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).

Production of influenza antibodies by lymphocytes of the respiratory tract.

Antibody production against influenza virus by lymphocytes of the human nasopharyneal tonsils and the tracheal wall of rats was investigated. Atibody-...
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