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Migration inhibition of normal rat thymocytes as an in vitro method for detecting cell-mediated immunity in rat and mouse
Journal o f l mmu n o l o g i c a l Methods, 51 (1982) 231--239
231
Elsevier Biomedical Press
M I G R A T I O N I N H I B I T I O N O F N O R M A L R A T T H Y M O C Y T E S AS A N IN V I T R O M E T H O D F O R D E T E C T I N G C E L L - M E D I A T E D I M M U N I T Y IN RAT AND MOUSE
SMITA TEWARI, M. SESHADRI and T.B. PODUVAL 1 Medical Group, Bhabha A t o m i c Research Centre, Mod. Labs., Trombay, Bombay 400085, India
(Received 12 August 1981, accepted 4 December 1981)
Normal rat thymocytes were used as a migrating population to study the effect of migration inhibition factor (MIF) released by sheep red blood cell (SRBC) immune lymphocytes. Normal thymocytes (migrating cells), SRBC (target antigen) and SRBC immune lymphocytes (effector cells) were packed in capillary tubes and their migration studied. This migration inhibition assay proved to be a very good in vitro correlate of specific delayed type hypersensitivity against SRBC, a particulate antigen, in rats. Normal rat thymocytes could also be used to study the specific immune reactivity of murine lymphocytes sensitised to SRBC. SRBC immune rat thymocytes functioned as both the migrating cell population and MIF producing cells. This assay permitted study of the interaction between thymocytes and lymphocytes from rats in producing enhanced inhibition of cellular migration. A major advantage of the technique is the possibility of assaying a large number of replicates from abundant normal thymocytes. Key words: rat thymocytes to measure M I F - - CMI measured by rat t h ym o cyt e migration
INTRODUCTION M a c r o p h a g e m i g r a t i o n i n h i b i t i o n studies e m p l o y i n g the capillary t u b e m e t h o d give results t h a t c o r r e l a t e with cell m e d i a t e d i m m u n i t y (CMI) in b o t h e x p e r i m e n t a l animals and man. (George and V a u g h a n , 1 9 6 2 ; B l o o m and B e n n e t t , 1 9 6 6 ; David, 1 9 6 6 ; T h o r ~ e t al., 1 9 6 8 ) . This test, h o w e v e r , w h i c h involves the assay o f m i g r a t i o n i n h i b i t i o n f a c t o r (MIF) p r o d u c e d b y stimulated l y m p h o c y t e s , has limited e x p e r i m e n t a l and clinical use because o f the t e c h n i c a l c o m p l e x i t y o f p e r f o r m i n g relatively few tests. A t t e m p t s to resolve these limitations have been m a d e . T w o - s t e p M I F assay, using s u p e r n a t a n t fluids f r o m l y m p h o i d cells c u l t u r e d in the presence o f the sensitising antigen, is p e r f o r m e d b y m e a s u r i n g M I F activity with guinea pig p e r i t o n e a l e x u d a t e
1 Correspondence to : T.B. Poduval, Medical Division, RBOH Section, Mod. Labs., Bhabha Atomic Research Centre, Trombay, Bombay 400085, India. 0022-1759/82/0000--O000/$02.75 © 1982 Elsevier Biomedical Press
232 cells as the migrating population (Thor et al., 1968). Another m e t h o d is a direct test, wherein migration inhibition experiments are performed with a mixture of lymphokine producing lymphoid cells and normal peritoneal exudate cells (PEC) in the presence of specific antigens (Rajapakse and Glynn, 1970; Marsman et al., 1972). PEC from sensitised animals contain both MIF producing lymphoid cells and migrating cells (Churchill et al., 1972; Neiburger and Youmans, 1973). SCborg and Benedixen (1967) have described a direct m e t h o d for the detection of cellular hypersensitivity in man using peripheral blood leucocytes. Similarly, a m e t h o d involving inhibition of migration of purified T cells has been used with success (Kowalczyk et al., 1981). Rat and mouse t h y m o c y t e s from specifically sensitised animals have been used both as migrating population and producers of MIF (Bakker et al., 1975; Hughes et al., 1980). We describe here a m e t h o d using normal rat t h y m o c y t e s as the migrating population incubated in the capillary along with sensitised lymphocytes and specific antigen sheep red blood cells (SRBC) to study the specific hypersensitive reaction in mice and rats. MATERIALS AND METHODS Female Wistar rats (8--12 weeks old) and male inbred Swiss mice (8--10 weeks old) were used.
Immunisation The day on which rats and mice were injected intraperitoneally with SRBC was designated day 0. On day 5, rats received 0.1 ml of 50% SRBC in one footpad and 0.1 ml saline in the contralateral one. Mice received similar treatment except for the volume (0.05 ml) of injected material. Normal mice and rats also received the footpad challenge. Footpad thickness was measured on day 6 and animals were killed the same day to prepare spleen and t h y m u s cell suspensions. The difference in the thickness of SRBC-injected footpads and of saline-injected footpads was taken as a measure of the specific hypersensitive reaction. Spleen and t h y m u s were removed aseptically and used as the source of effector cells. Cell suspension Spleen and t h y m u s were removed and placed in cold M-199 containing 100 U of penicillin/ml and 100 pg streptomycin/ml. A spleen cell suspension was prepared as reported earlier (Seshadri et al., 1979). The t h y m u s was placed on a nylon mesh in a petri dish containing M-199 and was gently pressed to obtain a single cell suspension. Erythrocytes were removed from the spleen cell suspension by the m e t h o d of BSyum (1968) on a Ficoll-Hypaque gradient, density 1.077 g/ml (Lymphoprep, Nyegaard, Oslo, Norway). Cell viability was checked by trypan blue dye exclusion. Preparations with more than 95% viability were used in the experiments.
233
Migration inhibition test T h y m o c y t e s (migrating population), SRBC or rabbit red blood cells (RRBC) and l y m p h o c y t e s ( ef f ect or cells which produce MIF) were mixed so as to provide a migrating t h y m o c y t e cell density of 80 X 106 cells/ml. The resulting cell suspension was mixed well and drawn into a capillary tube. The tubing e m p l o y e d was appr oxi m at el y 7 cm in length and had a volume of 75--80 pl. One end of the capillary was sealed with soft model clay. The capillaries were centrifuged (200 Xg, 3 min) at room t e m p e r a t u r e and cut just below the cell-liquid interface and m o u n t e d in plastic migration plates (Sterilin Code 3085), being fixed in place with silicone grease. Wells were filled with M-199 containing 10% FCS and antibiotics and covered with coverslips. The plates were incubated horizontally at 37°C. For each set of experiments a m i n i m u m of 6 capillaries were used. After 18 h incubation the migration patterns were projected at fixed magnification and traced on a graph paper. The migration index (MI) was calculated as follows: MI-
mean area of migration of t h y m o c y t e s + SRBC + immune l y m p h o c y t e s mean area o f migration of t h y m o c y t e s + SRBC + normal l y m p h o c y t e s
Statistical analysis was done with Student's t test and regression analysis. Preliminary experiments indicated a direct relationship between initial cell c o n c e n t r a t i o n and the migration area of t h y m o c y t e s . The optimal n u m b e r of t h y m o c y t e s yielding an appreciable migration area was in the range of 50-100 X 106/ml. It was n o t e d that the area of migration of t h y m o c y t e s alone was reduced if these cells were packed into capillaries m ore than 2 h after preparation. RESULTS
Effect o f SRBC and normal lymphocytes on thymocyte migration Initial experiments were designed to study the effect of various concentrations o f SRBC and normal l y m p h o c y t e s on t h y m o c y t e migration. Table 1 shows the effect of various concentrations of SRBC on t h y m o c y t e migration. Increase in SRBC numbers enhanced the area of cellular migration. In TABLE 1 E f f e c t o f S R B C c o n c e n t r a t i o n o n relative t h y m o c y t e m i g r a t i o n . Relative migration a SRBC/ml
(x 10 -6 )
0 1.0
100 1.0
200 1.47
400 1.94
a R e l a t i v e m i g r a t i o n refers t o t h e r a t i o o f m e a n area o f m i g r a t i o n o f t h y m o c y t e s in p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f S R B C t o t h e m e a n area o f m i g r a t i o n o f t h y m o c y t e s alone.
234 TABLE 2 Effect of normal lymphocytes on t h y m o c y t e migration. Source of splenocytes a
Relative migration b
Lymphoprep separated unseparated
0.95 0.22
a Cell density of splenocytes was 16 X 106/ml. b Relative migration refers to the ratio of mean area of migration of thymocyte-SRBC mixture in the presence of splenocytes to the mean area of migration of thymocyte-SRBC mixture alone. SRBC density was 400 X 106/ml.
subsequent experiments 400 X 106/ml of SRBC were used to pack the capill a r i e s as a s o u r c e o f t a r g e t cells. U n s e p a r a t e d n o r m a l s p l e n o c y t e s s i g n i f i c a n t l y i n h i b i t e d t h y m o c y t e m i g r a t i o n ( T a b l e 2).
Effect of varying effector cell concentration T w o e f f e c t o r c e l l c o n c e n t r a t i o n s , 8 X 1 0 6 / m l a n d 16 X 1 0 6 / m l , w e r e t e s t e d f o r s p e c i f i c m i g r a t i o n i n h i b i t i o n . I n h i b i t i o n a t 16 X 106, as r e f l e c t e d in t h e m i g r a t i o n i n d e x w a s s i g n i f i c a n t l y h i g h e r (P < 0 . 0 1 ) t h a n a t t h e l o w e r d o s e o f e f f e c t o r cells. A t t h e h i g h e r e f f e c t o r cell c o n c e n t r a t i o n , s p l e n o c y t e s f r o m RRBC immune rats also specifically inhibit migration when RRBC were used as t a r g e t cells ( T a b l e 3). L a t e r e x p e r i m e n t s w e r e c a r r i e d o u t w i t h 16 X 106/ ml of effector lymphocytes.
Specificity of migration inhibition R R B C w a s u s e d as t h e t a r g e t c e l l s a l o n g w i t h S R B C s e n s i t i s e d s p l e n i c l y m p h o c y t e s t o t e s t t h e s p e c i f i c i t y o f m i g r a t i o n i n h i b i t i o n . A s s e e n in T a b l e 4, a t various target cell densities RRBC did not inhibit cell migration. It was also
TABLE 3 Effect of effector cell number on migration index. Migration index Immune effector cells/ml
SRBC immune a RRBC immune a
(8 x 106 )
( 1 6 x 106 )
0.66 + 0.06 N.D.
0.31 -+ 0.04 0.41 + 0.03
b
a SRBC and RRBC were used at 400 X 106/ml with corresponding effector cells. The values are mean + S.E. of 3 different experiments, each consisting of 6 capillaries. b Statistically significant from 8 X 106 group (P < 0.01).
235 1.0 x
0'9 0'8
~ o
0.7 o.6
0-5 I
115 20
/
I
2-5 3 0 ]ncreose in Foot Pod Thickness (ram)
Fig. 1. Regression line of the relation b e t w e e n f o o t p a d swelling and migration index; the correlation coefficient is 0.99 and is statistically significant (P < 0.001).
noticed that SRBC immune rats did not exhibit delayed type hypersensitivity to RRBC as assessed by footpad swelling. Correlation between in vitro migration test and in vivo footpad swelling Experiments were designed to test the degree of correlation between the in vitro test and its in vivo CMI correlate, i.e., footpad swelling. Rats were immunised with various doses of SRBC (ranging from l 0 s to 109). On the fourth day after injection the rats were subjected to footpad swelling assay. The 24 h footpad swelling of each rat was measured, and the splenic lymohocytes of the same rat were used as a source of effector cell in a t h y m o c y t e
TABLE 4 Specificity of the t h y m o c y t e migration inhibition assay. Target
Migration index a
Increase in f o o t p a d thickness (mean -+ S . E . ) m m b
Target cell density ( x l 0 -6) A 100 200 400 SRBC RRBC
0.87 1.01
0.58 1.07
0.59 1.0
P-value 2.47 -+ 0.18 <1.0
<0.01 N.S. c
a Normal splenic l y m p h o c y t e s and SRBC i m m u n e splenic l y m p h o c y t e s were used at a cell density of 16 × 106/ml. b Refers to the values in SRBC i m m u n e rats injected with respective target cells. Statistical analysis (t test) was of the c o m p a r i s o n with results in a group of normal rats injected with the respective target cells. c N o t significant.
236 TABLE 5 Thymocyte as both effector and migrating population. Target cell density/ml
Migration index a
400 × 106
0.35 -+ 0.04
a Migration index is the ratio of mean area of migration of SRBC immune thymocytes and SRBC mixture to the mean area of migration of normal thymocytes and SRBC mixture.
m i g r a t i o n assay. Fig. 1 shows the regression line o f the relation b e t w e e n f o o t p a d swelling and m i g r a t i o n index. There was a very g o o d c o r r e l a t i o n (r = 0.99) b e t w e e n the 2 p a r a m e t e r s .
T h y m o c y t e as both effector and migrating cell population A cell suspension c o n t a i n i n g 80 X 106/ml o f t h y m o c y t e s f r o m an SRBC i m m u n e rat and 400 × 106/ml o f SRBC was p a c k e d into capillaries and m i g r a t i o n c o m p a r e d with t h a t f r o m capillaries c o n t a i n i n g t h e same n u m b e r s o f n o r m a l t h y m o c y t e s and SRBC. Significant m i g r a t i o n i n h i b i t i o n o f i m m u n e t h y m o c y t e s c o m p a r e d with n o r m a l t h y m o c y t e s was observed (Table 5).
Effect of source of lymphocytes on migration index I m m u n e t h y m i c l y m p h o c y t e s and i m m u n e splenic l y m p h o c y t e s were used separately as e f f e c t o r cells along with n o r m a l m i g r a t o r y t h y m o c y t e s and SRBC. T h e m i g r a t i o n i n d e x s h o w e d t h a t f o r a given n u m b e r o f e f f e c t o r cells, i m m u n e s p l e n o c y t e s inhibited the t h y m o c y t e m i g r a t i o n to a greater e x t e n t (P ~ 0 . 0 5 ) t h a n i m m u n e t h y m o c y t e s (Table 6).
Interaction between immune thymocytes and immune splenic lymphocytes N o r m a l and i m m u n e t h y m o c y t e s were separately used as t h e migrating cell p o p u l a t i o n . With each t y p e o f migrating p o p u l a t i o n , either n o r m a l or i m m u n e splenic l y m p h o c y t e s were used as t h e source o f e f f e c t o r cells. As
TABLE 6 Effect of effector cell source on migration index. Effector cells (16 x 106/ml)
Migration index (mean -+ S.E.)
Thymocytes Splenocytes
0.73 -+ 0.03 0.59 -+ 0.03 a
a Migration indices were significantly different between the groups (P < 0.05). SRBC were used at 400 x 106/ml.
237 TABLE 7 Interaction between immune thymic and splenic lymphocytes. Relative migration a Effector cell b
Normal spleen Immune spleen
Migrating cell Normal thymocytes
Immune thymocytes
1.0 0.6
0.35 0.12
a Migration area of normal thymocyte. Normal spleen cell mixture was taken as unity and other areas were expressed as fractions of this. b Effector cell concentration 16 × 106/ml, and SRBC 400 x 106/ml. TABLE 8 Rat thymocyte migration inhibition as an index of specific CMI m mouse. Treatment of effector cell donor mice
Increase in footpad thickness (ram) a (mean -+ S.E.)
Migration index b
Nil 10 s SRBC 2 × 10 s SRBC 4 x l 0 s SRBC
0.52 1.16 1.37 1.39
1.0 0.21 0.16 0.07
-+ 0.08 -+ 0.11 -+ 0.25 + 0.27
a Each group consists of 4 mice. Footpad assay was done as described in Materials and Methods. b Murine splenic lymphocyte was used as the effector cell at a density of 16 x 106/ml and the SRBC target cell density was 400 X 106/ml.
s e e n i n T a b l e 7, i m m u n e t h y m o c y t e s a n d i m m u n e s p l e n i c l y m p h o c y t e s c o m plement each other, together bringing about enhanced inhibition of cellular migration.
R a t t h y m o c y t e migration inhibition as an i n d e x o f specific CMI in m o u s e Rat thymocytes were mixed with murine SRBC immune lymphocytes ( 1 6 X 1 0 6 / m l ) a n d S R B C ( 4 0 0 × 1 0 6 / m l ) , a n d t h e m i g r a t i o n i n d e x w a s calculated. It was noted that murine immune lymphocytes specifically inhibited t h e m i g r a t i o n o f n o r m a l r a t t h y m o c y t e s ( T a b l e 8). DISCUSSION T h y m o c y t e m i g r a t i o n f r o m c a p i l l a r i e s h a s b e e n r e p o r t e d ( F r i e d m a n e t al., 1 9 6 9 ) . O u r a s s a y s y s t e m is b a s e d o n t h e i n t e r a c t i o n o f l y m p h o k i n e p r o d u c e d by specifically sensitised lymphocytes in the presence of antigen, with the
238 normal migrating t h y m o c y t e s . The main purpose of the present investigation was to check the possibility of using normal rat t h y m o c y t e s as the migrating population in a direct assay system, to monitor the specific CMI response against SRBC and also to characterise some of the requirements for optimal migration. In the present investigation optimal SRBC and l y m p h o c y t e numbers were determined. The assay is reproducible and correlates well with an in vivo parameter of CMI, footpad swelling in response to the particulate antigen SRBC. This method was also used successfully to assess specific CMI in mice. The system is equally applicable to another particulate antigen, RRBC. A major advantage of the technique is the possibility of assaying a large number of replicates with abundant normal thymocytes. The assay also obviates the necessity of using an indirect assay in which MIF has to be generated separately. Peritoneal exudate cells (PEC) induced in sensitised mice have been used in the direct test but have the disadvantage of numerical variation and cell heterogeneity (Neiburger and Youmans, 1973). In contrast, normal t h y m o c y t e s are not subject to numerical variation, and are a much more homogeneous population. It was observed that both immune t h y m o c y t e s and immune splenocytes can act as effector cell in generating the soluble mediator capable of inhibiting t h y m o c y t e migration. Our studies confirm the findings of Bakker et al. (1975) that the t h y m u s does contain effector cells capable of releasing immunoregulators of specific CMI. The quantitative difference in the inhibitor response of t h y m o c y t e s and splenocytes (Table 6) depends on the composition of MIF producing lymphoid cells and migrating cells as well as the interaction of MIF with the migrating cell population. The use of a normal migrating cell population offers the possibility of quantitating and characterising the MIF producing cells. Further, the use of normal t h y m o c y t e s has the advantage of identifying separately the MIF producing cells and the migrating population. This aids study of the interaction between the 2 cell types. The heightened inhibition of migration of immune t h y m o c y t e s in the presence of immune lymphocytes suggests the possibility of collaborative interaction between these cell types. Production of t h y m o c y t e stimulating factor by sensitised l y m p h o c y t e and specific antigen has been reported in mice (Giovanni et al., 1978) and can lead to amplification of the immune response. Migration inhibition experiments with mixtures of human peripheral blood l y m p h o c y t e and guinea pig PEC have been used for both indirect and direct tests (Thor et al., 1968; Rajapakse and Glynn, 1970; Marsman et al., 1972). In view of this, it may be possible to use normal rat t h y m o c y t e s as a migrating cell population to study the lymphokines released by sensitised h u m a n lymphocytes in the presence of antigen capable of inhibiting rat t h y m o c y t e s . Further, the assay may allow the use of soluble antigen-tagged SRBC as the target cell to monitor specific CMI directed against the antigen tagged. While the exact nature of the soluble mediator which inhibits t h y m o c y t e
239 m i g r a t i o n in o u r s y s t e m r e m a i n s t o be characterised, t h e t e c h n i q u e described herein offers a simple, rapid and r e p r o d u c i b l e m e t h o d f o r assessment o f CMI in t h e m o u s e a n d rat. ACKNOWLEDGEMENT T h e a u t h o r s wish to t h a n k M/s Nyegaaxd and Co. AS, Oslo, f o r their kind gift o f L y m p h o p r e p . REFERENCES Bakker, W.W., J.J. Engelhart, I. Mulder and P.J. Hoedemaeker, 1975, Int. Arch. Allergy Appl. Immunol. 49,491. Bloom, B. and B. Bennett, 1966, Science 153, 80. BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77. Churchill, W.H., B. Zbar, J.A. Belli and J.R. David, 1972, J. Natl. Cancer Inst. 48,541. David, J.R., 1966, Proc. Natl. Acad. Sci. U.S.A. 56, 72. Friedman, H., M. Sanz, C. Combe, L. Mills and Y. Lee, 1969, Proc. Soc. Exp. Biol. Med. 132,849. George, M. and J.H. Vaughan, 1962, Proc. Soc. Exp. Biol. Med. 111,514. Giovanni, D.S., D.-M. Chen and M. Altin, 1978, Cell. Immunol. 35,340. Hughes, L., A.W. Preece and P.A. Light, 1980, Imniunol. Lett. 1,269. Kowalczyk, D., M. Zembala and E. Litwora, 1981, Cell. Immunol. 57,361. Marsman, A.J.W., M.V.D. Hart, C. Walig and V.P. Eijsvoogel, 1972, Eur. J. Immunol. 2, 546. Neiburger, R.G. and G.P. Youmans, 1973, Infect. Immun. 7,190. Rajapakse, D.A. and L.E. Glynn, 1970, Nature 226,857. Seshadri, M., T.B. Poduval and K. Sundaram, 1979, J. Natl. Cancer Inst. 63, 1205. S~bborg, M. and G. Benedixen, 1967, Acta Med. Scand. 181,247. Thor, D.E., R.E. Jureziz, S.R. Veach, E. Miller and D. Sheldon, 1968, Nature 219,755.
231
Elsevier Biomedical Press
M I G R A T I O N I N H I B I T I O N O F N O R M A L R A T T H Y M O C Y T E S AS A N IN V I T R O M E T H O D F O R D E T E C T I N G C E L L - M E D I A T E D I M M U N I T Y IN RAT AND MOUSE
SMITA TEWARI, M. SESHADRI and T.B. PODUVAL 1 Medical Group, Bhabha A t o m i c Research Centre, Mod. Labs., Trombay, Bombay 400085, India
(Received 12 August 1981, accepted 4 December 1981)
Normal rat thymocytes were used as a migrating population to study the effect of migration inhibition factor (MIF) released by sheep red blood cell (SRBC) immune lymphocytes. Normal thymocytes (migrating cells), SRBC (target antigen) and SRBC immune lymphocytes (effector cells) were packed in capillary tubes and their migration studied. This migration inhibition assay proved to be a very good in vitro correlate of specific delayed type hypersensitivity against SRBC, a particulate antigen, in rats. Normal rat thymocytes could also be used to study the specific immune reactivity of murine lymphocytes sensitised to SRBC. SRBC immune rat thymocytes functioned as both the migrating cell population and MIF producing cells. This assay permitted study of the interaction between thymocytes and lymphocytes from rats in producing enhanced inhibition of cellular migration. A major advantage of the technique is the possibility of assaying a large number of replicates from abundant normal thymocytes. Key words: rat thymocytes to measure M I F - - CMI measured by rat t h ym o cyt e migration
INTRODUCTION M a c r o p h a g e m i g r a t i o n i n h i b i t i o n studies e m p l o y i n g the capillary t u b e m e t h o d give results t h a t c o r r e l a t e with cell m e d i a t e d i m m u n i t y (CMI) in b o t h e x p e r i m e n t a l animals and man. (George and V a u g h a n , 1 9 6 2 ; B l o o m and B e n n e t t , 1 9 6 6 ; David, 1 9 6 6 ; T h o r ~ e t al., 1 9 6 8 ) . This test, h o w e v e r , w h i c h involves the assay o f m i g r a t i o n i n h i b i t i o n f a c t o r (MIF) p r o d u c e d b y stimulated l y m p h o c y t e s , has limited e x p e r i m e n t a l and clinical use because o f the t e c h n i c a l c o m p l e x i t y o f p e r f o r m i n g relatively few tests. A t t e m p t s to resolve these limitations have been m a d e . T w o - s t e p M I F assay, using s u p e r n a t a n t fluids f r o m l y m p h o i d cells c u l t u r e d in the presence o f the sensitising antigen, is p e r f o r m e d b y m e a s u r i n g M I F activity with guinea pig p e r i t o n e a l e x u d a t e
1 Correspondence to : T.B. Poduval, Medical Division, RBOH Section, Mod. Labs., Bhabha Atomic Research Centre, Trombay, Bombay 400085, India. 0022-1759/82/0000--O000/$02.75 © 1982 Elsevier Biomedical Press
232 cells as the migrating population (Thor et al., 1968). Another m e t h o d is a direct test, wherein migration inhibition experiments are performed with a mixture of lymphokine producing lymphoid cells and normal peritoneal exudate cells (PEC) in the presence of specific antigens (Rajapakse and Glynn, 1970; Marsman et al., 1972). PEC from sensitised animals contain both MIF producing lymphoid cells and migrating cells (Churchill et al., 1972; Neiburger and Youmans, 1973). SCborg and Benedixen (1967) have described a direct m e t h o d for the detection of cellular hypersensitivity in man using peripheral blood leucocytes. Similarly, a m e t h o d involving inhibition of migration of purified T cells has been used with success (Kowalczyk et al., 1981). Rat and mouse t h y m o c y t e s from specifically sensitised animals have been used both as migrating population and producers of MIF (Bakker et al., 1975; Hughes et al., 1980). We describe here a m e t h o d using normal rat t h y m o c y t e s as the migrating population incubated in the capillary along with sensitised lymphocytes and specific antigen sheep red blood cells (SRBC) to study the specific hypersensitive reaction in mice and rats. MATERIALS AND METHODS Female Wistar rats (8--12 weeks old) and male inbred Swiss mice (8--10 weeks old) were used.
Immunisation The day on which rats and mice were injected intraperitoneally with SRBC was designated day 0. On day 5, rats received 0.1 ml of 50% SRBC in one footpad and 0.1 ml saline in the contralateral one. Mice received similar treatment except for the volume (0.05 ml) of injected material. Normal mice and rats also received the footpad challenge. Footpad thickness was measured on day 6 and animals were killed the same day to prepare spleen and t h y m u s cell suspensions. The difference in the thickness of SRBC-injected footpads and of saline-injected footpads was taken as a measure of the specific hypersensitive reaction. Spleen and t h y m u s were removed aseptically and used as the source of effector cells. Cell suspension Spleen and t h y m u s were removed and placed in cold M-199 containing 100 U of penicillin/ml and 100 pg streptomycin/ml. A spleen cell suspension was prepared as reported earlier (Seshadri et al., 1979). The t h y m u s was placed on a nylon mesh in a petri dish containing M-199 and was gently pressed to obtain a single cell suspension. Erythrocytes were removed from the spleen cell suspension by the m e t h o d of BSyum (1968) on a Ficoll-Hypaque gradient, density 1.077 g/ml (Lymphoprep, Nyegaard, Oslo, Norway). Cell viability was checked by trypan blue dye exclusion. Preparations with more than 95% viability were used in the experiments.
233
Migration inhibition test T h y m o c y t e s (migrating population), SRBC or rabbit red blood cells (RRBC) and l y m p h o c y t e s ( ef f ect or cells which produce MIF) were mixed so as to provide a migrating t h y m o c y t e cell density of 80 X 106 cells/ml. The resulting cell suspension was mixed well and drawn into a capillary tube. The tubing e m p l o y e d was appr oxi m at el y 7 cm in length and had a volume of 75--80 pl. One end of the capillary was sealed with soft model clay. The capillaries were centrifuged (200 Xg, 3 min) at room t e m p e r a t u r e and cut just below the cell-liquid interface and m o u n t e d in plastic migration plates (Sterilin Code 3085), being fixed in place with silicone grease. Wells were filled with M-199 containing 10% FCS and antibiotics and covered with coverslips. The plates were incubated horizontally at 37°C. For each set of experiments a m i n i m u m of 6 capillaries were used. After 18 h incubation the migration patterns were projected at fixed magnification and traced on a graph paper. The migration index (MI) was calculated as follows: MI-
mean area of migration of t h y m o c y t e s + SRBC + immune l y m p h o c y t e s mean area o f migration of t h y m o c y t e s + SRBC + normal l y m p h o c y t e s
Statistical analysis was done with Student's t test and regression analysis. Preliminary experiments indicated a direct relationship between initial cell c o n c e n t r a t i o n and the migration area of t h y m o c y t e s . The optimal n u m b e r of t h y m o c y t e s yielding an appreciable migration area was in the range of 50-100 X 106/ml. It was n o t e d that the area of migration of t h y m o c y t e s alone was reduced if these cells were packed into capillaries m ore than 2 h after preparation. RESULTS
Effect o f SRBC and normal lymphocytes on thymocyte migration Initial experiments were designed to study the effect of various concentrations o f SRBC and normal l y m p h o c y t e s on t h y m o c y t e migration. Table 1 shows the effect of various concentrations of SRBC on t h y m o c y t e migration. Increase in SRBC numbers enhanced the area of cellular migration. In TABLE 1 E f f e c t o f S R B C c o n c e n t r a t i o n o n relative t h y m o c y t e m i g r a t i o n . Relative migration a SRBC/ml
(x 10 -6 )
0 1.0
100 1.0
200 1.47
400 1.94
a R e l a t i v e m i g r a t i o n refers t o t h e r a t i o o f m e a n area o f m i g r a t i o n o f t h y m o c y t e s in p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s o f S R B C t o t h e m e a n area o f m i g r a t i o n o f t h y m o c y t e s alone.
234 TABLE 2 Effect of normal lymphocytes on t h y m o c y t e migration. Source of splenocytes a
Relative migration b
Lymphoprep separated unseparated
0.95 0.22
a Cell density of splenocytes was 16 X 106/ml. b Relative migration refers to the ratio of mean area of migration of thymocyte-SRBC mixture in the presence of splenocytes to the mean area of migration of thymocyte-SRBC mixture alone. SRBC density was 400 X 106/ml.
subsequent experiments 400 X 106/ml of SRBC were used to pack the capill a r i e s as a s o u r c e o f t a r g e t cells. U n s e p a r a t e d n o r m a l s p l e n o c y t e s s i g n i f i c a n t l y i n h i b i t e d t h y m o c y t e m i g r a t i o n ( T a b l e 2).
Effect of varying effector cell concentration T w o e f f e c t o r c e l l c o n c e n t r a t i o n s , 8 X 1 0 6 / m l a n d 16 X 1 0 6 / m l , w e r e t e s t e d f o r s p e c i f i c m i g r a t i o n i n h i b i t i o n . I n h i b i t i o n a t 16 X 106, as r e f l e c t e d in t h e m i g r a t i o n i n d e x w a s s i g n i f i c a n t l y h i g h e r (P < 0 . 0 1 ) t h a n a t t h e l o w e r d o s e o f e f f e c t o r cells. A t t h e h i g h e r e f f e c t o r cell c o n c e n t r a t i o n , s p l e n o c y t e s f r o m RRBC immune rats also specifically inhibit migration when RRBC were used as t a r g e t cells ( T a b l e 3). L a t e r e x p e r i m e n t s w e r e c a r r i e d o u t w i t h 16 X 106/ ml of effector lymphocytes.
Specificity of migration inhibition R R B C w a s u s e d as t h e t a r g e t c e l l s a l o n g w i t h S R B C s e n s i t i s e d s p l e n i c l y m p h o c y t e s t o t e s t t h e s p e c i f i c i t y o f m i g r a t i o n i n h i b i t i o n . A s s e e n in T a b l e 4, a t various target cell densities RRBC did not inhibit cell migration. It was also
TABLE 3 Effect of effector cell number on migration index. Migration index Immune effector cells/ml
SRBC immune a RRBC immune a
(8 x 106 )
( 1 6 x 106 )
0.66 + 0.06 N.D.
0.31 -+ 0.04 0.41 + 0.03
b
a SRBC and RRBC were used at 400 X 106/ml with corresponding effector cells. The values are mean + S.E. of 3 different experiments, each consisting of 6 capillaries. b Statistically significant from 8 X 106 group (P < 0.01).
235 1.0 x
0'9 0'8
~ o
0.7 o.6
0-5 I
115 20
/
I
2-5 3 0 ]ncreose in Foot Pod Thickness (ram)
Fig. 1. Regression line of the relation b e t w e e n f o o t p a d swelling and migration index; the correlation coefficient is 0.99 and is statistically significant (P < 0.001).
noticed that SRBC immune rats did not exhibit delayed type hypersensitivity to RRBC as assessed by footpad swelling. Correlation between in vitro migration test and in vivo footpad swelling Experiments were designed to test the degree of correlation between the in vitro test and its in vivo CMI correlate, i.e., footpad swelling. Rats were immunised with various doses of SRBC (ranging from l 0 s to 109). On the fourth day after injection the rats were subjected to footpad swelling assay. The 24 h footpad swelling of each rat was measured, and the splenic lymohocytes of the same rat were used as a source of effector cell in a t h y m o c y t e
TABLE 4 Specificity of the t h y m o c y t e migration inhibition assay. Target
Migration index a
Increase in f o o t p a d thickness (mean -+ S . E . ) m m b
Target cell density ( x l 0 -6) A 100 200 400 SRBC RRBC
0.87 1.01
0.58 1.07
0.59 1.0
P-value 2.47 -+ 0.18 <1.0
<0.01 N.S. c
a Normal splenic l y m p h o c y t e s and SRBC i m m u n e splenic l y m p h o c y t e s were used at a cell density of 16 × 106/ml. b Refers to the values in SRBC i m m u n e rats injected with respective target cells. Statistical analysis (t test) was of the c o m p a r i s o n with results in a group of normal rats injected with the respective target cells. c N o t significant.
236 TABLE 5 Thymocyte as both effector and migrating population. Target cell density/ml
Migration index a
400 × 106
0.35 -+ 0.04
a Migration index is the ratio of mean area of migration of SRBC immune thymocytes and SRBC mixture to the mean area of migration of normal thymocytes and SRBC mixture.
m i g r a t i o n assay. Fig. 1 shows the regression line o f the relation b e t w e e n f o o t p a d swelling and m i g r a t i o n index. There was a very g o o d c o r r e l a t i o n (r = 0.99) b e t w e e n the 2 p a r a m e t e r s .
T h y m o c y t e as both effector and migrating cell population A cell suspension c o n t a i n i n g 80 X 106/ml o f t h y m o c y t e s f r o m an SRBC i m m u n e rat and 400 × 106/ml o f SRBC was p a c k e d into capillaries and m i g r a t i o n c o m p a r e d with t h a t f r o m capillaries c o n t a i n i n g t h e same n u m b e r s o f n o r m a l t h y m o c y t e s and SRBC. Significant m i g r a t i o n i n h i b i t i o n o f i m m u n e t h y m o c y t e s c o m p a r e d with n o r m a l t h y m o c y t e s was observed (Table 5).
Effect of source of lymphocytes on migration index I m m u n e t h y m i c l y m p h o c y t e s and i m m u n e splenic l y m p h o c y t e s were used separately as e f f e c t o r cells along with n o r m a l m i g r a t o r y t h y m o c y t e s and SRBC. T h e m i g r a t i o n i n d e x s h o w e d t h a t f o r a given n u m b e r o f e f f e c t o r cells, i m m u n e s p l e n o c y t e s inhibited the t h y m o c y t e m i g r a t i o n to a greater e x t e n t (P ~ 0 . 0 5 ) t h a n i m m u n e t h y m o c y t e s (Table 6).
Interaction between immune thymocytes and immune splenic lymphocytes N o r m a l and i m m u n e t h y m o c y t e s were separately used as t h e migrating cell p o p u l a t i o n . With each t y p e o f migrating p o p u l a t i o n , either n o r m a l or i m m u n e splenic l y m p h o c y t e s were used as t h e source o f e f f e c t o r cells. As
TABLE 6 Effect of effector cell source on migration index. Effector cells (16 x 106/ml)
Migration index (mean -+ S.E.)
Thymocytes Splenocytes
0.73 -+ 0.03 0.59 -+ 0.03 a
a Migration indices were significantly different between the groups (P < 0.05). SRBC were used at 400 x 106/ml.
237 TABLE 7 Interaction between immune thymic and splenic lymphocytes. Relative migration a Effector cell b
Normal spleen Immune spleen
Migrating cell Normal thymocytes
Immune thymocytes
1.0 0.6
0.35 0.12
a Migration area of normal thymocyte. Normal spleen cell mixture was taken as unity and other areas were expressed as fractions of this. b Effector cell concentration 16 × 106/ml, and SRBC 400 x 106/ml. TABLE 8 Rat thymocyte migration inhibition as an index of specific CMI m mouse. Treatment of effector cell donor mice
Increase in footpad thickness (ram) a (mean -+ S.E.)
Migration index b
Nil 10 s SRBC 2 × 10 s SRBC 4 x l 0 s SRBC
0.52 1.16 1.37 1.39
1.0 0.21 0.16 0.07
-+ 0.08 -+ 0.11 -+ 0.25 + 0.27
a Each group consists of 4 mice. Footpad assay was done as described in Materials and Methods. b Murine splenic lymphocyte was used as the effector cell at a density of 16 x 106/ml and the SRBC target cell density was 400 X 106/ml.
s e e n i n T a b l e 7, i m m u n e t h y m o c y t e s a n d i m m u n e s p l e n i c l y m p h o c y t e s c o m plement each other, together bringing about enhanced inhibition of cellular migration.
R a t t h y m o c y t e migration inhibition as an i n d e x o f specific CMI in m o u s e Rat thymocytes were mixed with murine SRBC immune lymphocytes ( 1 6 X 1 0 6 / m l ) a n d S R B C ( 4 0 0 × 1 0 6 / m l ) , a n d t h e m i g r a t i o n i n d e x w a s calculated. It was noted that murine immune lymphocytes specifically inhibited t h e m i g r a t i o n o f n o r m a l r a t t h y m o c y t e s ( T a b l e 8). DISCUSSION T h y m o c y t e m i g r a t i o n f r o m c a p i l l a r i e s h a s b e e n r e p o r t e d ( F r i e d m a n e t al., 1 9 6 9 ) . O u r a s s a y s y s t e m is b a s e d o n t h e i n t e r a c t i o n o f l y m p h o k i n e p r o d u c e d by specifically sensitised lymphocytes in the presence of antigen, with the
238 normal migrating t h y m o c y t e s . The main purpose of the present investigation was to check the possibility of using normal rat t h y m o c y t e s as the migrating population in a direct assay system, to monitor the specific CMI response against SRBC and also to characterise some of the requirements for optimal migration. In the present investigation optimal SRBC and l y m p h o c y t e numbers were determined. The assay is reproducible and correlates well with an in vivo parameter of CMI, footpad swelling in response to the particulate antigen SRBC. This method was also used successfully to assess specific CMI in mice. The system is equally applicable to another particulate antigen, RRBC. A major advantage of the technique is the possibility of assaying a large number of replicates with abundant normal thymocytes. The assay also obviates the necessity of using an indirect assay in which MIF has to be generated separately. Peritoneal exudate cells (PEC) induced in sensitised mice have been used in the direct test but have the disadvantage of numerical variation and cell heterogeneity (Neiburger and Youmans, 1973). In contrast, normal t h y m o c y t e s are not subject to numerical variation, and are a much more homogeneous population. It was observed that both immune t h y m o c y t e s and immune splenocytes can act as effector cell in generating the soluble mediator capable of inhibiting t h y m o c y t e migration. Our studies confirm the findings of Bakker et al. (1975) that the t h y m u s does contain effector cells capable of releasing immunoregulators of specific CMI. The quantitative difference in the inhibitor response of t h y m o c y t e s and splenocytes (Table 6) depends on the composition of MIF producing lymphoid cells and migrating cells as well as the interaction of MIF with the migrating cell population. The use of a normal migrating cell population offers the possibility of quantitating and characterising the MIF producing cells. Further, the use of normal t h y m o c y t e s has the advantage of identifying separately the MIF producing cells and the migrating population. This aids study of the interaction between the 2 cell types. The heightened inhibition of migration of immune t h y m o c y t e s in the presence of immune lymphocytes suggests the possibility of collaborative interaction between these cell types. Production of t h y m o c y t e stimulating factor by sensitised l y m p h o c y t e and specific antigen has been reported in mice (Giovanni et al., 1978) and can lead to amplification of the immune response. Migration inhibition experiments with mixtures of human peripheral blood l y m p h o c y t e and guinea pig PEC have been used for both indirect and direct tests (Thor et al., 1968; Rajapakse and Glynn, 1970; Marsman et al., 1972). In view of this, it may be possible to use normal rat t h y m o c y t e s as a migrating cell population to study the lymphokines released by sensitised h u m a n lymphocytes in the presence of antigen capable of inhibiting rat t h y m o c y t e s . Further, the assay may allow the use of soluble antigen-tagged SRBC as the target cell to monitor specific CMI directed against the antigen tagged. While the exact nature of the soluble mediator which inhibits t h y m o c y t e
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