120
Specialia
p i p e t t e t o a l e n g t h of precisely 15 m m in t h e p i p e t t e . E a c h p i p e t t e h a s a n a p e r a t u r e j u s t large e n o u g h to allow passage of a 21-gauge needle. T h e p i p e t t e s are p l a c e d in a t e s t tube rack with their tip suspended into a test tube and i n c u b a t e d a t 37 ~ V e n o u s b l o o d was d r a w n in h e p a r i n i z e d tubes from healthy human volunteers. Each volunteer serves as his o w n c o n t r o l in t h a t e a c h s a m p l e of b l o o d was d i v i d e d i n t o e q u a l p o r t i o n s . To e a c h m l of t e s t s a m p l e 0.01 m l of e p i n e p h r i n e (1:1000 dilution) was a d d e d . T h e b l o o d was t h e n i n c u b a t e d for 10 min, m i x e d b y g e n t l e t u r n i n g a n d 1 m l a l i q u o t s were a d d e d to t h e p i p e t t e s a n d allowed t o filter t h r o u g h o v e r 10 m i n . E a c h e x p e r i m e n t w i t h a n d w i t h o u t e p i n e p h r i n e was r u n in t r i p l i c a t e . T h e n u m b e r of g r a n u l o c y t e s in t h e b l o o d was m e a s u r e d before and after passage through the column and the percentage of g r a n u l o c y t e a d h e r e n c e was c a l c u l a t e d . Results. T h e a d h e r e n c e of g r a n u l o c y t e s was d e c r e a s e d b y 27.9% in t h e p r e s e n c e of e p i n e p h r i n e (table). T h i s was s t a t i s t i c a l l y s i g n i f i c a n t a t p < 0.0025 level a n a l y z e d b y the Student t-test. Discussion. M a n y clinical c o n d i t i o n s w i t h i n c r e a s e d susc e p t i b i l i t y to i n f e c t i o n c a n n o t be e x p l a i n e d b y decreases in g r a n u l o c y t e p h a g o c y t o s i s or c h e m o t a x i s a l o n e a n d
Effect of Epinephrine on granulocyte adhesion
EXPERIENTIA 34/1
a d d i t i o n a l g r a n u l o c y t e f u n c t i o n defects m u s t be considered. G r a n u l o c y t e a d h e r e n c e to n y l o n fibres or glass m a y be used as m e a s u r e of t h e i r a b i l i t y to a d h e r e t o e n d o t h e l i a l walls b u t t h i s r e q u i r e s f u r t h e r proof. I t h a s b e e n s h o w n b y M a c G r e g o r e t al. 3 t h a t a n t i - i n f l a m m a t o r y d r u g s i n h i b i t g r a n u l o c y t e a d h e r e n c e to n y l o n fibres a n d s u g g e s t e d t h a t t h i s m a y b e t h e e x p l a n a t i o n for t h e i r effects in vivo. O t h e r a g e n t s h a v e also b e e n s h o w n t o i n h i b i t g r a n u l o c y t e a d h e s i o n s u c h as e t h a n o l 3, i o d o a c e t a m i d e 5, a n d E D T A 3, ~, cyclic A M P 7, colchicine *, a n d glycolitic i n h i b i t o r s s, w h e r e a s o x i d a t i v e i n h i b i t o r s s h a d n o effect. T h e p r e s e n c e of c e r t a i n ions ( m a g n e s i u m a n d p e r h a p s calcium)3, 6 a p p e a r to b e necessary. A b n o r m a l g r a n u l o c y t e a d h e s i o n h a s b e e n d e m o n s t r a t e d in several clinical c o n d i t i t i o n s , i.e. l e u k e m i a s a n d a c u t e p o s t s t r e p t o c o c c a l g l o m e r u l 0 n e p h r i t i s 9. I n t h e l a t t e r , t h i s a b n o r m a l i t y c a n be followed as a m e a s u r e of r e s o l u t i o n of disease. T h e a d m i n i s t r a t i o n of e p i n e p h r i n e in vivo causes t h e g r a n u l o c y t e s to d e m a r g i n a t e a n d e n t e r t h e c i r c u l a t i o n . This study showed a decreased granulocyte adhesion to nylon fibres in t h e p r e s e n c e of e p i n e p h r i n e in v i t r o s u g g e s t i n g t h a t s i m i l a r m e c h a n i s m s m a y o p e r a t e in b o t h s y s t e m s . G r a n u l o c y t e a d h e s i v e n e s s s h o u l d be s t u d i e d in clinical s i t u a t i o n s associated w i t h a n i n c r e a s e d s u s c e p t i b i l i t y t o infection. I n f o r m a t i o n o b t a i n e d f r o m t h e s e s t u d i e s m a y p r o v i d e b e t t e r u n d e r s t a n d i n g of t h e role g r a n u l o c y t e a d h e s i o n p l a y s in p r e v e n t i n g infection.
Percent adhesion j_ SEM Control Epinephrine
75.7 • 5.0 47.8 :t: 6.4 p < 0.0025
Each value represents 8 experiments performed'on blood from healthy male volunteers.
4 R.R. MacGregor, Am. J. Med. 61, 597 (1976). 5 J . E . Garvin, J. expl. Med. 114, 51 (1961). 6 R.E. Bryant, R.M. DesPrez and D.E. Rogers, Yale J. Biol. Med. d0, 192 (1967). 7 R.E. Bryant and M. Suteliffe, Clin. Res. 21, 594 (1973). 8 R. Penny and D. A. G, Galton, Br. J. Haemat. 12, 633 (1966). 9 E.J. Ruley, S. Huang, J. Plant and N. Morris, J. Pedlar. 89, 748 (1976).
Accumulation of 2 - g m latex particles in mouse Peyer's patches during chronic latex feeding1, 2 M. E. L e F e v r e 3, J. W. V a n d e r h o f f 4, J. A. Laissue s a n d D. D. Joel
Medical Research Center, Brookhaven National Laboratory, Upton (New York 11973, USA), 1 July 1977 Summary. 2-~xm l a t e x p a r t i c l e s a c c u m u l a t e d in m a c r o p h a g e s in i n t e s t i n a l P e y e r ' s p a t c h e s of mice g i v e n l a t e x s u s p e n s i o n s as d r i n k i n g fluid for 2 m o n t h s . T h e n u m b e r of p a r t i c l e s a c c u m u l a t i n g was a d i r e c t ( b u t n o n l i n e a r ) f u n c t i o n of t h e n u m b e r ingested. S o m e of t h e l a t e x p a r t i c l e s were still p r e s e n t in P e y e r ' s p a t c h e s 6 weeks a f t e r t h e cessation of l a t e x feeding. T h e e p i t h e l i u m of g u t - a s s o c i a t e d l y m p h o i d tissues ( G A L T ; P e y e r ' s p a t c h e s , a p p e n d i x , sacculus r o t u n d u s ) a p p e a r s t o b e a r o u t e b y w h i c h s m a l l i n e r t p a r t i c u l a t e s pass t h e m u c o s a l b a r r i e r of t h e m a m m a l i a n i n t e s t i n a l tract6, L P r e v i o u s m o r p h o l o g i c a l s t u d i e s h a v e d e s c r i b e d t h e mig r a t i o n of c a r b o n , t r y p a n blue, a n d f e r r i t i n p a r t i c l e s i n t o t h e G A L T s-12. T h e s e p a r t i c u l a t e s are small, less t h a n 0.1 ~xm in d i a m e t e r , a n d were seen w i t h t h e l i g h t m i c r o scope b e c a u s e of t h e i r a c c u m u l a t i o n i n t o aggregates. T h e p r e s e n t c o m m u n i c a t i o n describes t h e p e n e t r a t i o n of t h e m o u s e P e y e r ' s p a t c h e p i t h e l i u m b y m u c h l a r g e r particles, n a m e l y l a t e x s p h e r e s of 2%tm d i a m e t e r . L a t e x p a r t i c l e s resist d e g r a d a t i o n in t h e i n t e s t i n e a n d are large e n o u g h to b e seen a n d c o u n t e d w i t h t h e l i g h t microscope, p e r m i t t i n g a s e m i q u a n t i t a t i v e a s s e s s m e n t of t h e i r u p t a k e a n d distrib u t i o n . T h e r e s u l t s i n d i c a t e t h a t m a n y t h o u s a n d s of l a t e x p a r t i c l e s a c c u m u l a t e d in P e y e r ' s p a t c h e s d u r i n g c h r o n i c feeding of mice w i t h l a t e x suspensions. F u r t h e r m o r e ,
m a n y of t h e p a r t i c l e s were r e t a i n e d for m o r e t h a n 6 weeks a f t e r t h e c e s s a t i o n of l a t e x feeding. Materials and methods. 3 w a t e r s u s p e n s i o n s of p o l y v i n y l t o l u e n e l a t e x ( m e a n p a r t i c l e d i a m e t e r • SD, 2.02 d0.014 ~ m ; p a r t i c l e d e n s i t y , 1.027; i d e n t i f i c a t i o n No. LS-1078-B, T h e D o w C h e m i c a l Co.) c o n t a i n i n g 1.0, 0.1 a n d 0.01% solids were g i v e n as d r i n k i n g fluid to 3 g r o u p s of 11-week-old f e m a l e Swiss mice. T h e mice were g i v e n free access to t h e s u s p e n s i o n s a n d to s t a n d a r d p e l l e t e d m o u s e food. T h e l a t e x s u s p e n s i o n s were given for 61 d a y s followed b y a period of 2 t o 6 weeks d u r i n g w h i c h t h e mice r e c e i v e d latex-free w a t e r . A c o n t r o l g r o u p was g i v e n t a p w a t e r t o d r i n k . No a t t e m p t was m a d e to d e t e r m i n e t h e a m o u n t of l a t e x i n g e s t e d b y e a c h m o u s e ; h o w e v e r , t h e v o l u m e s of fluid c o n s u m e d b y t h e 4 g r o u p s did n o t a p p e a r to differ. All mice g a i n e d w e i g h t n o r m a l l y a n d a p p e a r e d healthy.
15, 1. 1978
Specialia
U p t a k e a n d r e t e n t i o n of l a t e x in P e y e r ' s p a t c h e s w e r e demonstrated h i s t o l o g i c a l l y i n p r e p a r a t i o n s of w h o l e P e y e r ' s p a t c h e s c l e a r e d i n K O H a n d g l y c e r o l la a n d in methacrylate-embedded sections. In addition latex was r e c o v e r e d f r o m d i g e s t s of i n t e s t i n a l t i s s u e s of l a t e x - f e d m i c e . T h e e n t i r e s m a l l i n t e s t i n e w a s r i n s e d in s a l i n e , s e p a r a t e d i n t o P e y e r ' s p a t c h a n d n o n - P e y e r ' s p a t c h (rem a i n d e r ) t i s s u e , w e i g h e d , m i n c e d , a n d p l a c e d in t e s t t u b e s c o n t a i n i n g 10 m l 3 % K O H . T h e t u b e s w e r e a g i t a t e d u n t i l the tissue was completely digested. The material was then
Fig. 1. 2g.xn latex particles within the dome of a cleared Peyer's patch follicle from a mouse sacrificed 2 weeks after termination of latex feeding. Black circles are latex particles above the plane of focus. Dark shadows are large aggregates of latex below the plane (~f focus. • 520.
c e n t r i f u g e d , w a s h e d in w a t e r , a n d t h e l a t e x r e c o v e r e d b y u l t r a c e n t r i f u g a t i o n of t h e s e d i m e n t t h r o u g h a l i n e a r d e n s i t y g r a d i e n t of 0 - 2 0 % u r e a . P a r t i c l e s w h i c h b a n d e d a t the same level as known latex were recovered by syringe and an aliquot counted using a Neubauer chamber and an optical microscope. Additional aliquots were deposited on 0.8-b~m N u c l e o p o r e filters, r i n s e d w i t h w a t e r , a n d e x a m i n e d a t 5000 • b y s c a n n i n g e l e c t r o n m i c r o s c o p y . T h e recovered material contained spheres identical to the stock latex ; these were not found elsewhere in the gradient. Results and discussion. 80 w h o l e c l e a r e d P e y e r ' s p a t c h e s were examined with a wide-field inverted microscope. Latex was unaffected by the clearing process, and indiv i d u a l p a r t i c l e s c o u l d b e s e e n a t d i f f e r e n t l e v e l s in t h e t i s s u e . E a c h p a t c h c o n s i s t e d of 2 - 8 follicles; t h e d o m e ~* of e a c h follicle of l a t e x - f e d m i c e c h a r a c t e r i s t i c a l l y s h o w e d a n a c c u m u l a t i o n of l a t e x p a r t i c l e s in t h e c e n t r a l p o r t i o n . F i g u r e 1 s h o w s s u c h a n a c c u m u l a t i o n in a P e y e r ' s p a t c h f r o m a m o u s e f e d t h e h i g h e s t c o n c e n t r a t i o n of l a t e x . P a r ticles near the mucosal surface were distributed singly or in s m a l l c l u s t e r s w h e r e a s p a r t i c l e s c l o s e t o t h e s e r o s a l s u r f a c e w e r e m o s t l y in a g g r e g a t e s n e a r t h e p e r i p h e r y of t h e follicle. P a t c h e s f r o m m i c e g i v e n l o w e r c o n c e n t r a t i o n s of latex had fewer particles and smaller aggregates. Cleared p a t c h e s f r o m c o n t r o l m i c e c o n t a i n e d n o p a r t i c u l a t e s res e m b l i n g 2-b~m l a t e x . L a t e x p a r t i c l e s w e r e still v i s i b l e in d i m i n i s h e d n u m b e r s in c l e a r e d P e y e r ' s p a t c h e s 6 w e e k s a f t e r t e r m i n a t i o n of l a t e x i n g e s t i o n . T h e l o c a t i o n of l a t e x in r e l a t i o n t o c e l l u l a r e l e m e n t s c o u l d n o t b e d e t e r m i n e d in c l e a r e d p r e p a r a t i o n s , b u t in p l a s t i c e m b e d d e d s e c t i o n s t h e particles were seen to be associated with macrophages ( f i g u r e 2). T h e t a b l e l i s t s n u m b e r s of l a t e x p a r t i c l e s r e c o v e r e d a f t e r d i g e s t i o n of i n t e s t i n a l t i s s u e s a n d u l t r a c e n t r i f u g a t i o n of t h e s e d i m e n t t h r o u g h a d e n s i t y g r a d i e n t . S e d i m e n t s of control tissues centrifuged through the density gradient s h o w e d n o b a n d i n g of p a r t i c l e s a t t h e l a t e x level. T h e table shows that Peyer's patch tissue comprised only a s m a l l f r a c t i o n of t h e w e i g h t of t h e s m a l l i n t e s t i n e , b u t c o n t a i n e d t h e m a j o r p o r t i o n of t h e l a t e x . T h e p a t t e r n of u p t a k e w a s t h a t of i n c o m p l e t e s a t u r a t i o n . T h e l o c a t i o n of t h e l a t e x f o u n d in t h e r e m a i n d e r of t h e i n t e s t i n e h a s not yet been determined. l
2 3 4 5 6 7 8 9 10 Fig. 2. 2btm latex particles (arrows) within a macrophage process in a mouse Peyer's patch. Distortion of the particles from spherical shape is due to the action of methyl methacrylate which partially dissolves latex during the embedding process. Dark lymphocyte nuclei are visible together with several pale maerophages containing other partially visible latex particles. Section was taken near the serosal surface of a Peyer's patch from a mouse given a suspension of latex as drinking fluid for 2 months. H. & E. • 1500.
121
11 12 13 14
Acknowledgments. Research supported by the U. S. Energy Research and Development Administration. W~e thank Mr W. Marin, Division of Photography and Graphic Arts, Brookhaven National Laboratory, for figure 2. The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. Correspondence should be addressed to: M. E. LeFevre, Medical Research Center, Brookhaven National Laboratory, Upton, N. Y. 11973. Center for Surface and Coatings Research, Lehigh University, Bethlehem, Pa. 18015. Pathologisches Institut, Kantonsspital, CH 6000 Luzern (Switzerland). M . E . LeFevre and D. D. Joel, Life Sci., in press. R . L . Owen, Gastroenterology 72, 440 (1977}. D . E . Bockman and M. D. Cooper, Am. J. Anat. 136, 455 (1973). M. Hanaoka, R. Williams and B. Waksman, Lab. Invest. 2d, 31 (1971). M.W. Hess, H. Cottier, B. Sordat, D. D. Joel and A. D. Chanana, in: Nonspecifie Factors Influencing Host Resistance. A Reexamination, p. 447. Karger, Basel 1973. D . D . Joel, B. Sordat, M. Hess and H. Cottier, Experientia 26, 694 (1970). M. Kagan, Z. Zellforsch. 14, 544 (1931). G.L. Humanson, Animal Tissue Techniques, 3rd ed., p. 195. Freeman, San Francisco 1972. B. H. Waksinan, O. Ozer and H. E. Blythman, Lab. Invest. 28, 614 (1973).
122
EXPERIENTIA34/1
Specialia
Quantitative determination of latex recovered from Peyer's patches and remainder of small intestine No. Latex suspension fed* (% solids)
N
1.0 0.I 0.01
15 15 15
Peyer's patches Tissue weight (g)
No. particles • 105
Remainder Tissue weight (g)
No particles • 105
0.054::t=0.016 0.056-4-0.013 0.045-[ 0,008
2.13• 1.424-0.98 0.18-[-0.14
1.062• 1.078=k0.227 1.005•
1.78+0.58 1.04•
Specialia
p i p e t t e t o a l e n g t h of precisely 15 m m in t h e p i p e t t e . E a c h p i p e t t e h a s a n a p e r a t u r e j u s t large e n o u g h to allow passage of a 21-gauge needle. T h e p i p e t t e s are p l a c e d in a t e s t tube rack with their tip suspended into a test tube and i n c u b a t e d a t 37 ~ V e n o u s b l o o d was d r a w n in h e p a r i n i z e d tubes from healthy human volunteers. Each volunteer serves as his o w n c o n t r o l in t h a t e a c h s a m p l e of b l o o d was d i v i d e d i n t o e q u a l p o r t i o n s . To e a c h m l of t e s t s a m p l e 0.01 m l of e p i n e p h r i n e (1:1000 dilution) was a d d e d . T h e b l o o d was t h e n i n c u b a t e d for 10 min, m i x e d b y g e n t l e t u r n i n g a n d 1 m l a l i q u o t s were a d d e d to t h e p i p e t t e s a n d allowed t o filter t h r o u g h o v e r 10 m i n . E a c h e x p e r i m e n t w i t h a n d w i t h o u t e p i n e p h r i n e was r u n in t r i p l i c a t e . T h e n u m b e r of g r a n u l o c y t e s in t h e b l o o d was m e a s u r e d before and after passage through the column and the percentage of g r a n u l o c y t e a d h e r e n c e was c a l c u l a t e d . Results. T h e a d h e r e n c e of g r a n u l o c y t e s was d e c r e a s e d b y 27.9% in t h e p r e s e n c e of e p i n e p h r i n e (table). T h i s was s t a t i s t i c a l l y s i g n i f i c a n t a t p < 0.0025 level a n a l y z e d b y the Student t-test. Discussion. M a n y clinical c o n d i t i o n s w i t h i n c r e a s e d susc e p t i b i l i t y to i n f e c t i o n c a n n o t be e x p l a i n e d b y decreases in g r a n u l o c y t e p h a g o c y t o s i s or c h e m o t a x i s a l o n e a n d
Effect of Epinephrine on granulocyte adhesion
EXPERIENTIA 34/1
a d d i t i o n a l g r a n u l o c y t e f u n c t i o n defects m u s t be considered. G r a n u l o c y t e a d h e r e n c e to n y l o n fibres or glass m a y be used as m e a s u r e of t h e i r a b i l i t y to a d h e r e t o e n d o t h e l i a l walls b u t t h i s r e q u i r e s f u r t h e r proof. I t h a s b e e n s h o w n b y M a c G r e g o r e t al. 3 t h a t a n t i - i n f l a m m a t o r y d r u g s i n h i b i t g r a n u l o c y t e a d h e r e n c e to n y l o n fibres a n d s u g g e s t e d t h a t t h i s m a y b e t h e e x p l a n a t i o n for t h e i r effects in vivo. O t h e r a g e n t s h a v e also b e e n s h o w n t o i n h i b i t g r a n u l o c y t e a d h e s i o n s u c h as e t h a n o l 3, i o d o a c e t a m i d e 5, a n d E D T A 3, ~, cyclic A M P 7, colchicine *, a n d glycolitic i n h i b i t o r s s, w h e r e a s o x i d a t i v e i n h i b i t o r s s h a d n o effect. T h e p r e s e n c e of c e r t a i n ions ( m a g n e s i u m a n d p e r h a p s calcium)3, 6 a p p e a r to b e necessary. A b n o r m a l g r a n u l o c y t e a d h e s i o n h a s b e e n d e m o n s t r a t e d in several clinical c o n d i t i t i o n s , i.e. l e u k e m i a s a n d a c u t e p o s t s t r e p t o c o c c a l g l o m e r u l 0 n e p h r i t i s 9. I n t h e l a t t e r , t h i s a b n o r m a l i t y c a n be followed as a m e a s u r e of r e s o l u t i o n of disease. T h e a d m i n i s t r a t i o n of e p i n e p h r i n e in vivo causes t h e g r a n u l o c y t e s to d e m a r g i n a t e a n d e n t e r t h e c i r c u l a t i o n . This study showed a decreased granulocyte adhesion to nylon fibres in t h e p r e s e n c e of e p i n e p h r i n e in v i t r o s u g g e s t i n g t h a t s i m i l a r m e c h a n i s m s m a y o p e r a t e in b o t h s y s t e m s . G r a n u l o c y t e a d h e s i v e n e s s s h o u l d be s t u d i e d in clinical s i t u a t i o n s associated w i t h a n i n c r e a s e d s u s c e p t i b i l i t y t o infection. I n f o r m a t i o n o b t a i n e d f r o m t h e s e s t u d i e s m a y p r o v i d e b e t t e r u n d e r s t a n d i n g of t h e role g r a n u l o c y t e a d h e s i o n p l a y s in p r e v e n t i n g infection.
Percent adhesion j_ SEM Control Epinephrine
75.7 • 5.0 47.8 :t: 6.4 p < 0.0025
Each value represents 8 experiments performed'on blood from healthy male volunteers.
4 R.R. MacGregor, Am. J. Med. 61, 597 (1976). 5 J . E . Garvin, J. expl. Med. 114, 51 (1961). 6 R.E. Bryant, R.M. DesPrez and D.E. Rogers, Yale J. Biol. Med. d0, 192 (1967). 7 R.E. Bryant and M. Suteliffe, Clin. Res. 21, 594 (1973). 8 R. Penny and D. A. G, Galton, Br. J. Haemat. 12, 633 (1966). 9 E.J. Ruley, S. Huang, J. Plant and N. Morris, J. Pedlar. 89, 748 (1976).
Accumulation of 2 - g m latex particles in mouse Peyer's patches during chronic latex feeding1, 2 M. E. L e F e v r e 3, J. W. V a n d e r h o f f 4, J. A. Laissue s a n d D. D. Joel
Medical Research Center, Brookhaven National Laboratory, Upton (New York 11973, USA), 1 July 1977 Summary. 2-~xm l a t e x p a r t i c l e s a c c u m u l a t e d in m a c r o p h a g e s in i n t e s t i n a l P e y e r ' s p a t c h e s of mice g i v e n l a t e x s u s p e n s i o n s as d r i n k i n g fluid for 2 m o n t h s . T h e n u m b e r of p a r t i c l e s a c c u m u l a t i n g was a d i r e c t ( b u t n o n l i n e a r ) f u n c t i o n of t h e n u m b e r ingested. S o m e of t h e l a t e x p a r t i c l e s were still p r e s e n t in P e y e r ' s p a t c h e s 6 weeks a f t e r t h e cessation of l a t e x feeding. T h e e p i t h e l i u m of g u t - a s s o c i a t e d l y m p h o i d tissues ( G A L T ; P e y e r ' s p a t c h e s , a p p e n d i x , sacculus r o t u n d u s ) a p p e a r s t o b e a r o u t e b y w h i c h s m a l l i n e r t p a r t i c u l a t e s pass t h e m u c o s a l b a r r i e r of t h e m a m m a l i a n i n t e s t i n a l tract6, L P r e v i o u s m o r p h o l o g i c a l s t u d i e s h a v e d e s c r i b e d t h e mig r a t i o n of c a r b o n , t r y p a n blue, a n d f e r r i t i n p a r t i c l e s i n t o t h e G A L T s-12. T h e s e p a r t i c u l a t e s are small, less t h a n 0.1 ~xm in d i a m e t e r , a n d were seen w i t h t h e l i g h t m i c r o scope b e c a u s e of t h e i r a c c u m u l a t i o n i n t o aggregates. T h e p r e s e n t c o m m u n i c a t i o n describes t h e p e n e t r a t i o n of t h e m o u s e P e y e r ' s p a t c h e p i t h e l i u m b y m u c h l a r g e r particles, n a m e l y l a t e x s p h e r e s of 2%tm d i a m e t e r . L a t e x p a r t i c l e s resist d e g r a d a t i o n in t h e i n t e s t i n e a n d are large e n o u g h to b e seen a n d c o u n t e d w i t h t h e l i g h t microscope, p e r m i t t i n g a s e m i q u a n t i t a t i v e a s s e s s m e n t of t h e i r u p t a k e a n d distrib u t i o n . T h e r e s u l t s i n d i c a t e t h a t m a n y t h o u s a n d s of l a t e x p a r t i c l e s a c c u m u l a t e d in P e y e r ' s p a t c h e s d u r i n g c h r o n i c feeding of mice w i t h l a t e x suspensions. F u r t h e r m o r e ,
m a n y of t h e p a r t i c l e s were r e t a i n e d for m o r e t h a n 6 weeks a f t e r t h e c e s s a t i o n of l a t e x feeding. Materials and methods. 3 w a t e r s u s p e n s i o n s of p o l y v i n y l t o l u e n e l a t e x ( m e a n p a r t i c l e d i a m e t e r • SD, 2.02 d0.014 ~ m ; p a r t i c l e d e n s i t y , 1.027; i d e n t i f i c a t i o n No. LS-1078-B, T h e D o w C h e m i c a l Co.) c o n t a i n i n g 1.0, 0.1 a n d 0.01% solids were g i v e n as d r i n k i n g fluid to 3 g r o u p s of 11-week-old f e m a l e Swiss mice. T h e mice were g i v e n free access to t h e s u s p e n s i o n s a n d to s t a n d a r d p e l l e t e d m o u s e food. T h e l a t e x s u s p e n s i o n s were given for 61 d a y s followed b y a period of 2 t o 6 weeks d u r i n g w h i c h t h e mice r e c e i v e d latex-free w a t e r . A c o n t r o l g r o u p was g i v e n t a p w a t e r t o d r i n k . No a t t e m p t was m a d e to d e t e r m i n e t h e a m o u n t of l a t e x i n g e s t e d b y e a c h m o u s e ; h o w e v e r , t h e v o l u m e s of fluid c o n s u m e d b y t h e 4 g r o u p s did n o t a p p e a r to differ. All mice g a i n e d w e i g h t n o r m a l l y a n d a p p e a r e d healthy.
15, 1. 1978
Specialia
U p t a k e a n d r e t e n t i o n of l a t e x in P e y e r ' s p a t c h e s w e r e demonstrated h i s t o l o g i c a l l y i n p r e p a r a t i o n s of w h o l e P e y e r ' s p a t c h e s c l e a r e d i n K O H a n d g l y c e r o l la a n d in methacrylate-embedded sections. In addition latex was r e c o v e r e d f r o m d i g e s t s of i n t e s t i n a l t i s s u e s of l a t e x - f e d m i c e . T h e e n t i r e s m a l l i n t e s t i n e w a s r i n s e d in s a l i n e , s e p a r a t e d i n t o P e y e r ' s p a t c h a n d n o n - P e y e r ' s p a t c h (rem a i n d e r ) t i s s u e , w e i g h e d , m i n c e d , a n d p l a c e d in t e s t t u b e s c o n t a i n i n g 10 m l 3 % K O H . T h e t u b e s w e r e a g i t a t e d u n t i l the tissue was completely digested. The material was then
Fig. 1. 2g.xn latex particles within the dome of a cleared Peyer's patch follicle from a mouse sacrificed 2 weeks after termination of latex feeding. Black circles are latex particles above the plane of focus. Dark shadows are large aggregates of latex below the plane (~f focus. • 520.
c e n t r i f u g e d , w a s h e d in w a t e r , a n d t h e l a t e x r e c o v e r e d b y u l t r a c e n t r i f u g a t i o n of t h e s e d i m e n t t h r o u g h a l i n e a r d e n s i t y g r a d i e n t of 0 - 2 0 % u r e a . P a r t i c l e s w h i c h b a n d e d a t the same level as known latex were recovered by syringe and an aliquot counted using a Neubauer chamber and an optical microscope. Additional aliquots were deposited on 0.8-b~m N u c l e o p o r e filters, r i n s e d w i t h w a t e r , a n d e x a m i n e d a t 5000 • b y s c a n n i n g e l e c t r o n m i c r o s c o p y . T h e recovered material contained spheres identical to the stock latex ; these were not found elsewhere in the gradient. Results and discussion. 80 w h o l e c l e a r e d P e y e r ' s p a t c h e s were examined with a wide-field inverted microscope. Latex was unaffected by the clearing process, and indiv i d u a l p a r t i c l e s c o u l d b e s e e n a t d i f f e r e n t l e v e l s in t h e t i s s u e . E a c h p a t c h c o n s i s t e d of 2 - 8 follicles; t h e d o m e ~* of e a c h follicle of l a t e x - f e d m i c e c h a r a c t e r i s t i c a l l y s h o w e d a n a c c u m u l a t i o n of l a t e x p a r t i c l e s in t h e c e n t r a l p o r t i o n . F i g u r e 1 s h o w s s u c h a n a c c u m u l a t i o n in a P e y e r ' s p a t c h f r o m a m o u s e f e d t h e h i g h e s t c o n c e n t r a t i o n of l a t e x . P a r ticles near the mucosal surface were distributed singly or in s m a l l c l u s t e r s w h e r e a s p a r t i c l e s c l o s e t o t h e s e r o s a l s u r f a c e w e r e m o s t l y in a g g r e g a t e s n e a r t h e p e r i p h e r y of t h e follicle. P a t c h e s f r o m m i c e g i v e n l o w e r c o n c e n t r a t i o n s of latex had fewer particles and smaller aggregates. Cleared p a t c h e s f r o m c o n t r o l m i c e c o n t a i n e d n o p a r t i c u l a t e s res e m b l i n g 2-b~m l a t e x . L a t e x p a r t i c l e s w e r e still v i s i b l e in d i m i n i s h e d n u m b e r s in c l e a r e d P e y e r ' s p a t c h e s 6 w e e k s a f t e r t e r m i n a t i o n of l a t e x i n g e s t i o n . T h e l o c a t i o n of l a t e x in r e l a t i o n t o c e l l u l a r e l e m e n t s c o u l d n o t b e d e t e r m i n e d in c l e a r e d p r e p a r a t i o n s , b u t in p l a s t i c e m b e d d e d s e c t i o n s t h e particles were seen to be associated with macrophages ( f i g u r e 2). T h e t a b l e l i s t s n u m b e r s of l a t e x p a r t i c l e s r e c o v e r e d a f t e r d i g e s t i o n of i n t e s t i n a l t i s s u e s a n d u l t r a c e n t r i f u g a t i o n of t h e s e d i m e n t t h r o u g h a d e n s i t y g r a d i e n t . S e d i m e n t s of control tissues centrifuged through the density gradient s h o w e d n o b a n d i n g of p a r t i c l e s a t t h e l a t e x level. T h e table shows that Peyer's patch tissue comprised only a s m a l l f r a c t i o n of t h e w e i g h t of t h e s m a l l i n t e s t i n e , b u t c o n t a i n e d t h e m a j o r p o r t i o n of t h e l a t e x . T h e p a t t e r n of u p t a k e w a s t h a t of i n c o m p l e t e s a t u r a t i o n . T h e l o c a t i o n of t h e l a t e x f o u n d in t h e r e m a i n d e r of t h e i n t e s t i n e h a s not yet been determined. l
2 3 4 5 6 7 8 9 10 Fig. 2. 2btm latex particles (arrows) within a macrophage process in a mouse Peyer's patch. Distortion of the particles from spherical shape is due to the action of methyl methacrylate which partially dissolves latex during the embedding process. Dark lymphocyte nuclei are visible together with several pale maerophages containing other partially visible latex particles. Section was taken near the serosal surface of a Peyer's patch from a mouse given a suspension of latex as drinking fluid for 2 months. H. & E. • 1500.
121
11 12 13 14
Acknowledgments. Research supported by the U. S. Energy Research and Development Administration. W~e thank Mr W. Marin, Division of Photography and Graphic Arts, Brookhaven National Laboratory, for figure 2. The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. Correspondence should be addressed to: M. E. LeFevre, Medical Research Center, Brookhaven National Laboratory, Upton, N. Y. 11973. Center for Surface and Coatings Research, Lehigh University, Bethlehem, Pa. 18015. Pathologisches Institut, Kantonsspital, CH 6000 Luzern (Switzerland). M . E . LeFevre and D. D. Joel, Life Sci., in press. R . L . Owen, Gastroenterology 72, 440 (1977}. D . E . Bockman and M. D. Cooper, Am. J. Anat. 136, 455 (1973). M. Hanaoka, R. Williams and B. Waksman, Lab. Invest. 2d, 31 (1971). M.W. Hess, H. Cottier, B. Sordat, D. D. Joel and A. D. Chanana, in: Nonspecifie Factors Influencing Host Resistance. A Reexamination, p. 447. Karger, Basel 1973. D . D . Joel, B. Sordat, M. Hess and H. Cottier, Experientia 26, 694 (1970). M. Kagan, Z. Zellforsch. 14, 544 (1931). G.L. Humanson, Animal Tissue Techniques, 3rd ed., p. 195. Freeman, San Francisco 1972. B. H. Waksinan, O. Ozer and H. E. Blythman, Lab. Invest. 28, 614 (1973).
122
EXPERIENTIA34/1
Specialia
Quantitative determination of latex recovered from Peyer's patches and remainder of small intestine No. Latex suspension fed* (% solids)
N
1.0 0.I 0.01
15 15 15
Peyer's patches Tissue weight (g)
No. particles • 105
Remainder Tissue weight (g)
No particles • 105
0.054::t=0.016 0.056-4-0.013 0.045-[ 0,008
2.13• 1.424-0.98 0.18-[-0.14
1.062• 1.078=k0.227 1.005•
1.78+0.58 1.04•
