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Antigenic surface determinants of chicken thrombocytoid cells
DEVELOPMENTAL AND COMLPARATIVE ~ O L O G Y , Vol. 4, pp. 123-135, 1980. 0145-305X/80/010123-13502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.
ANTIGENIC
SURFACE
H. Janzarik,
DETERMINANTS
OF C H I C K E N
THROMBOCYTOID
CELLS
K. S c h a u e n s t e i n +, H. W o l f + , and G. W i c k +
D e p a r t m e n t of I n t e r n a l M e d i c i n e , J u s t u s L i e b i g - U n i v e r s i t ~ t , Giessen, FRG, and + I n s t i t u t e for G e n e r a l and E x p e r i m e n t a l P a t h o l o g y , U n i v e r s i t y of Innsbruck, Austria.
ABSTRACT
This p a p e r d e s c r i b e s a first a t t e m p t to c o r r e l a t e m o r p h o l o g i c a l data of c h i c k e n t h r o m b o c y t o i d cells o b t a i n e d by p h a s e c o n t r a s t m i c r o s c o p y w i t h the exp r e s s i o n of surface d e t e r m i n a n t s as r e c o g n i z e d by h e t e r o l o g o u s a n t i - c h i c k e n l y m p h o c y t e sera. The following three m o r p h o l o g i c a l types of m o n o n u c l e a r cells in the p e r i p h e r a l b l o o d of c h i c k e n s have been d i s t i n g u i s h e d : (I) small round cells, (II) small star s h a p e d cells w i t h short p s e u d o p o d i a ; both these types r e s e m b l i n g l y m p h o i d cells, and (III) platelet-like cells w i t h the c a p a c i t y for i n s t a n t a n e o u s a d h e s i o n and s u b s e q u e n t spreading, i.e.,"chicken t h r o m b o c y t e s " . F l u i d t r a n s i t i o n s of cells type I to type III have been o b s e r v e d to o c c u r in e a r l i e r studies. In a c o m b i n e d p h a s e c o n t r a s t and m e m b r a n e i m m u n o f l u o r e s c e n c e assay on living cells using turkey a n t i - b u r s a cell and a n t i - t h y m u s cell sera (ABS and ATS) and a r a b b i t a n t i - c h i c k e n i m m u n o g l o b u l i n serum, cells of type I and II b o t h turned out to be p o s i t i v e l y s t a i n e d in 95% w i t h ATS, w h e r e a s the v a s t m a j o r i t y (98%) of t h r o m b o c y t o i d cells reacted w i t h ABS. The i m p a c t of these first d a t a for a p o s s i b l e relat i o n s h i p b e t w e e n c h i c k e n B cells and t h r o m b o c y t e s w i l l be discussed.
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INTRODUCTION C h i c k e n t h r o m b o c y t e s have been d e s c r i b e d in early p u b l i c a tions as nucleated, oval s h a p e d cells of about 8x5 ~ m d i a m e t e r (1,2). These studies refer to c o n v e n t i o n a l h i s t o l o g i c a l inv e s t i g a t i o n s w i t h fixed and s t a i n e d smears of p e r i p h e r a l blood. A c c o r d i n g to Lucas and J a m r o z (2) t h r o m b ~ c y t e s are found at c o n c e n t r a t i o n s of 30.000 - 60.000 per m m of p e r i p h e r a l blood. In the m e a n t ± m e several e l e c t r o n m i c r o s c o p i c a l studies on this cell type have b e e n p e r f o r m e d (3 - 11), but only a few deal with living cells u s i n g phase c o n t r a s t m i c r o s c o p y (12 - 15). Both these latter t e c h n i q u e s have r e v e a l e d v a r i o u s a d d i t i o n a l shapes of t h r o m b o c y t e s d i f f e r e n t from the "classical" forms found in smear p r e p a r a t i o n s . Our own i n v e s t i g a t i o n s u s i n g phase c o n t r a s t m i c r o s c o p y (16) have p r o v i d e d e v i d e n c e that the char a c t e r i s t i c a l t h r o m b o c y t e shapes g r a d u a l l y arise from r o u n d cells, w h i c h cannot be d i f f e r e n t i a t e d f r o m l y m p h o i d cells by m o r p h o l o g i c a l criteria. T h e s e findings w e r e f u r t h e r s u b s t a n t i a ted by e l e c t r o n m i c r o s c o p i c a l d a t a (17). A c c o r d i n g l y , it can be assumed that any p r e v i o u s w o r k d e a l i n g w i t h the s e r o l o g i c a l properties of p e r i p h e r a l c h i c k e n l y m p h o i d cells - i n c l u d i n g our own studies (18 - 21) - m i g h t have i n c l u d e d a c o n s i d e r a b l e p r o p o r t i o n of cells b e l o n g i n g to the t h r o m b o c y t o i d system. T h i s p r o b l e m is also r e f l e c t e d by the fact that i m m u n o l o g i c a l i n v e s t i g a t i o n s refer to l y m p h o i d cell counts of 6 3 - 90% of total l e u k o c y t e s (22 - 24), w h i c h is in obvious c o n t r a s t to the m o r p h o l o g i c a l d a t a of Lucas and J a m r o z (2), where the n u m b e r of t h r o m b o c y t e s is d e s c r i b e d to be 1.4 to 2.9 times that of the l y m p h o c y t e count. It was t h e r e f o r e d e e m e d of i n t e r e s t to i n v e s t i g a t e , (a) w h e ther cells w i t h the f e a t u r e s of t h r o m b o c y t e s do react w i t h h e t e r o logous a n t i s e r a a g a i n s t v a r i o u s chicken l y m p h o c y t e surface determinants, and (b) to find a p o s s i b l e c o r r e l a t i o n b e t w e e n the m o r p h o l o g i c a l a p p e a r a n c e of the cells and a p r e f e r e n t i a l reactivity w i t h any of the d i f f e r e n t types of r e a g e n t s u s e d - i.e. turkey a n t i - b u r s a cell and a n t i - t h y m u s cell sera (ABS and ATS), as w e l l as f l u o r e s c e i n a t e d rabbit a n t i b o d y s p e c i f i c a l l y r e a c t i n g w i t h chicken i m m u n o g l o b u l i n . Finally, (c) the i n f l u e n c e of the various m a n i p u l a t i o n s r e q u i r e d for i n d i r e c t and d i r e c t m e m b r a n e immunofluorescence (MeIF) assays on m o r p h o l o g y and r e l a t i v e dist r i b u t i o n in the d i f f e r e n t i a l cell count was evaluated.
MATERIALS
AND M E T H O D S
Animals Adult, r a n d o m bred, c l i n i c a l l y healthy, female N o r m a l W h i t e L e g h o r n (NWL) chickens w e r e o b t a i n e d from a local c o m m e r c i a l source. T h e y were k e p t under s t a n d a r d e n v i r o n m e n t a l and d i e t a r y conditions as d e s c r i b e d (20). B l o o d c o l l e c t i o n and p r e p a r a t i o n of l e u k o c y t e s u s p e n s i o n s Six ml of b l o o d w e r e c o l l e c t e d from the w i n g vein d i r e c t l y into s i l i c o n i z e d p l a s t i c tubes c o n t a i n i n g s o d i u m c i t r a t e as ant i c o a g u l a n t (3.8%, 1/10, v/v). E x a c t p u n c t u r e w i t h o u t h e m a t o m a is crucial for o p t i m a l l y r e p r o d u c i b l e results. For the p r e p a r a -
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tion of l e u k o c y t e - r i c h p l a s m a the a n t i c o a g u l a t e d b l o o d was cent r i f u g e d at 100 x g for 3 min at r o o m t e m p e r a t u r e and the buffy coat r e m o v e d t o g e t h e r w i t h the s u p e r n a t a n t plasma. The cell susp e n s i o n was then w a s h e d 3 times at 700 x g and a d j u s t e d to about 4 x 107 living w h i t e cells p e r ml. T h e s e s u s p e n s i o n s c o n t a i n mainly small m o n o n u c l e a r cells and are only n e g l i g i b l y c o n t a m i n a t e d w i t h e r y t h r o c y t e s , g r a n u l o c y t e s , and monocytes. For all d i l u t i o n s and w a s h i n g s m e d i u m RPMI 1640 (LS, L a b o r s e r v i c e , MHnchen, W e s t Germany) was employed. Antisera The p r o d u c t i o n and p r o p e r t i e s of s p e c i f i c turkey ABS and ATS have b e e n d e s c r i b e d in d e t a i l p r e v i o u s l y (18,19). A r a b b i t antichicken Ig s e r u m was also p r e p a r e d as o u t l i n e d e l s e w h e r e (25). Conjugates A c h i c k e n a n t i - t u r k e y Ig g l o b u l i n f r a c t i o n was l a b e l l e d w i t h f l u o r e s c e i n i s o t h i o c y a n a t e (FITC) (Baltimore B i o l o g i c a l L a b o r a t o ries, Md, USA) a c c o r d i n g to s t a n d a r d p r o c e d u r e s (26) and charact e r i z e d f o l l o w i n g the g u i d e l i n e s r e c o m m e n d e d by B e u t n e r et al. (27). It had 32 s t a n d a r d p r e c i p i t a t i o n units, a m o l a r f l u o r o c h r o m e / p r o t e i n ratio (F/P) of 6.9 and was u s e d in a d i l u t i o n of 1:32 for i n d i r e c t M e I F tests d e t e r m i n e d to the o p t i m a l by c h e s s b o a r d t i t r a t i o n s (conjugate code Nr. CW 10). The c h a r a c t e r i s t i c s of a r a b b i t a n t i - c h i c k e n Ig F I T C c o n j u g a t e (code Nr. CWW 13) also m a n u f a c t u r e d in this l a b o r a t o r y were 16 s t a n d a r d p r e c i p i t a t i o n units, a m o l a r F/P of 2.5 and a w o r k i n g d i l u t i o n of 1:32 for d i r e c t M e I F tests. Procedure D i r e c t and i n d i r e c t M e I F tests w e r e p e r f o r m e d a c c o r d i n g to the o r i g i n a l m e t h o d as d e s c r i b e d by M 6 1 1 e r (28). S e r a from u n i m m u n i z e d n o r m a l t u r k e y s (NTS) and m e d i u m alone s e r v e d as n e g a t i v e controls. NaN 3 (final c o n c e n t r a t i o n 0.O1 M) was added to the m e d i u m for the last w a s h i n g b e f o r e and the final three w a s h i n g s after ~ n c u b a t i o n w i t h the conjugate. All s e r o l o g i c a l reagents w e r e u l t r a c e n t r i f u g e d (1OO.000 x g, 20 min) in a B e c k m a n A i r f u g e (Beckman Instruments, Irvine, Cal., USA) just p r i o r to the tests. Microscopy All r e a d i n g s were done w i t h a Zeiss S t a n d a r d RA m i c r o s c o p e (Carl Zeiss, O b e r k o c h e m , W e s t Germany) e q u i p p e d for t r a n s m i t t e d light p h a s e c o n t r a s t o b s e r v a t i o n and s i m u l t a n e o u s i n c i d e n t light fluorescence. The i n c i d e n t light f l u o r e s c e n c e unit c o n s i s t e d of a H B O - 5 0 h i g h p r e s s u r e m e r c u r y v a p o u r bulb, a 455-490 e x c i t a t i o n filter, a d i c h r o i c m i r r o r (wedge 510 nm), and a 520 nm b a r r i e r filter. Readin@ All o b s e r v a t i o n s w e r e m a d e on living cells under n a i l p o l i s h s e a l e d c o v e r s l i p s i m m e d i a t e l y after the last washing. T h e s e susp e n s i o n s p e c i m e n s p r o v i d e the p o s s i b i l i t y to follow all transitions of cell shape in the course of s e d i m e n t a t i o n onto the glass surface. A t least 100 white b l o o d cells (PBL) w e r e e v a l u a t e d p e r slide, the cells were chosen at random, and c l a s s i f i e d m o r p h o l o g i c a l l y under phase c o n t r a s t and s e r o l o g i c a l l y by i n c i d e n t light
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fluorescence. A f t e r the r e p e a t e d w a s h i n g s r e q u i r e d for the M e I F p r o c e d u r e the cell p r e p a r a t i o n s c o n t a i n e d only very low n u m b e r s of g r a n u l o c y t e s and m o n o c y t e s (both less than 5%), w h i c h can be clearly i d e n t i f i e d in the p h a s e c o n t r a s t (H.Janzarik, to be published) and have b e e n ruled o u t in this study. For the c o r r e l a tion of m o r p h o l o g y and s e r o l o g i c a l r e a c t i v i t y 100 cells of each m o r p h o l o g i c a l type (see results) were s e l e c t e d in a s e c o n d r e a d i n g under phase c o n t r a s t and s u b s e q u e n t l y e v a l u a t e d for M e I F staining. B l o o d cell counts PBL counts w e r e m a d e e x c l u s i v e l y on u n s t a i n e d v i t a l cells in the c o u n t i n g chamber. C i t r a t e d b l o o d w a s drawn into an erythrocyte p i p e t t e to the 0.5 m a r k and filled up to the 101 m a r k w i t h u n b u f f e r e d 0.9% N a C I s o l u t i o n w i t h s u b s e q u e n t mixing. After s e d i m e n t a t i o n for 30 min in the c o u n t i n g c h a m b e r the v a r i o u s cell types are d i f f e r e n t i a t e d and c o u n t e d in a b s o l u t e n u m b e r s per m m J (+ 10% due to the d i l u t i o n w i t h a n t i c o a g u l a n t ) .
RESULTS I. M o r p h o l o g y Living, small m o n o n u c l e a r cells, w h i c h m a k e up the m a j o r i t y of PBL of chickens can be c l a s s i f i e d u n d e r p h a s e c o n t r a s t into 3 m a j o r groups, the third group b e i n g s u b d i v i d e d into 2 subclasses (Fig. 1): I. R o u n d m o n o n u c l e a r cells W i t h small c y t o p l a s m i c rim and r a t h e r smooth o u t l i n e - i.e., the c l a s s i c a l f e a t u r e s of small l y m p h o i d cells. II. S t a r - s h a p e d a s y m m e t r i c a l cells w i t h short p s e u d o p o d i a and i r r e g u l a r outline. III. P l a t e l e t - l i k e cells, "chicken t h r o m b o c y t e s " , (a) s p i n d l e - s h a p e d cells w i t h two long p s e u d o p o d i a on each cell pole; oval cells w i t h v a c u o l e s , (b) p l a t e l e t - l i k e s p r e a d cells w i t h a c o m p l e t e l y or partially e x t e n d e d c y t o p l a s m i c "veil"; cells w i t h several long, thin p s e u d o p o d i a . Type IIIa r e p r e s e n t s the shape of t h r o m b o c y t e s in s u s p e n s i o n not given the o p p o r t u n i t y to adhere to surfaces. In the p r e s e n c e of an a p p r o p r i a t e s u p p o r t they are able to t r a n s f o r m into cells of type IIIb w i t h i n a few seconds or minutes. I r r e s p e c t i v e of the fact that avian t h r o m b o c y t e s are n u c l e a t e d , cells of type IIIa and IIIb look like giant m a m m a l i a n p l a t e l e t s (for the less known spind l e - s h a p e d human p l a t e l e t form see B a r n h a r t et al. (29)). Cells of types IIIa and IIIb, like m a m m a l i a n p l a t e l e t s , show a h i g h l y s p e c i a l i z e d form of i n s t a n t a n e o u s a d h e s i o n and c o n s e c u t i v e spreading over an area several times that of the o r i g i n a l outline.
2. C o r r e l a t i o n of m o r p h o l o g i c a l and s e r o l o g i c a l findings. R e a c t i v i t y of m o n o n u c l e a r cells of types I, II, and III w i t h ABS, ATS, and anti-Ig in i n d i r e c t and d i r e c t MeIF. Table I depicts the q u a n t i t a t i v e d a t a of M e I F s t a i n i n g s of the m o n o n u c l e a r cells of 10 adult chickens w i t h ABS, ATS, and
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127
0
Type I
0
o Type II
0
Q
Type I I I a
Type III b
Fig.
I. M o r p h o l o g i c a l
@ classification
in the phase contrast.
Magn.
of chicken m o n o n u c l e a r x 800, oil immersion.
PBL
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CHICKEN THROM~OCYTES
TABLE
Vol. 4, No. 1
I
Results of Direct (DIF) and Indirect (IIF) MeIF Tests on M o n o n u c l e a r PBL of 10 Adult NWL Chickens °
% reacting
ABS
living mononuclear PBL (excluding granulocytes monocytes) o
(IIF)
30.9~15.4
ATS
cells
(IIF)
70.7~15.7
(mean ~ SD) with
NTS
(IIF)
O.4~0.6
anti-Ig
(DIF)
4.4~3.8
and
19 experiments
TABLE
2
Serological C h a r a c t e r i s t i c s of M o r p h o l o g i c a l l y Classified PBL Types I - I I I in Direct (DIF) and Indirect (IIF) MeIF Tests °
% reacting
ABS
(IIF)
ATS
cells
(IIF)
+ (mean - SD) with
ABS+ATS
anti-Ig
(DIF)
mononuclear PBL Type I
5.9~4.9
95.3~3.O
IO1.2~5.4
5.4~5.1
Type II
5.4~3.5
94.7~4.2
IOO.I~4.1
7.O~7.9
98.8~1.2
3.6~5.3
IO2.4~5.7
4.1~6.6
Type III
o
IO animals,
19 experiments
anti-Ig conjugate irrespective of morphology. The respective percentages obtained with ABS and ATS are in agreement with the values of earlier studies (21,30), whereas the values for anti-Ig staining are likewise low. The respective reactivity of each m o r p h o l o g i c a l cell type with the three kinds of serological reagents is shown in Table 2.
Vol.
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It t u r n e d out that cells of type I and II b o t h r e a c t e d p r e d o m i nantly w i t h ATS, w h e r e a s the large m a j o r i t y of the p l a t e l e t - l i k e cells type III w e r e p o s i t i v e w i t h ABS. In all i n s t a n c e s the additive values for ABS and ATS s t a i n i n g a m o u n t e d to about 100%. The m e a n p e r c e n t a g e s of small m o n o n u c l e a r cells types I and II c a r r y i n g surface Ig c o r r e s p o n d e d to the p e r c e n t a g e of ABS positive cells, w h e r e a s o n l y few cells of type III (mean 4%), a l b e i t s t a i n e d w i t h ABS (mean 98%), were found to be p o s i t i v e w i t h the anti-Ig conjugate. The a p p e a r a n c e of the v a r i o u s cell types in the c o m b i n e d M e I F / p h a s e c o n t r a s t assay is d o c u m e n t e d in Fig. 2.
3. I n f l u e n c e of c o n c e n t r a t i o n and w a s h i n g p r o c e d u r e s on the r e l a t i v e d i s t r i b u t i o n of m o n o n u c l e a r cells of types I - I I I in MeIF tests T h e reasons for the o b v i o u s d i s c r e p a n c i e s b e t w e e n the numbers for l y m p h o i d cells and t h r o m b o c y t e s of p e r i p h e r a l chicken b l o o d as given by Lucas and Jamroz (2) .and that of n u m e r o u s s e r o l o g i cal studies have t u r n e d out to be of t e c h n i c a l nature. By the u n a v o i d a b l e m a n i p u l a t i o n s d u r i n g M e I F tests - i.e., l e u k o c y t e isolation, c o n c e n t r a t i o n and w a s h i n g , the r e l a t i v e d i s t r i b u t i o n of m o n o n u c l e a r cells types I - I I I was found to u n d e r g o char a c t e r i s t i c a l m o d i f i c a t i o n s from the o r i g i n a l b l o o d cell count to that in the final specimen. C o m p a r i s o n of the r e l a t i v e values before and after the p r e p a r a t i o n p r o c e d u r e s for the M e I F tests d e m o n s t r a t e d d i f f e r e n t s t a b i l i t i e s of the d i f f e r e n t cell types. As it is o b v i o u s from Fig. 3 the n u m b e r s of small round
%
n=19
Fig.
80 70 60 I
50 40 30 20I0O-
ab
ab
ab
Type I
II
III
Lymphoid cells
3. R e l a t i v e numbers of c h i c k e n P B L types I - I I I as c o u n t e d in w h o l e b l o o d b e f o r e (a, open bars), and in the cell s u s p e n s i o n a{ter p r e p a r a t i o n for M e I F tests (b, h a t c h e d bars).
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cells of type I and n o t a b l y those of type II w e r e found to increase c o n s i d e r a b l y , w h e r e a s a large n u m b e r of p l a t e l e t - l i k e cells of type III, w h i c h c o n s t i t u t e the largest p o r t i o n in the c h i c k e n native b l o o d cell count, were o b v i o u s l y lost d u r i n g the cell p r e p a r a t i o n , thus e n t a i l i n g the r e l a t i v e increase of the other types. T h e s e data w o u l d be in a g r e e m e n t w i t h phase contrast o b s e r v a t i o n s s h o w i n g p l a t e l e t - l i k e cells p a r t i c u l a r l y prone to cell lysis. C o n s e q u e n t l y , the last s p e c i m e n for serological e v a l u a t i o n c o n t a i n s less cells type III (predominantly ABS positive) and more cells of type I and II (predominantly ATS positive) than the animals native blood.
DISCUSSION The m o r p h o l o g i c a l and f u n c t i o n a l c h a r a c t e r i s t i c s of chicken t h r o m b o c y t e s have b e e n w e l l d o c u m e n t e d in the literature (I-15, 31-33). R e c e n t e l e c t r o n m i c r o s c o p i c a l and phase c o n t r a s t studies on living cells have s u g g e s t e d fluid t r a n s i t i o n s of small, lymp h o c y t e - l i k e m o n o n u c l e a r cells to those w i t h the known features of thrombocytes. The p u r p o s e of the p r e s e n t i n v e s t i g a t i o n was to s u p p l e m e n t these m o r p h o l o g i c a l o b s e r v a t i o n s by the use of immun o l o g i c a l r e a g e n t s known to r e c o g n i z e surface d e t e r m i n a n t s on s u b p o p u l a t i o n s of c h i c k e n l y m p h o i d cells. It should be e m p h a s i z e d that the s e r o l o g i c a l c h a r a c t e r i z a t i o n of p e r i p h e r a l b l o o d l e u k o c y t e s by m e a n s of d i r e c t and i n d i r e c t M e I F was p e r f o r m e d at e x a c t l y the same c o n d i t i o n s and using the same r e a g e n t s as in our e a r l i e r w o r k (18-21,30). The only difference in the p r o c e d u r e was the s i m u l t a n e o u s e v a l u a t i o n of living cells in the phase c o n t r a s t and for M e I F r e a c t i v i t y after a sedi m e n t a t i o n time of 30 min. W i t h the c o n v e n t i o n a l m e t h o d s for serological c h a r a c t e r i z a t i o n of c h i c k e n PBL as h i t h e r t o a p p l i e d the cells were dried, fixed, and c o v e r e d w i t h b u f f e r e d g l y c e r o l before evaluation. A f t e r this t r e a t m e n t in the last step of the p r o c e d u r e all cells appear more or less u n i f o r m and of c i r c u l a r shape due to m e m b r a n e damage, thus p r e v e n t i n g a m o r p h o l o g i c a l c l a s s i f i c a t i o n . The o v e r a l l p e r c e n t a g e s of ABS and ATS p o s i t i v e cells c o r r e s p o n d e d w e l l to the values as e x p e c t e d from e a r l i e r studies. The r e a c t i v i t y w i t h the a n t i - I g conjugate, however, a p p e a r e d m a r k e d l y d e c r e a s e d in c o m p a r i s o n to our own data and those of other authors, w h e r e the n u m b e r of surface Ig p o s i t i v e cells q u a n t i t a t i v e l y c o r r e s p o n d e d the n u m b e r of ABS p o s i t i v e cells. C o m p a r i s o n of the m o r p h o l o g i c a l a p p e a r a n c e and serological findings r e v e a l e d that this d i s c r e p a n c y is only a c c o u n t e d for by spread cells of type III, w h i c h are almost q u a n t i t a t i v e ly s t a i n e d by ABS, but only to a very low degree w i t h anti-Ig. In contrast, cells of type I and II reveal the same numbers for ABS and anti-Ig staining. It can, therefore, be a s s u m e d that the low i n c i d e n c e of d i r e c t M e I F s t a i n i n g w i t h anti-Ig m a y be the r e s u l t of a d e c r e a s e d d e n s i t y of a n t i g e n i c d e t e r m i n a n t s due to the increase of cell surface during the s p r e a d i n g process, w h i c h is not o b s e r v e d w i t h d r i e d cells. The fact that the s t a i n i n g with ABS is o b v i o u s l y not a f f e c t e d by these p h y s i c a l events could be e x p l a i n e d e i t h e r by a p o s s i b l e h i g h e r d e n s i t y of ABS r e a c t i v e surface d e t e r m i n a n t s or by the use of the i n d i r e c t tech-
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nique, w h i c h is known to be more s e n s i t i v e than the d i r e c t assay. In essence, the results of this w o r k can be s u m m a r i z e d as follows: (a) chicken t h r o m b o c y t o i d cells r e a c t p o s i t i v e l y w i t h h e t e r o l o g o u s a n t i s e r a s p e c i f i c for c h i c k e n l y m p h o i d cells, (b) the m a j o r i t y of cells e x h i b i t i n g the features of l y m p h o i d cells (type I and II) was found to be p o s i t i v e w i t h ATS, whereas cells of type III ( t h r o m b o c y t o i d cells) r e a c t e d largely w i t h ABS. (c) a small p e r c e n t a g e of these latter cells seem to express surface Ig. The r e a c t i v i t y of t h r o m b o c y t o i d cells w i t h ABS w o u l d e i t h e r imply that this a n t i s e r u m is not s t r i c t l y s p e c i f i c for the bursad e r i v e d l y m p h o i d cell s y s t e m or the e x i s t e n c e of common cell surface d e t e r m i n a n t s on B l y m p h o c y t e s and t h r o m b o c y t o i d cells. Conc e r n i n g specifity, the sera used in the p r e s e n t e x p e r i m e n t s have been shown to be h i g h l y s p e c i f i c in vitro and in v i v o (20). N e v e r t h e l e s s , we cannot e x c l u d e that ABS m a y als--o con-----tain a n t i b o dies to antigens on t h r o m b o c y t o i d cells in a d d i t i o n to anti-B cell a n t i b o d i e s proper. However, t h y m o c y t e s u s p e n s i o n s are k n o w n to be c o n t a m i n a t e d w i t h p e r i p h e r a l b l o o d to a m u c h h i g h e r degree than b u r s a c y t e suspensions. A c c o r d i n g l y , one w o u l d e x p e c t ATS to contain c o n s i d e r a b l y h i g h e r amounts of c o n t a m i n a t i n g a n t i - t h r o m b o cyte a n t i b o d i e s than ABS. This is o b v i o u s l y not the case. In order to clarify this issue we are c u r r e n t l y p r o d u c i n g a n t i s e r a to p e r i p h e r a l s p r e a d t h r o m b o c y t o i d cells. R e c e n t e x p e r i m e n t s using ABS and ATS d e r i v e d from m a m m a l i a n species (i.e. goats) y i e l d e d e s s e n t i a l l y i d e n t i c a l results, w h i c h further s u p p o r t our initial data and e x c l u d e the i n v o l v e m e n t of Fc b i n d i n g since m a m m a l i a n Ig is known not to be b o u n d by chicken Fc r e c e p t o r s (34). The a p p a r e n t a n t i g e n i c s i m i l a r i t y of c h i c k e n B cells and t h r o m b o c y t o i d cells m a y r e f l e c t a close r e l a t i o n s h i p or p e r h a p s identity of both cell systems, thus s u p p o r t i n g the initial morp h o l o g i c a l o b s e r v a t i o n s . In this c o n t e x t m o r p h o l o g i c a l d a t a of Stokes and F i r k i n (35) on fish t h r o m b o c y t e s are of interest, w h i c h s u g g e s t an i d e n t i t y of l y m p h o c y t e s and t h r o m b o c y t e s in this species. For the f u r t h e r c l a r i f i c a t i o n of this m a i n q u e s t i o n functional and c o m b i n e d m o r p h o l o g i c a l and i m m u n o l o g i c a l e x p e r i ments are u n d e r w a y w i t h b u r s e c t o m i z e d and t h y m e c t o m i z e d animals, respectively.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e t by the A u s t r i a n C a n c e r R e s e a r c h Found. The i n c i d e n t light f l u o r e s c e n c e m i c r o s c o p i c e q u i p m e n t was kindly p r o v i d e d from C.ZEISS, F r a n k f u r t branch, F r a n k f u r t / M a i n , FRG. We also a c k n o w l e d g e the free supply of m e d i a from LS, L a b o r service,
M~nchen,
FRG.
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STOKES, E.E. and FIRKIN, B.G. Studies of the p e r i p h e r a l b l o o d of the P o r t J a c k s o n shark (Heterodontus p o r t u s j a c k soni) w i t h p a r t i c u l a r r e f e r e n c e to the thrombocyte. Brit. J. Haematol. 20, 427, 1971.
in the chicken and turkey.
Fed.
ANTIGENIC
SURFACE
H. Janzarik,
DETERMINANTS
OF C H I C K E N
THROMBOCYTOID
CELLS
K. S c h a u e n s t e i n +, H. W o l f + , and G. W i c k +
D e p a r t m e n t of I n t e r n a l M e d i c i n e , J u s t u s L i e b i g - U n i v e r s i t ~ t , Giessen, FRG, and + I n s t i t u t e for G e n e r a l and E x p e r i m e n t a l P a t h o l o g y , U n i v e r s i t y of Innsbruck, Austria.
ABSTRACT
This p a p e r d e s c r i b e s a first a t t e m p t to c o r r e l a t e m o r p h o l o g i c a l data of c h i c k e n t h r o m b o c y t o i d cells o b t a i n e d by p h a s e c o n t r a s t m i c r o s c o p y w i t h the exp r e s s i o n of surface d e t e r m i n a n t s as r e c o g n i z e d by h e t e r o l o g o u s a n t i - c h i c k e n l y m p h o c y t e sera. The following three m o r p h o l o g i c a l types of m o n o n u c l e a r cells in the p e r i p h e r a l b l o o d of c h i c k e n s have been d i s t i n g u i s h e d : (I) small round cells, (II) small star s h a p e d cells w i t h short p s e u d o p o d i a ; both these types r e s e m b l i n g l y m p h o i d cells, and (III) platelet-like cells w i t h the c a p a c i t y for i n s t a n t a n e o u s a d h e s i o n and s u b s e q u e n t spreading, i.e.,"chicken t h r o m b o c y t e s " . F l u i d t r a n s i t i o n s of cells type I to type III have been o b s e r v e d to o c c u r in e a r l i e r studies. In a c o m b i n e d p h a s e c o n t r a s t and m e m b r a n e i m m u n o f l u o r e s c e n c e assay on living cells using turkey a n t i - b u r s a cell and a n t i - t h y m u s cell sera (ABS and ATS) and a r a b b i t a n t i - c h i c k e n i m m u n o g l o b u l i n serum, cells of type I and II b o t h turned out to be p o s i t i v e l y s t a i n e d in 95% w i t h ATS, w h e r e a s the v a s t m a j o r i t y (98%) of t h r o m b o c y t o i d cells reacted w i t h ABS. The i m p a c t of these first d a t a for a p o s s i b l e relat i o n s h i p b e t w e e n c h i c k e n B cells and t h r o m b o c y t e s w i l l be discussed.
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INTRODUCTION C h i c k e n t h r o m b o c y t e s have been d e s c r i b e d in early p u b l i c a tions as nucleated, oval s h a p e d cells of about 8x5 ~ m d i a m e t e r (1,2). These studies refer to c o n v e n t i o n a l h i s t o l o g i c a l inv e s t i g a t i o n s w i t h fixed and s t a i n e d smears of p e r i p h e r a l blood. A c c o r d i n g to Lucas and J a m r o z (2) t h r o m b ~ c y t e s are found at c o n c e n t r a t i o n s of 30.000 - 60.000 per m m of p e r i p h e r a l blood. In the m e a n t ± m e several e l e c t r o n m i c r o s c o p i c a l studies on this cell type have b e e n p e r f o r m e d (3 - 11), but only a few deal with living cells u s i n g phase c o n t r a s t m i c r o s c o p y (12 - 15). Both these latter t e c h n i q u e s have r e v e a l e d v a r i o u s a d d i t i o n a l shapes of t h r o m b o c y t e s d i f f e r e n t from the "classical" forms found in smear p r e p a r a t i o n s . Our own i n v e s t i g a t i o n s u s i n g phase c o n t r a s t m i c r o s c o p y (16) have p r o v i d e d e v i d e n c e that the char a c t e r i s t i c a l t h r o m b o c y t e shapes g r a d u a l l y arise from r o u n d cells, w h i c h cannot be d i f f e r e n t i a t e d f r o m l y m p h o i d cells by m o r p h o l o g i c a l criteria. T h e s e findings w e r e f u r t h e r s u b s t a n t i a ted by e l e c t r o n m i c r o s c o p i c a l d a t a (17). A c c o r d i n g l y , it can be assumed that any p r e v i o u s w o r k d e a l i n g w i t h the s e r o l o g i c a l properties of p e r i p h e r a l c h i c k e n l y m p h o i d cells - i n c l u d i n g our own studies (18 - 21) - m i g h t have i n c l u d e d a c o n s i d e r a b l e p r o p o r t i o n of cells b e l o n g i n g to the t h r o m b o c y t o i d system. T h i s p r o b l e m is also r e f l e c t e d by the fact that i m m u n o l o g i c a l i n v e s t i g a t i o n s refer to l y m p h o i d cell counts of 6 3 - 90% of total l e u k o c y t e s (22 - 24), w h i c h is in obvious c o n t r a s t to the m o r p h o l o g i c a l d a t a of Lucas and J a m r o z (2), where the n u m b e r of t h r o m b o c y t e s is d e s c r i b e d to be 1.4 to 2.9 times that of the l y m p h o c y t e count. It was t h e r e f o r e d e e m e d of i n t e r e s t to i n v e s t i g a t e , (a) w h e ther cells w i t h the f e a t u r e s of t h r o m b o c y t e s do react w i t h h e t e r o logous a n t i s e r a a g a i n s t v a r i o u s chicken l y m p h o c y t e surface determinants, and (b) to find a p o s s i b l e c o r r e l a t i o n b e t w e e n the m o r p h o l o g i c a l a p p e a r a n c e of the cells and a p r e f e r e n t i a l reactivity w i t h any of the d i f f e r e n t types of r e a g e n t s u s e d - i.e. turkey a n t i - b u r s a cell and a n t i - t h y m u s cell sera (ABS and ATS), as w e l l as f l u o r e s c e i n a t e d rabbit a n t i b o d y s p e c i f i c a l l y r e a c t i n g w i t h chicken i m m u n o g l o b u l i n . Finally, (c) the i n f l u e n c e of the various m a n i p u l a t i o n s r e q u i r e d for i n d i r e c t and d i r e c t m e m b r a n e immunofluorescence (MeIF) assays on m o r p h o l o g y and r e l a t i v e dist r i b u t i o n in the d i f f e r e n t i a l cell count was evaluated.
MATERIALS
AND M E T H O D S
Animals Adult, r a n d o m bred, c l i n i c a l l y healthy, female N o r m a l W h i t e L e g h o r n (NWL) chickens w e r e o b t a i n e d from a local c o m m e r c i a l source. T h e y were k e p t under s t a n d a r d e n v i r o n m e n t a l and d i e t a r y conditions as d e s c r i b e d (20). B l o o d c o l l e c t i o n and p r e p a r a t i o n of l e u k o c y t e s u s p e n s i o n s Six ml of b l o o d w e r e c o l l e c t e d from the w i n g vein d i r e c t l y into s i l i c o n i z e d p l a s t i c tubes c o n t a i n i n g s o d i u m c i t r a t e as ant i c o a g u l a n t (3.8%, 1/10, v/v). E x a c t p u n c t u r e w i t h o u t h e m a t o m a is crucial for o p t i m a l l y r e p r o d u c i b l e results. For the p r e p a r a -
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tion of l e u k o c y t e - r i c h p l a s m a the a n t i c o a g u l a t e d b l o o d was cent r i f u g e d at 100 x g for 3 min at r o o m t e m p e r a t u r e and the buffy coat r e m o v e d t o g e t h e r w i t h the s u p e r n a t a n t plasma. The cell susp e n s i o n was then w a s h e d 3 times at 700 x g and a d j u s t e d to about 4 x 107 living w h i t e cells p e r ml. T h e s e s u s p e n s i o n s c o n t a i n mainly small m o n o n u c l e a r cells and are only n e g l i g i b l y c o n t a m i n a t e d w i t h e r y t h r o c y t e s , g r a n u l o c y t e s , and monocytes. For all d i l u t i o n s and w a s h i n g s m e d i u m RPMI 1640 (LS, L a b o r s e r v i c e , MHnchen, W e s t Germany) was employed. Antisera The p r o d u c t i o n and p r o p e r t i e s of s p e c i f i c turkey ABS and ATS have b e e n d e s c r i b e d in d e t a i l p r e v i o u s l y (18,19). A r a b b i t antichicken Ig s e r u m was also p r e p a r e d as o u t l i n e d e l s e w h e r e (25). Conjugates A c h i c k e n a n t i - t u r k e y Ig g l o b u l i n f r a c t i o n was l a b e l l e d w i t h f l u o r e s c e i n i s o t h i o c y a n a t e (FITC) (Baltimore B i o l o g i c a l L a b o r a t o ries, Md, USA) a c c o r d i n g to s t a n d a r d p r o c e d u r e s (26) and charact e r i z e d f o l l o w i n g the g u i d e l i n e s r e c o m m e n d e d by B e u t n e r et al. (27). It had 32 s t a n d a r d p r e c i p i t a t i o n units, a m o l a r f l u o r o c h r o m e / p r o t e i n ratio (F/P) of 6.9 and was u s e d in a d i l u t i o n of 1:32 for i n d i r e c t M e I F tests d e t e r m i n e d to the o p t i m a l by c h e s s b o a r d t i t r a t i o n s (conjugate code Nr. CW 10). The c h a r a c t e r i s t i c s of a r a b b i t a n t i - c h i c k e n Ig F I T C c o n j u g a t e (code Nr. CWW 13) also m a n u f a c t u r e d in this l a b o r a t o r y were 16 s t a n d a r d p r e c i p i t a t i o n units, a m o l a r F/P of 2.5 and a w o r k i n g d i l u t i o n of 1:32 for d i r e c t M e I F tests. Procedure D i r e c t and i n d i r e c t M e I F tests w e r e p e r f o r m e d a c c o r d i n g to the o r i g i n a l m e t h o d as d e s c r i b e d by M 6 1 1 e r (28). S e r a from u n i m m u n i z e d n o r m a l t u r k e y s (NTS) and m e d i u m alone s e r v e d as n e g a t i v e controls. NaN 3 (final c o n c e n t r a t i o n 0.O1 M) was added to the m e d i u m for the last w a s h i n g b e f o r e and the final three w a s h i n g s after ~ n c u b a t i o n w i t h the conjugate. All s e r o l o g i c a l reagents w e r e u l t r a c e n t r i f u g e d (1OO.000 x g, 20 min) in a B e c k m a n A i r f u g e (Beckman Instruments, Irvine, Cal., USA) just p r i o r to the tests. Microscopy All r e a d i n g s were done w i t h a Zeiss S t a n d a r d RA m i c r o s c o p e (Carl Zeiss, O b e r k o c h e m , W e s t Germany) e q u i p p e d for t r a n s m i t t e d light p h a s e c o n t r a s t o b s e r v a t i o n and s i m u l t a n e o u s i n c i d e n t light fluorescence. The i n c i d e n t light f l u o r e s c e n c e unit c o n s i s t e d of a H B O - 5 0 h i g h p r e s s u r e m e r c u r y v a p o u r bulb, a 455-490 e x c i t a t i o n filter, a d i c h r o i c m i r r o r (wedge 510 nm), and a 520 nm b a r r i e r filter. Readin@ All o b s e r v a t i o n s w e r e m a d e on living cells under n a i l p o l i s h s e a l e d c o v e r s l i p s i m m e d i a t e l y after the last washing. T h e s e susp e n s i o n s p e c i m e n s p r o v i d e the p o s s i b i l i t y to follow all transitions of cell shape in the course of s e d i m e n t a t i o n onto the glass surface. A t least 100 white b l o o d cells (PBL) w e r e e v a l u a t e d p e r slide, the cells were chosen at random, and c l a s s i f i e d m o r p h o l o g i c a l l y under phase c o n t r a s t and s e r o l o g i c a l l y by i n c i d e n t light
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fluorescence. A f t e r the r e p e a t e d w a s h i n g s r e q u i r e d for the M e I F p r o c e d u r e the cell p r e p a r a t i o n s c o n t a i n e d only very low n u m b e r s of g r a n u l o c y t e s and m o n o c y t e s (both less than 5%), w h i c h can be clearly i d e n t i f i e d in the p h a s e c o n t r a s t (H.Janzarik, to be published) and have b e e n ruled o u t in this study. For the c o r r e l a tion of m o r p h o l o g y and s e r o l o g i c a l r e a c t i v i t y 100 cells of each m o r p h o l o g i c a l type (see results) were s e l e c t e d in a s e c o n d r e a d i n g under phase c o n t r a s t and s u b s e q u e n t l y e v a l u a t e d for M e I F staining. B l o o d cell counts PBL counts w e r e m a d e e x c l u s i v e l y on u n s t a i n e d v i t a l cells in the c o u n t i n g chamber. C i t r a t e d b l o o d w a s drawn into an erythrocyte p i p e t t e to the 0.5 m a r k and filled up to the 101 m a r k w i t h u n b u f f e r e d 0.9% N a C I s o l u t i o n w i t h s u b s e q u e n t mixing. After s e d i m e n t a t i o n for 30 min in the c o u n t i n g c h a m b e r the v a r i o u s cell types are d i f f e r e n t i a t e d and c o u n t e d in a b s o l u t e n u m b e r s per m m J (+ 10% due to the d i l u t i o n w i t h a n t i c o a g u l a n t ) .
RESULTS I. M o r p h o l o g y Living, small m o n o n u c l e a r cells, w h i c h m a k e up the m a j o r i t y of PBL of chickens can be c l a s s i f i e d u n d e r p h a s e c o n t r a s t into 3 m a j o r groups, the third group b e i n g s u b d i v i d e d into 2 subclasses (Fig. 1): I. R o u n d m o n o n u c l e a r cells W i t h small c y t o p l a s m i c rim and r a t h e r smooth o u t l i n e - i.e., the c l a s s i c a l f e a t u r e s of small l y m p h o i d cells. II. S t a r - s h a p e d a s y m m e t r i c a l cells w i t h short p s e u d o p o d i a and i r r e g u l a r outline. III. P l a t e l e t - l i k e cells, "chicken t h r o m b o c y t e s " , (a) s p i n d l e - s h a p e d cells w i t h two long p s e u d o p o d i a on each cell pole; oval cells w i t h v a c u o l e s , (b) p l a t e l e t - l i k e s p r e a d cells w i t h a c o m p l e t e l y or partially e x t e n d e d c y t o p l a s m i c "veil"; cells w i t h several long, thin p s e u d o p o d i a . Type IIIa r e p r e s e n t s the shape of t h r o m b o c y t e s in s u s p e n s i o n not given the o p p o r t u n i t y to adhere to surfaces. In the p r e s e n c e of an a p p r o p r i a t e s u p p o r t they are able to t r a n s f o r m into cells of type IIIb w i t h i n a few seconds or minutes. I r r e s p e c t i v e of the fact that avian t h r o m b o c y t e s are n u c l e a t e d , cells of type IIIa and IIIb look like giant m a m m a l i a n p l a t e l e t s (for the less known spind l e - s h a p e d human p l a t e l e t form see B a r n h a r t et al. (29)). Cells of types IIIa and IIIb, like m a m m a l i a n p l a t e l e t s , show a h i g h l y s p e c i a l i z e d form of i n s t a n t a n e o u s a d h e s i o n and c o n s e c u t i v e spreading over an area several times that of the o r i g i n a l outline.
2. C o r r e l a t i o n of m o r p h o l o g i c a l and s e r o l o g i c a l findings. R e a c t i v i t y of m o n o n u c l e a r cells of types I, II, and III w i t h ABS, ATS, and anti-Ig in i n d i r e c t and d i r e c t MeIF. Table I depicts the q u a n t i t a t i v e d a t a of M e I F s t a i n i n g s of the m o n o n u c l e a r cells of 10 adult chickens w i t h ABS, ATS, and
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0
Type I
0
o Type II
0
Q
Type I I I a
Type III b
Fig.
I. M o r p h o l o g i c a l
@ classification
in the phase contrast.
Magn.
of chicken m o n o n u c l e a r x 800, oil immersion.
PBL
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CHICKEN THROM~OCYTES
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Vol. 4, No. 1
I
Results of Direct (DIF) and Indirect (IIF) MeIF Tests on M o n o n u c l e a r PBL of 10 Adult NWL Chickens °
% reacting
ABS
living mononuclear PBL (excluding granulocytes monocytes) o
(IIF)
30.9~15.4
ATS
cells
(IIF)
70.7~15.7
(mean ~ SD) with
NTS
(IIF)
O.4~0.6
anti-Ig
(DIF)
4.4~3.8
and
19 experiments
TABLE
2
Serological C h a r a c t e r i s t i c s of M o r p h o l o g i c a l l y Classified PBL Types I - I I I in Direct (DIF) and Indirect (IIF) MeIF Tests °
% reacting
ABS
(IIF)
ATS
cells
(IIF)
+ (mean - SD) with
ABS+ATS
anti-Ig
(DIF)
mononuclear PBL Type I
5.9~4.9
95.3~3.O
IO1.2~5.4
5.4~5.1
Type II
5.4~3.5
94.7~4.2
IOO.I~4.1
7.O~7.9
98.8~1.2
3.6~5.3
IO2.4~5.7
4.1~6.6
Type III
o
IO animals,
19 experiments
anti-Ig conjugate irrespective of morphology. The respective percentages obtained with ABS and ATS are in agreement with the values of earlier studies (21,30), whereas the values for anti-Ig staining are likewise low. The respective reactivity of each m o r p h o l o g i c a l cell type with the three kinds of serological reagents is shown in Table 2.
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It t u r n e d out that cells of type I and II b o t h r e a c t e d p r e d o m i nantly w i t h ATS, w h e r e a s the large m a j o r i t y of the p l a t e l e t - l i k e cells type III w e r e p o s i t i v e w i t h ABS. In all i n s t a n c e s the additive values for ABS and ATS s t a i n i n g a m o u n t e d to about 100%. The m e a n p e r c e n t a g e s of small m o n o n u c l e a r cells types I and II c a r r y i n g surface Ig c o r r e s p o n d e d to the p e r c e n t a g e of ABS positive cells, w h e r e a s o n l y few cells of type III (mean 4%), a l b e i t s t a i n e d w i t h ABS (mean 98%), were found to be p o s i t i v e w i t h the anti-Ig conjugate. The a p p e a r a n c e of the v a r i o u s cell types in the c o m b i n e d M e I F / p h a s e c o n t r a s t assay is d o c u m e n t e d in Fig. 2.
3. I n f l u e n c e of c o n c e n t r a t i o n and w a s h i n g p r o c e d u r e s on the r e l a t i v e d i s t r i b u t i o n of m o n o n u c l e a r cells of types I - I I I in MeIF tests T h e reasons for the o b v i o u s d i s c r e p a n c i e s b e t w e e n the numbers for l y m p h o i d cells and t h r o m b o c y t e s of p e r i p h e r a l chicken b l o o d as given by Lucas and Jamroz (2) .and that of n u m e r o u s s e r o l o g i cal studies have t u r n e d out to be of t e c h n i c a l nature. By the u n a v o i d a b l e m a n i p u l a t i o n s d u r i n g M e I F tests - i.e., l e u k o c y t e isolation, c o n c e n t r a t i o n and w a s h i n g , the r e l a t i v e d i s t r i b u t i o n of m o n o n u c l e a r cells types I - I I I was found to u n d e r g o char a c t e r i s t i c a l m o d i f i c a t i o n s from the o r i g i n a l b l o o d cell count to that in the final specimen. C o m p a r i s o n of the r e l a t i v e values before and after the p r e p a r a t i o n p r o c e d u r e s for the M e I F tests d e m o n s t r a t e d d i f f e r e n t s t a b i l i t i e s of the d i f f e r e n t cell types. As it is o b v i o u s from Fig. 3 the n u m b e r s of small round
%
n=19
Fig.
80 70 60 I
50 40 30 20I0O-
ab
ab
ab
Type I
II
III
Lymphoid cells
3. R e l a t i v e numbers of c h i c k e n P B L types I - I I I as c o u n t e d in w h o l e b l o o d b e f o r e (a, open bars), and in the cell s u s p e n s i o n a{ter p r e p a r a t i o n for M e I F tests (b, h a t c h e d bars).
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cells of type I and n o t a b l y those of type II w e r e found to increase c o n s i d e r a b l y , w h e r e a s a large n u m b e r of p l a t e l e t - l i k e cells of type III, w h i c h c o n s t i t u t e the largest p o r t i o n in the c h i c k e n native b l o o d cell count, were o b v i o u s l y lost d u r i n g the cell p r e p a r a t i o n , thus e n t a i l i n g the r e l a t i v e increase of the other types. T h e s e data w o u l d be in a g r e e m e n t w i t h phase contrast o b s e r v a t i o n s s h o w i n g p l a t e l e t - l i k e cells p a r t i c u l a r l y prone to cell lysis. C o n s e q u e n t l y , the last s p e c i m e n for serological e v a l u a t i o n c o n t a i n s less cells type III (predominantly ABS positive) and more cells of type I and II (predominantly ATS positive) than the animals native blood.
DISCUSSION The m o r p h o l o g i c a l and f u n c t i o n a l c h a r a c t e r i s t i c s of chicken t h r o m b o c y t e s have b e e n w e l l d o c u m e n t e d in the literature (I-15, 31-33). R e c e n t e l e c t r o n m i c r o s c o p i c a l and phase c o n t r a s t studies on living cells have s u g g e s t e d fluid t r a n s i t i o n s of small, lymp h o c y t e - l i k e m o n o n u c l e a r cells to those w i t h the known features of thrombocytes. The p u r p o s e of the p r e s e n t i n v e s t i g a t i o n was to s u p p l e m e n t these m o r p h o l o g i c a l o b s e r v a t i o n s by the use of immun o l o g i c a l r e a g e n t s known to r e c o g n i z e surface d e t e r m i n a n t s on s u b p o p u l a t i o n s of c h i c k e n l y m p h o i d cells. It should be e m p h a s i z e d that the s e r o l o g i c a l c h a r a c t e r i z a t i o n of p e r i p h e r a l b l o o d l e u k o c y t e s by m e a n s of d i r e c t and i n d i r e c t M e I F was p e r f o r m e d at e x a c t l y the same c o n d i t i o n s and using the same r e a g e n t s as in our e a r l i e r w o r k (18-21,30). The only difference in the p r o c e d u r e was the s i m u l t a n e o u s e v a l u a t i o n of living cells in the phase c o n t r a s t and for M e I F r e a c t i v i t y after a sedi m e n t a t i o n time of 30 min. W i t h the c o n v e n t i o n a l m e t h o d s for serological c h a r a c t e r i z a t i o n of c h i c k e n PBL as h i t h e r t o a p p l i e d the cells were dried, fixed, and c o v e r e d w i t h b u f f e r e d g l y c e r o l before evaluation. A f t e r this t r e a t m e n t in the last step of the p r o c e d u r e all cells appear more or less u n i f o r m and of c i r c u l a r shape due to m e m b r a n e damage, thus p r e v e n t i n g a m o r p h o l o g i c a l c l a s s i f i c a t i o n . The o v e r a l l p e r c e n t a g e s of ABS and ATS p o s i t i v e cells c o r r e s p o n d e d w e l l to the values as e x p e c t e d from e a r l i e r studies. The r e a c t i v i t y w i t h the a n t i - I g conjugate, however, a p p e a r e d m a r k e d l y d e c r e a s e d in c o m p a r i s o n to our own data and those of other authors, w h e r e the n u m b e r of surface Ig p o s i t i v e cells q u a n t i t a t i v e l y c o r r e s p o n d e d the n u m b e r of ABS p o s i t i v e cells. C o m p a r i s o n of the m o r p h o l o g i c a l a p p e a r a n c e and serological findings r e v e a l e d that this d i s c r e p a n c y is only a c c o u n t e d for by spread cells of type III, w h i c h are almost q u a n t i t a t i v e ly s t a i n e d by ABS, but only to a very low degree w i t h anti-Ig. In contrast, cells of type I and II reveal the same numbers for ABS and anti-Ig staining. It can, therefore, be a s s u m e d that the low i n c i d e n c e of d i r e c t M e I F s t a i n i n g w i t h anti-Ig m a y be the r e s u l t of a d e c r e a s e d d e n s i t y of a n t i g e n i c d e t e r m i n a n t s due to the increase of cell surface during the s p r e a d i n g process, w h i c h is not o b s e r v e d w i t h d r i e d cells. The fact that the s t a i n i n g with ABS is o b v i o u s l y not a f f e c t e d by these p h y s i c a l events could be e x p l a i n e d e i t h e r by a p o s s i b l e h i g h e r d e n s i t y of ABS r e a c t i v e surface d e t e r m i n a n t s or by the use of the i n d i r e c t tech-
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nique, w h i c h is known to be more s e n s i t i v e than the d i r e c t assay. In essence, the results of this w o r k can be s u m m a r i z e d as follows: (a) chicken t h r o m b o c y t o i d cells r e a c t p o s i t i v e l y w i t h h e t e r o l o g o u s a n t i s e r a s p e c i f i c for c h i c k e n l y m p h o i d cells, (b) the m a j o r i t y of cells e x h i b i t i n g the features of l y m p h o i d cells (type I and II) was found to be p o s i t i v e w i t h ATS, whereas cells of type III ( t h r o m b o c y t o i d cells) r e a c t e d largely w i t h ABS. (c) a small p e r c e n t a g e of these latter cells seem to express surface Ig. The r e a c t i v i t y of t h r o m b o c y t o i d cells w i t h ABS w o u l d e i t h e r imply that this a n t i s e r u m is not s t r i c t l y s p e c i f i c for the bursad e r i v e d l y m p h o i d cell s y s t e m or the e x i s t e n c e of common cell surface d e t e r m i n a n t s on B l y m p h o c y t e s and t h r o m b o c y t o i d cells. Conc e r n i n g specifity, the sera used in the p r e s e n t e x p e r i m e n t s have been shown to be h i g h l y s p e c i f i c in vitro and in v i v o (20). N e v e r t h e l e s s , we cannot e x c l u d e that ABS m a y als--o con-----tain a n t i b o dies to antigens on t h r o m b o c y t o i d cells in a d d i t i o n to anti-B cell a n t i b o d i e s proper. However, t h y m o c y t e s u s p e n s i o n s are k n o w n to be c o n t a m i n a t e d w i t h p e r i p h e r a l b l o o d to a m u c h h i g h e r degree than b u r s a c y t e suspensions. A c c o r d i n g l y , one w o u l d e x p e c t ATS to contain c o n s i d e r a b l y h i g h e r amounts of c o n t a m i n a t i n g a n t i - t h r o m b o cyte a n t i b o d i e s than ABS. This is o b v i o u s l y not the case. In order to clarify this issue we are c u r r e n t l y p r o d u c i n g a n t i s e r a to p e r i p h e r a l s p r e a d t h r o m b o c y t o i d cells. R e c e n t e x p e r i m e n t s using ABS and ATS d e r i v e d from m a m m a l i a n species (i.e. goats) y i e l d e d e s s e n t i a l l y i d e n t i c a l results, w h i c h further s u p p o r t our initial data and e x c l u d e the i n v o l v e m e n t of Fc b i n d i n g since m a m m a l i a n Ig is known not to be b o u n d by chicken Fc r e c e p t o r s (34). The a p p a r e n t a n t i g e n i c s i m i l a r i t y of c h i c k e n B cells and t h r o m b o c y t o i d cells m a y r e f l e c t a close r e l a t i o n s h i p or p e r h a p s identity of both cell systems, thus s u p p o r t i n g the initial morp h o l o g i c a l o b s e r v a t i o n s . In this c o n t e x t m o r p h o l o g i c a l d a t a of Stokes and F i r k i n (35) on fish t h r o m b o c y t e s are of interest, w h i c h s u g g e s t an i d e n t i t y of l y m p h o c y t e s and t h r o m b o c y t e s in this species. For the f u r t h e r c l a r i f i c a t i o n of this m a i n q u e s t i o n functional and c o m b i n e d m o r p h o l o g i c a l and i m m u n o l o g i c a l e x p e r i ments are u n d e r w a y w i t h b u r s e c t o m i z e d and t h y m e c t o m i z e d animals, respectively.
ACKNOWLEDGEMENTS This w o r k was s u p p o r t e t by the A u s t r i a n C a n c e r R e s e a r c h Found. The i n c i d e n t light f l u o r e s c e n c e m i c r o s c o p i c e q u i p m e n t was kindly p r o v i d e d from C.ZEISS, F r a n k f u r t branch, F r a n k f u r t / M a i n , FRG. We also a c k n o w l e d g e the free supply of m e d i a from LS, L a b o r service,
M~nchen,
FRG.
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