378
Specialia
Table III. Response of C57B1/6 mouse thymus cells separated on BSA diseontinucms gradients to LPS CelI fraction Control ~
Band A
BandB
BandC
BandD
Pellet
Unstimulated cells (counts/rain)
Stimulated ceils (counts/rain)
Stimulation index
370 zt= 49 u 116~c 09 224~: 04 1522 ~ 225 4405 ~ 359 702 ~= 99 253 ~ 19 158 :~= 20 166 2~ 35 93 ~ 18 48 ~_~ 06 173 ~ 13 1 3 6 i 24 8 4 i 11 249 ~ 52
398 4- 50 173 ~ 44 243 ~ 30 1336 -t- 108 1994 =E 451 295 ~ 62 528 4- 15 311 J= 78 395 4- 32 99 4- 12 74 =/= 15 221 -4- 40 167 q- 30 89 j= 22 184 -4- 29
1.08 1.48 1.08 0.88 0.45 0.42 2.09 1.96 2.38 1.09 1.55 1.28 1.23
276 ~ 105 160 ~ 21 129 ~c 14
192 ~ 07 159-4- 48 124 -1- 08
0.69 0.99 1.09
1.05 0.74
Nonseparated thymoeytes, b Data for 3 individual experiments is given. Values listed are the mean epm of 4 replica tubes plus the standard deviation.
f r o m b a n d I3 e x h i b i t e d t h e g r e a t e s t m i t o g e n i c response. T h e m i t o g e n i c r e s p o n s e s of n o n - s e p a r a t e d cells w a s h i g h e r t h a n t h e r e s p o n s e s of b a n d s A, C, D, or t h e pellet. T h y m u s cell s u b p o p u l a t i o n s were s t i m u l a t e d w i t h t h e t h y m u s i n d e p e n d e n t m i t o g e n E. colt L P S (10 ~g). T h i s d a t a (Table I I I ) shows t h a t cells f r o m b a n d 13 e x h i b i t e d t h e h i g h e s t degree of s t i m u l a t i o n . Cells ill t h e o t h e r b a n d s a n d p e l l e t did n o t r e s p o n d to L P S . T h e cell s u b p o p u l a t i o n i s o l a t e d f r o m t h e lowest d e n s i t y a r e a of t h e g r a d i e n t ( b a n d A) was r i c h i n large b l a s t - l i k e cells d i s p l a y i n g a n a b u n d a n t a m o u n t of c y t o p l a s m a n d large r o u n d nuclei. T h e s e cells were n o t s t i m u l a t e d b y a n y of t h e m i t o g e n s b u t d i d d i s p l a y t h e h i g h e s t b a s a l u p t a k e of t r i t i a t e d t h y m i d i n e (Tables, I, I I I ) w h e n i n c u b a t e d w i t h o u t m i t o g e n . D u e t o t h e h i g h e r level of t r i t i a t e d t h y m i d i n e u p t a k e in t h i s cell p o p u l a t i o n i t is felt t h a t t h e s e cells c o r r e s p o n d to t h e h i g h l y m i t o t i c ceils of t h e o u t e r c o r t e x t of t h e i n t a c t t h y m u s ~t. T h e r e was a s i g n i f i c a n t e n r i c h m e n t of cells in b a n d ]3 r e s p o n d i n g to m i t o g e n i c s t i m u l a t i o n w h e n c o m p a r e d to t h e o t h e r b a n d s a n d u n s e p a r a t e d cells. T h i s r e a c t i v e s u b p o p u l a t i o n w a s not, h o w e v e r , m o r p h o l o g i c a l l y h o m o g enous. I t is n o t clear a t t h e p r e s e n t t i m e w h i c h of t h e
EXPERIEN*TIA31/3
m o r p h o l o g i c a l cell t y p e s r e s p o n d to t h e m i t o g e n s . P r e v i o u s investigations have shown that cortisone resistant t h y m o c y t e s are r e s p o n s i v e t o P H A a n d Con A. Therefore, it is p r o b a b l e t h a t t h e cells i s o l a t e d f r o m b a n d B correspond to the cortisone resistant immunologically c o m p e t e n t cells of t h e t h y m u s m e d u l l a ~2. T h e m o r p h o logical h e t e r o g e n e i t y of t h i s r e a c t i v e s u b p o p u l a t i o n m a y b e d u e t o c o n t a m i n a t i o n w i t k n o n r e a c t i n g ceils. H o w e v e r , a m o r e p l a u s i b l e e x p l a n a t i o n w o u l d be t h a t t h i s s u b p o p u l a t i o n c o n t a i n s cells t h a t are in v a r i o u s stages of i m m u n o logical m a t u r a t i o n a n d t h u s a p p e a r m o r p h o l o g i c a l l y dissimilar. O u r d a t a (Table I I I ) show a s m a l l n u m b e r of t h y m u s cells i s o l a t e d f r o m b a n d B t h a t are c a p a b l e of r e s p o n d i n g t o t h e t h y m u s i n d e p e n d e n t m i t o g e n E. coli L P S . H o w e v e r , u n s e p a r a t e d t h y m u s cells e x h i b i t e d no m i t o g e n i c r e s p o n s e to L P S , w h i c h is in a g r e e m e n t w i t h t h e d a t a of AI~DERSSON et al. ~a. D e n s i t y g r a d i e n t s e p a r a t i o n of t h y m u s cells allowed t h e i s o l a t i o n of m i t o g e n r e s p o n s i v e cells i n a single s u b p o p u l a t i o n . S p e c u l a t i o n on t h e f u n c t i o n a l i t y of t h e t h y m u s cells r e s p o n d i n g t o L P S m a y lead t o t h e following s u p p o s i t i o n s . First, t h e cells m a y b e t3 cells i n v o l v e d in h u m o r a l i m m u n i t y a n d c o r r e s p o n d t o t h e a n t i b o d y p r o d u c i n g cells d e s c r i b e d b y VITETTA et al. ~4 O n t h e o t h e r h a n d t h e y m a y r e p r e s e n t 13 cells t h a t are d e s t i n e d t o b e c o m e T cells u n d e r t h e i n f l u e n c e of t h e t h y m u s . A t h i r d p o s s i b i l i t y is t h a t t h e L P S r e a c t i v e cells are c o n t a m i n a n t s f r o m t h e circulation.
Zusammen/assung. F r a k t i o n e n y o n T h y m u s l y m p h o z y t e n d e r Maus, die i m d i s k o n t i n u i e r l i c h e n 13SA-Dichteg r a d i e n t e n a u f g e t r e n n t w o r d e n w a r e n , w u r d e n a u f ihre S t i m u l i e r b a r k e i t d u r c h die
Specialia
Table III. Response of C57B1/6 mouse thymus cells separated on BSA diseontinucms gradients to LPS CelI fraction Control ~
Band A
BandB
BandC
BandD
Pellet
Unstimulated cells (counts/rain)
Stimulated ceils (counts/rain)
Stimulation index
370 zt= 49 u 116~c 09 224~: 04 1522 ~ 225 4405 ~ 359 702 ~= 99 253 ~ 19 158 :~= 20 166 2~ 35 93 ~ 18 48 ~_~ 06 173 ~ 13 1 3 6 i 24 8 4 i 11 249 ~ 52
398 4- 50 173 ~ 44 243 ~ 30 1336 -t- 108 1994 =E 451 295 ~ 62 528 4- 15 311 J= 78 395 4- 32 99 4- 12 74 =/= 15 221 -4- 40 167 q- 30 89 j= 22 184 -4- 29
1.08 1.48 1.08 0.88 0.45 0.42 2.09 1.96 2.38 1.09 1.55 1.28 1.23
276 ~ 105 160 ~ 21 129 ~c 14
192 ~ 07 159-4- 48 124 -1- 08
0.69 0.99 1.09
1.05 0.74
Nonseparated thymoeytes, b Data for 3 individual experiments is given. Values listed are the mean epm of 4 replica tubes plus the standard deviation.
f r o m b a n d I3 e x h i b i t e d t h e g r e a t e s t m i t o g e n i c response. T h e m i t o g e n i c r e s p o n s e s of n o n - s e p a r a t e d cells w a s h i g h e r t h a n t h e r e s p o n s e s of b a n d s A, C, D, or t h e pellet. T h y m u s cell s u b p o p u l a t i o n s were s t i m u l a t e d w i t h t h e t h y m u s i n d e p e n d e n t m i t o g e n E. colt L P S (10 ~g). T h i s d a t a (Table I I I ) shows t h a t cells f r o m b a n d 13 e x h i b i t e d t h e h i g h e s t degree of s t i m u l a t i o n . Cells ill t h e o t h e r b a n d s a n d p e l l e t did n o t r e s p o n d to L P S . T h e cell s u b p o p u l a t i o n i s o l a t e d f r o m t h e lowest d e n s i t y a r e a of t h e g r a d i e n t ( b a n d A) was r i c h i n large b l a s t - l i k e cells d i s p l a y i n g a n a b u n d a n t a m o u n t of c y t o p l a s m a n d large r o u n d nuclei. T h e s e cells were n o t s t i m u l a t e d b y a n y of t h e m i t o g e n s b u t d i d d i s p l a y t h e h i g h e s t b a s a l u p t a k e of t r i t i a t e d t h y m i d i n e (Tables, I, I I I ) w h e n i n c u b a t e d w i t h o u t m i t o g e n . D u e t o t h e h i g h e r level of t r i t i a t e d t h y m i d i n e u p t a k e in t h i s cell p o p u l a t i o n i t is felt t h a t t h e s e cells c o r r e s p o n d to t h e h i g h l y m i t o t i c ceils of t h e o u t e r c o r t e x t of t h e i n t a c t t h y m u s ~t. T h e r e was a s i g n i f i c a n t e n r i c h m e n t of cells in b a n d ]3 r e s p o n d i n g to m i t o g e n i c s t i m u l a t i o n w h e n c o m p a r e d to t h e o t h e r b a n d s a n d u n s e p a r a t e d cells. T h i s r e a c t i v e s u b p o p u l a t i o n w a s not, h o w e v e r , m o r p h o l o g i c a l l y h o m o g enous. I t is n o t clear a t t h e p r e s e n t t i m e w h i c h of t h e
EXPERIEN*TIA31/3
m o r p h o l o g i c a l cell t y p e s r e s p o n d to t h e m i t o g e n s . P r e v i o u s investigations have shown that cortisone resistant t h y m o c y t e s are r e s p o n s i v e t o P H A a n d Con A. Therefore, it is p r o b a b l e t h a t t h e cells i s o l a t e d f r o m b a n d B correspond to the cortisone resistant immunologically c o m p e t e n t cells of t h e t h y m u s m e d u l l a ~2. T h e m o r p h o logical h e t e r o g e n e i t y of t h i s r e a c t i v e s u b p o p u l a t i o n m a y b e d u e t o c o n t a m i n a t i o n w i t k n o n r e a c t i n g ceils. H o w e v e r , a m o r e p l a u s i b l e e x p l a n a t i o n w o u l d be t h a t t h i s s u b p o p u l a t i o n c o n t a i n s cells t h a t are in v a r i o u s stages of i m m u n o logical m a t u r a t i o n a n d t h u s a p p e a r m o r p h o l o g i c a l l y dissimilar. O u r d a t a (Table I I I ) show a s m a l l n u m b e r of t h y m u s cells i s o l a t e d f r o m b a n d B t h a t are c a p a b l e of r e s p o n d i n g t o t h e t h y m u s i n d e p e n d e n t m i t o g e n E. coli L P S . H o w e v e r , u n s e p a r a t e d t h y m u s cells e x h i b i t e d no m i t o g e n i c r e s p o n s e to L P S , w h i c h is in a g r e e m e n t w i t h t h e d a t a of AI~DERSSON et al. ~a. D e n s i t y g r a d i e n t s e p a r a t i o n of t h y m u s cells allowed t h e i s o l a t i o n of m i t o g e n r e s p o n s i v e cells i n a single s u b p o p u l a t i o n . S p e c u l a t i o n on t h e f u n c t i o n a l i t y of t h e t h y m u s cells r e s p o n d i n g t o L P S m a y lead t o t h e following s u p p o s i t i o n s . First, t h e cells m a y b e t3 cells i n v o l v e d in h u m o r a l i m m u n i t y a n d c o r r e s p o n d t o t h e a n t i b o d y p r o d u c i n g cells d e s c r i b e d b y VITETTA et al. ~4 O n t h e o t h e r h a n d t h e y m a y r e p r e s e n t 13 cells t h a t are d e s t i n e d t o b e c o m e T cells u n d e r t h e i n f l u e n c e of t h e t h y m u s . A t h i r d p o s s i b i l i t y is t h a t t h e L P S r e a c t i v e cells are c o n t a m i n a n t s f r o m t h e circulation.
Zusammen/assung. F r a k t i o n e n y o n T h y m u s l y m p h o z y t e n d e r Maus, die i m d i s k o n t i n u i e r l i c h e n 13SA-Dichteg r a d i e n t e n a u f g e t r e n n t w o r d e n w a r e n , w u r d e n a u f ihre S t i m u l i e r b a r k e i t d u r c h die
