were prepared as previously outlined (Eremin et al., 1976). Blood l y m p h o c y t e s were isolated from heparinised venous blood on a FicollH y p a q u e gradient (B¢yum, 1968), washed and made up in tissue culture medium RPMI 1640 (TCM), containing 100 IU penicillin/ml, 100 ~g streptomycin/ml, 0.7 g sodium bicarbonate/l, 25 mM Hepes buffer/ml and 10% heat-inactivated foetal calf serum. Tonsillar l y m p h o c y t e s were isolated from fresh operative specimens, washed and made up in TCM. Carbonyl iron was used to remove phagocytic cells. Treated lymphocyte preparation Deionised water. A b o u t 0.3 ml of deionised water (10 drops) was added to the l y m p h o c y t e pellet (3 X 107 cells), mixed and allowed to stand for 20 sec at 20°C {room temperature). A similar volume of twice concent rat ed phosphate-buffered saline was then added, followed by an excess volume of TCM. The red cell ghosts were removed by centrifuging at 100 X g for 3 min, twice. Isotonic ammonium chloride (KHCO3 0.01 M, NH4C1 0.15 M, 0.1 mM EDTA -- pH 7.4} *. 7 ml of a m m o n i u m chloride was added to the lymphocyte pellet (3 X 10 ~ cells), mixed and allowed to stand for 7 min. This was then neutralized with an excess volume of TCM and centrifuged to remove the red cell ghosts. This procedure was used as it had previously been found to give the most efficient (virtually total) removal of contaminating red blood c e l l s - - c o m p a r a b l e with deionised water. To minimize l y m p h o c y t e cell loss, with o ut diminishing the lytic capacity for red blood cells, heatinactivated foetal calf serum (5%) was added to the a m m o n i u m chloride solution just prior to use. The lytic process was carried out at 20°C and 4°C. Lymphocyte markers L y m p h o c y t e rosetting assays were carried out as previously described (Eremin et al., 1976}. For all rosetting tests the l y m p h o c y t e s were made up to 2 X 10~/ml in TCM. Sheep red cell rosettes were used as markers for T l y m p h o c y t e s . Fc receptor-bearing l y m p h o c y t e s were detected by opsonic adherence o f ox red blood cells, sensitized with a subagglutinating dose of rabbit IgG anti-ox red blood cell antiserum. L y m p h o c y t e s with receptors for the third c o m p o n e n t o f c o m p l e m e n t were detected by rosette formation
* NHaCI, ammonium chloride; KHC03, potassium hydrogen carbonate; EDTA, ethylenediaminetetraacetic acid.
259 with ox red blood cells sensitized with rabbit IgM anti-ox red blood cell antiserum and mouse complement.
Cytotoxicity assays The details of the Slchromium (Cr) release cytotoxicity assays, used to measure ADCC and natural c y t o t o x i c i t y , have been published elsewhere (Eremin et al., 1977a, 1978a). CLA-4, a lymphoblastoid cell not lysed by phagocytic cells, was used as the target cell and l y m p h o c y t e c y t o t o x i c i t y was expressed in terms of S~Cr released. Killer (K) cell activity, responsible for ADCC, was measured 3--4 h after commencing the assay, whilst the natural killer (NK) cell, responsible for natural cytotoxicity was measured after an 18--24 h incubation period. Varying effector cell to target cell ratios were used: 50/1 and 25/1 in the case of blood, and 200/1 and 100/1 in the case of tonsil. Statistical analysis Percentage isotope released was calculated and assays were statistically analysed by analysis of variance on the logarithm of the percentage isotope released, using the Genstat computer package (Nelder, 1975). The significance of the results, at the 5% level, was determined using Duncan's multiple range test. RESULTS
L y m p h o c y t e preparation Following the standard preparation of blood and tonsil lymphoid cell suspensions, 98% of the cells were viable (phase contrast), containing 3% or less of monocytes and polymorphs. Pre-treatment of these lymphoid cell suspensions with deionised water and with a m m o n i u m chloride resulted in varying but comparable amounts of l y m p h o c y t e loss, usually less than 20%, the residual l y m p h o c y t e population being 98% viable (phase contrast). In the absence of foetal calf serum a much larger percentage of l y m p h o c y t e loss occurred with a m m o n i u m chloride treatment. L y m p h o c y t e markers No selective depletion of l y m p h o c y t e subpopulations occurred after treating the l y m p h o c y t e suspensions with a m m o n i u m chloride and with deionised w a t e r - - t h e T l y m p h o c y t e , Fc receptor and C3 receptor-bearing l y m p h o c y t e percentages being unaltered (see Table 1). A n tibody-dependen t cellular cy to toxicity (ADCC) As we have shown previously that K cell activity is minimal or absent in tonsil (Eremin et al., 1977a), data dealing with ADCC in blood only are presented here. It can be seen from Fig. 1 that there was a virtual abolition of ADCC after treatment with a m m o n i u m chloride at 20°C, but no altera-
TABLE 1 LACK O F E F F E C T O F T H E T R E A T M E N T S SUBPOPULATIONS Lymphocyte
Lymphocyte treatment
ON T H E D I F F E R E N T
LYMPHOCYTE
L y m p h o c y t e s u b p o p u l a t i o n s (%)
source
'F lymphocytes
Fc receptorbearing lymphocytes
CB receptorbearing lymphocytes
Blood
Standard preparation Deionised water I s o t o n i c a m m o n i u m chloride
66 68 62
31 29 28
2O 22 22
Tonsil
Standard preparation Deionised w a t e r Isotonic ammonium chloride
33 32 29
33 32 30
75 74 77
90 ~
go
80
80
70
70
~s
.
60 X
SO
5O
////#/
t.O
30
3O ~ ~ 0
20
20
f J
10
}:
o
10
~a
[Al m
!
!
25:1
50:1
0
[BI I
!
25:1
50:1
Lymphocyte- target celt ratio
Fig. 1. E f f e c t o f t r e a t m e n t w i t h a m m o n i u m c h l o r i d e a n d deionised w a t e r o n a n t i b o d y d e p e n d e n t cellular c y t o t o x i c i t y o f h u m a n b l o o d l y m p h o c y t e s against SlCr-labelled CLA-4 target cells c o a t e d w i t h h u m a n a n t i - H L A IgG. I n c u b a t i o n t i m e o f 3 h. U n t r e a t e d l y m p h o c y t e s (o c)); u n t r e a t e d l y m p h o c y t e s , b u t k e p t at 4°C for 45 rain (o o); l y m p h o c y t e s t r e a t e d w i t h i s o t o n i c a m m o n i u m c h l o r i d e at 20°C (:!-- -- ~ : . ) and at 4~C (x . . . . . x); l y m p h o c y t e s t r e a t e d w i t h deionised w a t e r (/,- . . . . . ;.). Statistically signific a n t r e d u c t i o n o f ADCC ( P < 0 . 0 5 ) w i t h a m m o n i u m c h l o r i d e t r e a t e d l y m p h o c y t e s at 20°C (A), (B) a n d at 4°C (B).
261 tion o f A D C C after t r e a t m e n t with deionised w a t e r at 20°C. If h o w e v e r , the e f f e c t o r l y m p h o c y t e s were p r e - t r e a t e d with a m m o n i u m chloride at 4°C, the r e d u c t i o n o f A D C C , a l t h o u g h significant, was n o t so m a r k e d . Fig. 4 shows t h a t after a 24 h i n c u b a t i o n period at 37°C, the l y m p h o c y t e s pre-treated with a m m o n i u m chloride at 4°C had fully recovered their K cell activity, whilst t h o s e pre-treated at 20°C, a l t h o u g h s h o w i n g p r o m i n e n t K cell activity, had n o t y e t fully recovered. This r e c o v e r y period was p r o l o n g e d f u r t h e r if the a m m o n i u m chloride treated l y m p h o c y t e s (4°C o r 20°C) were i n c u b a t e d at r o o m t e m p e r a t u r e (data n o t shown}.
90 ~
90
80
80
?0
70 6O
./.4/:INSJ
5O ~l
;i
i
I
"°
f
/
v /,//
/]
/ j
Q
/ n/
3O
30 20
20
'/
10
10
[AI I
!
0
25:1
[B] 50:1
0
!
!
25:1
50:1
Lymphocyte-target cell ratio
Fig. 2. Effect of treatment with ammonium chloride and deionised water on natural cytotoxicity of human blood lymphocytes against SlCr-labelled CLA-4 target cells. Incubation time of 18--24 h. Untreated lymphocytes (c~ ©); untreated lymphocytes, but kept at 4°C for 45 rains (v, ~); lymphocytes treated with isotonic ammonium chloride at 20°C ( L 1 - - - - ~ t : ] ) and at 4°C (x-- -- --x); lymphocytes treated with deionised water (/, . . . . . . A). Statistically significant reduction of natural cytotoxicity (P < 0.05) with ammonium chloride treated lymphocytes at 20°C (B), (A), except where marked NS -- 25/1(A).
262
Natural cytotoxicity Natural c y t o t o x i c i t y is s h o w n b y l y m p h o c y t e p r e p a r a t i o n s from b o t h b l o o d and tonsil ( E r e m i n et al., 1978a). T h e b l o o d NK cell appears to be similar t o the b l o o d K c e l l - - a n o n - t h y m u s - d e p e n d e n t , Ig-bearing l y m p h o c y t e with Fc r e c e p t o r s , b u t lacking C3 r e c e p t o r s (Eremin et al., 1977b, 1 9 7 8 b ) . T h e tonsil NK cell, on the o t h e r hand, appears to be a non-Igbearing T l y m p h o c y t e , lacking r e c e p t o r s for b o t h Fc and C3 (Eremin et al., 1 9 7 8 b ) . T h e e f f e c t o f the various t r e a t m e n t s will t h e r e f o r e be c o n s i d e r e d separately f o r b l o o d and tonsil l y m p h o c y t e preparations. Blood. F r o m Fig. 2 it can be seen t h a t natural c y t o t o x i c i t y is partially impaired after treating the b l o o d l y m p h o c y t e s with a m m o n i u m chloride at 20°C, b u t appears t o be u n a f f e c t e d if this t r e a t m e n t is carried o u t at 4°C.
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