90

Specialia

f r a c t i o n 1251-18 (48% for t h e u n a b s o r b e d a n d 3 5 % for t h e a b s o r b e d a n t i s e r u m ) . T h a t for a n t i - P M was h i g h w i t h f r a c t i o n s 1251-15 a n d ~51-16 (21%) a n d f r a c t i o n ~51-19 (23~ a n d lower (15%) w i t h f r a c t i o n 1251-18. T h e b i n d i n g c a p a c i t y for a n t i - C E A was 9 % w i t h fract i o n s 1~51-13 a n d 1~51-14 a n d nil w i t h f r a c t i o n s 1251-18 (figure 3b). No b i n d i n g c a p a c i t y was d e t e c t e d w h e n u n a b s o r b e d a n d a b s o r b e d a n t i - P M K a n d a n t i - P M were ass a y e d w i t h 125I-CEA. Conclusion. I n t h e d o u b l e diffusion test, p e a k 5 o b t a i n e d b y c h r o m a t o g r a p h y of M K o n D E A E - c e l l u l o s e r e a c t s w i t h a b s o r b e d a n t i - P M K m i d w a y b e t w e e n t h e 2 wells. T h e failure of p e a k 5 to p r o d u c e p r e c i p i t a t i o n lines w i t h a n t i - C E A and, a f t e r f u r t h e r p u r i f i c a t i o n , w i t h a b s o r b e d a n t i - P M K c a n b e a t t r i b u t e d t o t h e p o o r e r s e n s i t i v i t y of this method compared to radioimmunology. T h e r e s u l t s of t h e d o u b l e diffusion t e s t t h u s i n d i c a t e t h a t t h e p r e c i p i t a t i o n line b e t w e e n p e a k 5 a n d a b s o r b e d a n t i P M K is d u e t o a n t i g e n or a n t i g e n s associated w i t h b r e a s t t u m o r s t h a t do n o t c r o s s r e a c t w i t h a n t i - C E A in t h i s

EXPERIENTIA 33/1

m e t h o d , in spite of t h e c o n s i d e r a b l e c r o s s r e a c t i v i t y obs e r v e d in t h e R. I. A. F r a c t i o n 18 o b t a i n e d b y gel f i l t r a t i o n of p e a k 5 of M K was c h o s e n b e c a u s e of its h i g h r e s p o n s e t o a n t i - C E A in t h e r a d i o i m m u n o l o g i c a l s t u d y . F r a c t i o n 18 was labeled a n d b y gel f i l t r a t i o n y i e l d e d f r a c t i o n s (1251-13 a n d 1251-14) t h a t c r o s s r e a c t e d w i t h a n t i - C E A a n d also r e a c t e d w i t h anti-PMK. A n t i g e n ( s ) in t h e s e f r a c t i o n s do n o t , h o w e v e r , a p p e a r to b e CEA, since a n t i - P M K , w h i c h b i n d s 1~51-13 a n d 1~51-14 t o a fair e x t e n t (13% b i n d i n g c a p a c i t y ) , does n o t b i n d I~sI-CEA, a v e r y p u r e m a t e r i a l . On t h e o t h e r h a n d , fract i o n 1251-18 r e a c t s o n l y w i t h a n t i - P M K . I t is t h e r e f o r e p r o b a b l e t h a t f r a c t i o n s 1~51-13 a n d 1251-14 c o n t a i n a n t i gens a s s o c i a t e d w i t h b r e a s t c a r c i n o m a t h a t crossreact w i t h anti-C]?;A. I t c a n i n s t e a d b e p o s t u l a t e d t h a t fract i o n 1251-18 c o n t a i n s a n t i g e n s a s s o c i a t e d w i t h b r e a s t c a r c i n o m a t h a t do n o t c r o s s r e a c t w i t h a n t i - C E A a n d react only with an antibody produced using antigens e x t r a c t e d f r o m t h i s t y p e of t u m o r .

I m m u n o - e l e c t r o n m i c r o s c o p i c study of h u m a n EA rosettes L. FONTANA a n d G. TONIETTI 1

I. Patologia Medica and I I . Clinica Medica dell' Universitg, Policlinico Umberto, 1-00167, Roma (Italy), 29 June 7976 Summary. T h e i m m u n o - e l e c t r o n m i c r o s c o p i c s t u d y of h u m a n E A rosettes, u s i n g f e r r i t i n - l a b e l l e d a n t i - h u m a n I g G a n t i s e r u m , s h o w e d clusters of I e r r i t i n g r a n u l e s a t t h e p o i n t s of c o n t a c t b e t w e e n e r y t h r o c y t e s a n d l y m p h o i d ceils, ind i c a t i n g t h a t t h e links b e t w e e n F c f r a g m e n t of I g G o n t h e surface of e r y t h r o c y t e s a n d specific r e c e p t o r on t h e surface of l y m p h o i d cell c o r r e s p o n d to t h e sites of m o r p h o l o g i c a l i n t e r a c t i o n b e t w e e n t h e 2 cell types. I n p r e v i o u s studies, we d e s c r i b e d t h e u l t r a s t r u c t u r a l a p p e a r a n c e of t h e i n t e r a c t i o n b e t w e e n r o s e t t i n g l y m p h o i d cells a n d e r y t h r o c y t e s in d i f f e r e n t t y p e s of rosettes. T h e h i g h e s t degree of i n t e r a c t i o n , c o n s i s t i n g of i n t e r d i g i t a t i o n s b e t w e e n t h e 2 cell types, was o b s e r v e d in E A r o s e t t e s 3. I n t h i s article, we r e p o r t t h e results of a n i m m u n o e l e c t r o n m i c r o s c o p i c s t u d y of h u m a n E A r o s e t t e s u s i n g ferritin-labelled anti-human IgG antibody. Materials and methods. G r o u p 0 R h p o s i t i v e h u m a n n o r m a l e r y t h r o c y t e s sensitised w i t h a h u m a n a n t i - D antiserum and peripheral blood normal human lympho-

c y t e s were used for t h e p r e p a r a t i o n of h u m a n E A r o s e t t e s a c c o r d i n g to t e c h n i q u e s a l r e a d y described for E A ox r o s e t t e s 2. A f t e r p r e p a r a t i o n of rosettes, t h e cell m i x t u r e was i n c u b a t e d for 15 m i n a t r o o m t e m p e r a t u r e w i t h a ferritin-labelled anti-human IgG antibody, washed 3 t i m e s w i t h b u f f e r e d saline a n d t h e n processed for elect r o n m i c r o s c o p y . F o r control, t h e s a m e e r y t h r o c y t e s , e i t h e r u n t r e a t e d or sensitised w i t h t h e a n t i - D a n t i s e r u m , were i n c u b a t e d w i t h f e r r i t i n alone or w i t h ferritinlabelled a n t i - m o u s e I g G a n t i b o d y . Results, R o s e t t i n g l y m p h o c y t e s , in several experim e n t s , a v e r a g e d 12%. E A rosettes, o b s e r v e d in t h e elec-

Fig. 1. Parts of 2 erythroeytes and of a lymphoid cell which are not part of a EA rosette. Clusters of ferritin granules, arranged at fairly regular intervals, are present at the erythrocytes surface. No ferritin granules are visible on the surface of the lymphoid cell. • 29000.

Fig. 2. Part of EA rosette. Interdigitations between arl erythrocyte and a lymphoid cell are visible. The points of contact between the 2 cell types are heavily labelled with ferritin. • 46000,

15.1. 1977

Specialia

i r o n microscope, s h o w e d t h e f e a t u r e s a l r e a d y described, with interdigitations between erythrocytes and lymphoid cells ~. R e s e t t i n g l y m p h o i d cells were in no i n s t a n c e labelled w i t h ferritin. O n l y 4 % of n o n - r e s e t t i n g l y m p h o c y t e s s h o w e d scarce ferritin g r a n u l e s a t t h e i r surface. All e r y t h r o c y t e s were h e a v i l y labelled w i t h ferritin granules, g a t h e r e d in clusters a r r a n g e d a t fairly regular intervals. I n all r o s e t t e s observed, clusters of ferritin were seen a t t h e p o i n t s of c o n t a c t b e t w e e n e r y t h r o c y t e s a n d l y m p h o i d cells. N e i t h e r e r y t h r o c y t e s nor l y m p h o i d cells were labelled w i t h ferritin in t h e c o n t r o l e x p e r i m e n t s . Discussion. Our i m m u n o - e l e c t r o m i c r o s c o p i c s t u d y confirms, on m o r p h o l o g i c a l grounds, t h a t in lEA r o s e t t e s sensitized e r y t h r o c y t e s a n d l y m p h o i d cells are held tog e t h e r b y t h e link b e t w e e n t h e Fc f r a g m e n t of igG,

91

p r e s e n t on t h e surface of t h e former, a n d specific rec e p t o r s on t h e surface of t h e latter. F u r t h e r m o r e , this s t u d y seems to i n d i c a t e t h a t in h u m a n E A rosettes, t h e r e s e t t i n g l y m p h o i d cells lack surface IgG which, if present, would be labelled w i t h ferritin. F u r t h e r i m m u n o e l e c t r o n m i c r o s c o p i c studies are n o w in p r o g r e s s in o r d e r to elucidate o t h e r a s p e c t s of c e l l - m e d i a t e d i m m u n i t y in vitro.

Institute (ff Clinical Immunology, University of Aquile, Italy; recipient of a Grant of the Italian Consiglio Nazionale Ricerche. 2 G. TONIETTI, G. PEcci, G. D'AcuNTo, E. Lio,~, M. E. MERCAI~LI, R. PERRICONE and L. FONTANA,Experientia 31, 1464 (1975).

A d h e s i o n and a g g r e g a t i o n of h u m a n platelets to rabbit s u b e n d o t h e l i u r n . A n e w a p p r o a c h for i n v e s t i g a t i o n : Specific a n t i b o d i e s J. P. Caen, H. Michel, G. Tobelem, E. B o d e v i n and S. L e v y - T o l e d a n o INSERM, U. R. 150, C. N . R. S., E. R. A . 335, H @ i t a l Lariboisi~re, 2, rue Ambroise Pard, F 75475 Paris-Cedex lO (France), 5 J u l y 1976

S u m m a r y . A n IgG a n t i b o d y occurring in a r e c e n t l y t r a n s f u s e d t h r o m b a s t h e n i c p a t i e n t i n h i b i t e d all t h e A D P - m e d i a t e d aggregations and p l a t e l e t - p l a t e l e t i n t e r a c t i o n ( t h r o m b u s f o r m a t i o n ) on r a b b i t a o r t a s u b e n d o t h e l i u m ; a n o t h e r IgG a n t i b o d y occurring in a m u l t i t r a n s f u s e d B e r n a r d - S o u l i e r p a t i e n t i n h i b i t e d r i s t o c e t i n and b o v i n e f a c t o r V I I I m e d i a t e d a g g r e g a t i o n and p l a t e l e t - s u b e n d o t h e l i u m interaction. The i n t e r a c t i o n of p l a t e l e t s w i t h r a b b i t s u b e n d o t h e l i u m in t h r o m b a s t h e n i a a n d in t h e B e r n a r d - S o u l i e r s y n d r o m e has a l r e a d y been s t u d i e d ~, 2. W h e r e a s t h e t h r o m b u s form a t i o n was normal, i m p a i r e d a d h e s i o n was f o u n d in t h e B e r n a r d Soulier s y n d r o m e , and it was c o n s i d e r e d t h a t an abnormal interaction between von \u factor a n d t h e p l a t e l e t m e m b r a n e could be responsible for this abn o r m a l p l a t e l e t a d h e s i o n 2. I n c o n t r a s t , a d h e s i o n was e i t h e r n o r m a l or o n l y slightly d e c r e a s e d in t h r o m b a s t h e n i a w i t h a s u b e n d o t h e l i a l surface covered only b y a

Table 1. Effect of the 1.... serum on AI)P-induced aggregatiol~ and interaction with rabbit subendothelimn of normal hmnan platelets. Results are expressed in percent Final serum dilution

ADP-induced aggregation

Interaction with rabbit subendothelium Adhesion Thrombi

0 1:40 1:20

100 70 30

46 53 34

13.75 0.30 0

Table 2. Effect of the P... IgG on ristocetin induced aggregation and interaction with rabbit subendothelium of normal human platelets. Results are expressed in percent Final lgG dilution

Ristocetin-induced aggregation

Interaction with rabbit subendothelium Adhesion Thrombi

0 1:80 1:40

100 50 0

46 48 24

13.75 9 0

m o n o l a y e r of p l a t e l e t s ; t h e m o s t s t r i k i n g d e f e c t on this last disease was t h e absence of t h r o m b i ~,3. The d e f e c t associated w i t h b o t h t h o s e disorders of p l a t e l e t f u n c t i o n has been r e p o r t e d to be a b n o r m a l i t i e s in t h e p l a t e l e t surface g l y c o p r o t e i n s 4-~. R e c e n t l y we h a v e h a d the o p p o r t u n i t y of s t u d y i n g 2 diff e r e n t a n t i p l a t e l e t antibodies. The first one o c c u r r e d in a p o l y t r a n s f u s e d t h r o m b a s t h e n i c p a t i e n t ( L . . . ) a n d inh i b i t e d in v i t r o t h e a g g r e g a t i o n of n o r m a l p l a t e l e t s ind u c e d b y A D P L The second a n t i b o d y o c c u r r e d in a p o l y t r a n s f u s e d B e r n a r d Soulier p a t i e n t ( P . . . ) a n d inh i b i t e d in v i t r o t h e a g g r e g a t i o n of n o r m a l p l a t e l e t s b y r i s t o c e t i n w i t h o u t a n y effect on t h e A D P m e d i a t e d aggregationsS. The aim of this s t u d y was t o correlate t h e effect of b o t h t h o s e p l a t e l e t a n t i b o d i e s on p l a t e l e t funct i o n s t e s t e d in t h e a g g r e g o m e t e r a n d on t h e i n t e r a c t i o n of p l a t e l e t s w i t h s u b e n d o t h e l i a l surface of r a b b i t aorta. F o r t h e d e t e r m i n a t i o n of this i n t e r a c t i o n , we h a v e used the morphometric technique described by Baumgartner". C i t r a t e d whole blood f r o m n o r m a l s u b j e c t s was c i r c u l a t e d in t h e p r e s e n c e or a b s e n c e of s e r u m f r o m p a t i e n t L . . . a n d of purified IgG f r o m p a t i e n t P . . . The c o n t r o l whole blood was c o m p a t i b l e in t h e A, B, 0, Rhesus, s y s t e m w i t h

1 Th. B. Tsehopp, H. J. Weiss and H. R. Baumgartner, Experientia 31, 113 (1975). 2 H. J. Weiss, Th. B. Tschopp and H. R. Baumgartner, Am. J. Med. 57, 920 (1974). 3 H. Michel, E. Bodevin and J. P. Caen, Thromb. Diath. haemorrh. 3/1, 600 (1975). 4 A. Nurden and J. P. Caen, Br. J. Haemat 28, 253 (1974). 5 A. Nurden and J. P. Caen, Nature 255, 720 (1975). 6 J. P. Caen, A. T. Nurden, C. Jeanneau, H. Michel, G. Tobelem, S. Levy-Toledano, Y. Sultan, F. Valensi and J. Bernard, J. Lab. Clin. Med. 87, 586 (1976). 7 S. Levy-Toledano, R. Bredoux, F. Rendu, G. Tobelem, L. Degos and J. P. Caen, Thromb. Diath. haemorrh. 34, 921 (1975). 8 G. Tobelem, S. Levy-Toledano, L. Degos, H. Michel, A. Nurden, R. Bredoux and J. P. Caen, Nature 263, 427 (1976). 9 H. R. Baumgartner, Microvasc. Res. 5, 167 (1973).

Immuno-electronmicroscopic study of human EA rosettes.

90 Specialia f r a c t i o n 1251-18 (48% for t h e u n a b s o r b e d a n d 3 5 % for t h e a b s o r b e d a n t i s e r u m ) . T h a t for a n...
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