PRODUCTION LYMPHOCYTES
OF
INFLUENZA
OF
THE
E . N. A g r a n o v s k a y a , Y a . S. S h v a r t s m a n ,
ANTIBODIES
RESPIRATORY
BY
TRACT
A. I. M a l ' t s e v a , a n d L. V. S o l i l o v a - B o z h e n k o
UDC 616.988.75-097.5-031.84:611.2
Antibody production against influenza v i r u s by l y m p h o c y t e s of the human n a s o p h a r y n g e a l tonsils and the t r a c h e a l wall of r a t s was investigated. Antibody-producing c e l l s w e r e found in p r e p a r a t i o n s of lymphadenoid tissue r e m o v e d in the p o s t e p i d e m i c period. I n t r a n a s a l i m m u nization of r a t s with living influenza vaccine led to the accmnulation of p r o d u c e r c e l l s in the t r a c h e a l walt and of antibodies in the s e c r e t i o n s of the r e s p i r a t o r y tract. R e i m m u n i z a t i o n was followed by a w e l l - m a r k e d s e c o n d a r y response. A c h a r a c t e r i s t i c feature of s e c r e t o r y immunity is the slower f o r m a t i o n of p r o d u c e r c e l l s and of antibodies than is o b s e r v e d in the s y s t e m of general immunity, KEY WORDS: antibodies; lymphoeytes; influenza; r e s p i r a t o r y tract.
The concept of two s y s t e m s of specific h u m o r a l immunity has been f o r m e d in r e c e n t y e a r s . The f i r s t s y s t e m is r e s p o n s i b l e for the production of antibodies contained in the blood s e r u m and o t h e r t i s s u e s of the internal milieu, the second for the synthesis of antibodies p r e s e n t in the e x t e r n a l s e c r e t i o n s and e x c r e t i o n s [4-7]. Most of the s e c r e t o r y antibodies a r e synthesized by l y m p h o c y t e s located in the walls of o r g a n s c o m municating d i r e c t l y with the e x t e r n a l e n v i r o n m e n t [3, 41. Meanwhile, the development of the p l a s m a - c e l l r e s p o n s e and the d y n a m i c s of accumulation of the antibody-producing c e l l s in the m u c o s a and s u b m u c o s a of the r e s p i r a t o r y and a l i m e n t a r y t r a c t s have been inadequately studied. The production of influenza antibodies by the cells of the lymphadenoid tissue of unimmunized children and the t r a c h e a of a n i m a l s immunized with influenza v i r u s was investigated. EXPERIMENTAL
METHOD
Lymphadenoid tissue was obtained from children undergoing o p e r a t i o n s for h y p e r t r o p h y of the n a s o pharyngeal tonsils; 140 W i s t a r r a t s weighing 120-150 g w e r e immunized i n t r a n a s a l l y under s u p e r f i c i a l e t h e r a n e s t h e s i a with 0.8 ml of an atlantoic culture of A/Hong Kong/1/68/21 (H3N2) influenza virus diluted 1 : 10 with 6 % peptone solution. Instead of the virus, the r a t s of the control group r e c e i v e d peptone solution. The a n i m a l s w e r e s a c r i f i c e d in groups of 8-10 before and at various t i m e s a f t e r immunization. Blood and m u cus f r o m the r e s p i r a t o r y o r g a n s w e r e collected, and the t r a c h e a , the lymph gland and its bifurcation, and the spleen were removed. The t r a c h e a was divided longitudinally and the m u c o s a and s u b m u c o s a w e r e r e moved with a scalpel and t r a n s f e r e d into medium No. 199. To obtain cell suspensions the tissue was teased a p a r t with dissecting needles and then filtered through Kapron gauze. Human (for testing lymphadenoid t i s sue cells) or s h e e p ' s (for testing the animal cells) red cells, conjugated with influenza v i r u s by c h e m i c a l bonds [1], were used as the test antigen for d e t e r m i n i n g the n u m b e r of p r o d u c e r cells. The s a m e p r e p a r a tion was ~sed to d e t e r m i n e antibodies in the indirect hemagglutination test (IHT). To detect p r o d u c e r c e l l s L a b o r a t o r y of Immunology and I m m u n o c h e m i s t r y and L a b o r a t o r y of Pathomorphology of Acute Virus R e s p i r a t o r y D i s e a s e s , All-Union S c i e n t i f i c - R e s e a r c h Institute of Influenza, Ministry of Health of the USSR, Leningrad. (Presented by A c a d e m i c i a n of the A c a d e m y of Medical Sciences of the USSR A. A. S m o r o d i n tsev.) T r a n s l a t e d f r o m Byulleten' t~ksperimental'noi Biologii i Meditsiny, Vol. 78, No. 7, pp, 81-84, July, 1974. Original a r t i c l e submitted October 29, 1973. 9 1974 Consultants Bureau, a division of Plenum Publishing Corporation, 227 West 17th Street, New York, N. Y. 10011. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission of the publisher. A copy of this article is available from the publisher for $15.00.
798
TABLE I . D e t e r m i n a t i o n of C e l l s Producing Antibodies against A / H o n g K o n g / 1 / 6 8 Influenza V i r u s in Human L y m p h a d e n o i d T i s s u e Time of operation
No. of cells }No. of active cells a-bs~ -~
Iinvestigated
Before influ: enza epidemic
Af~er infl~lenza epidemic
a d r o p of s u s p e n s i o n containing l0 s c e l l s / m l was added to a d r o p of a b r o t h c u l t u r e of a nonpathogentc s t r e p t o c o c c u s and an equal v o l u m e of a s u s p e n s i o n of r e d c e l l s e i t h e r s e n s i t i z e d o r not s e n s i tized with the v i r u s . The m i x t u r e was kept for 30 rain at r o o m t e m p e r a t u r e and i n t e r a c t i o n of the c e l l s with the t e s t antigen s t u d led with the aid of a p h a s e - c o n t r a s t o p t i c a l s y s t e m [2]. C e l l s with the m o r p h o l o g i c a l c h a r a c t e r i s t i c s of p l a s m a c e l l s but without phagocytic a c t i v i t y and fixing not l e s s than 3 r e d c e l l s on t h e i r s u r f a c e w e r e taken as p r o d u c e r s . When the r e s u l t s of the e x p e r i m e n t s w e r e r e a d the n u m b e r of c e l l s i n t e r a c t i n g with u n s e n s i t i z e d e r y t h r o c y t e s was s u b t r a c t e d from the n u m b e r of c e l l s fixing s e n s i t i z e d r e d c e l l s . The d i f f e r e n c e thus obtained gave the n u m b e r of c e l l s p r o d u c i n g a n t i b o d i e s a g a i n s t influenza v i r u s in the m a t e r i a l studied,
1280 1556 900 1300 1194 1550 1820 1264
0,3 0,[ 0,4
0,3
5 14 13 10
0,4 0,9 0,7 0,8
To study the p l a s m a - c e l l r e s p o n s e in the t r a c h e a and l y m phoid t i s s u e the o r g a n s w e r e fixed in C a r n o y ' s fluid. Sections w e r e s t a i n e d by B r a c h e t ' s method. The i n t e n s i t y of d e v e l o p m e n t of the p l a s m a - c e l l r e s p o n s e s was e s t i m a t e d in s e c t i o n s through the lymphoid o r g a n s and t r a c h e a at the s i t e s of the g r e a t e s t c o n c e n t r a t i o n s of p l a s m a c e l l s in 50 f i e l d s of vision. The r e s u l t s w e r e s u b j e c t e d to s t a t i s t i c a l a n a l y s i s , using W i l c o x o n ' s c r i t e r i o n , by the Minsk-22 c o m p u t e r in the L a b o r a ~ r y of G e n e r a l E p i d e m i o l o g y of the I n s t i t u t e .
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Days after injection of antigen Fig. 1. D y n a m i c s of antibody a c c u m u l a t i o n in s e c r e t i o n of r e s p i r a t o r y tract and blood serum, and of producer cells and plasma cells in the tracheal wall and lymphoid organs of rats immunized with A/Hong Kong/1/68 influenza virus: A) producer cells and plasma cells in the spleen; B) producer cells and plasma cells in the lymph gland at the tracheal bifurcation and antibodies in the blood serum; C) producer cells and plasma cells in the tracheal wall, antibodies in the secretion of the respiratory tract. 1) Antibody-producingcells; 2) antibody titers. Columns denote plasma cellsArrow marks injection of antigen.
799
EXPERIMENTAL
RESULTS
Investigation of 8 p r e p a r a t i o n s of lymphadenoid tissue r e v e a l e d a significant (P < 0.05) i n c r e a s e in the n u m b e r of active ceils in the m a t e r i a l obtained a f t e r the end of an influenza epidemic (Table 1). A m a r k e d i n c r e a s e in the antibody concentration in the blood s e r u m of the immunized r a t s was o b s e r v e d a f t e r the 5th day, coinciding with the maximal i n c r e a s e in the n u m b e r of active ceils in the lymph gland draining the t r a c h e a and in the spleen (Fig. 1). By c o n t r a s t , a significant i n c r e a s e in the antibody concentrations in the s e c r e t i o n a f t e r a single i m munization was o b s e r v e d for the f i r s t time only on the 10th day; the n u m b e r of p l a s m a c e i l s in the t r a c h e a l wall was mxchanged and the r a t e of i n c r e a s e in the n u m b e r of p r o d u c e r cells was slower. This index r e a c h e d its m a x i m u m by the 15th day. Repeated antigen stimulation led to a much m o r e active accumulation of p l a s m a ceils and p r o d u c e r c e l l s in the t r a c h e a l m u c o s a and s u b m u c o s a and of antibodies in the s e c r e t i o n f r o m the mucous glands of the trachea. Meanwhile the n u m b e r of p r o d u c e r c e l l s in the tissue of the lymph gland at the bifurcation of the t r a c h e a and of the spleen i n c r e a s e d and there was a s h a r p r i s e in the a n t i body concentration in the blood s e r u m . The r e s u l t s thus c o n f i r m e d o b s e r v a t i o n s concerning the local synthesis of all o r m o s t of the s e c r e tory antibodies~ Evidence in support of this conclusion is given by the good a g r e e m e n t between the dynamics of their accumulation in the lumen of the t r a c h e a and the formation of p l a s m a c e l l s and p r o d u c e r c e l l s in the wall of that organ. The p r e s e n c e of a s e c o n d a r y r e s p o n s e in the s y s t e m for the s y n t h e s i s of s e c r e t o r y antibodies indicates the value of repeated injections of v i r a l and b a c t e r i a l vaccines in intranasal, a e r o s o l , and e n t e r a l immunization. In o r d e r to e s t a b l i s h the optimal intervals for this revaccination, the pattern of development o[ the immunologic m e m o r y in the s y s t e m of s e c r e t o r y immunity m u s t be investigated. A m o r e intensive study is also required of the c a u s e s of the much g r e a t e r r,~lative n u m b e r of antibody~producing c e l l s in the t r a c h e a than in the lymph gland and spleen. This is evidently explained by migration of p r e c u r s o r c e l l s capable of interacting with the immunogen into the focus of antigenic stimulation. LITERATURE 1. 2. 3. 4. 5. 6. 7.
800
CITED
E . P . Korneeva, Ya. S. S h v a r t s m a n , and A. A. Smorodintsev, Zh. Mikrobiol., No. 3, 51 (1972). Ya. S. S h v a r t s m a n , Byull. t~ksperim. Biol. i Med., No. 12, 75 (1966). P . A . Crabbe, D. R. Nash, D. H. Bazin, et al., J. Exp. Med., 130, 723 (1969). V. Pesak, Folia Microbiol. (Prague), 16._., 323 (1971). R . D . Rossen, W. T. Butler, R. H. Waldman, et al., J. Clin. Invest., 45, 768 (1966). R . D . Rossen, J. A. Kasel, and R. B. Couch, Prog. Med. Virol., 13, 194 (1971). T . B . T o m a s i and J. Bienenstock, Adv, Immunol., 9_, 2 (1968).