Electron-Histochemical Investigations on Clara Cells and Type II Pneumocytes of Normal Rat Lungs U. A c h t e r r a t h
Pathologisches
and S. B l i ~ m c k e
Institut, Universit~tsklinikum
Essen,
Fed. Rep. Germany
Ab s t r act. C l a r a c e l l s and t y p e II p n e u m o c y t e s of n o r m a l r a t l u n g s w e r e i n v e s t i g a t e d w i t h an e l e c t r o n m i c r o s c o p e a f t e r i n c u b a t i o n f o r acid phosphatase, endogenous peroxidase (catalase), osmium-zinciodide positive material, phosphoglycerides, and periodic acid-thiocarbazide-OsO 4 positive material. Comparison of the two cells revealed that secretion vacuoles like lamellar bodies are secondary lysosomes. Whereas lamellated bodies show a strong reaction with osmium-zinciodide and phosphoglycerides, no r e a c t i o n of C l a r a c e l l s e c r e t i o n v a c u o l e s c o u l d b e o b s e r v e d . In b o t h c e l l s t h e O s O 4 - Z n I 2 p r o c e d u r e r e v e a l e d a s t r o n g r e a c t i o n of E R m e m b r a n e s a n d a f t e r f i x a t i o n a r e a c t i o n of E R c i s t e r n a e and p e r i n u c l e a r c l e f t w a s o b s e r v e d . S e c r e t i o n v a c u o l e s of C l a r a c e l l s t h e r e f o r e do not a p p e a r to c o n t a i n s u r f a c e a c t i v e m a t e r i a l but it i s s u p p o s e d t h a t t h i s m a t e r i a l i s s y n t h e s i z e d and s t o r e d b y t h e ER. Peroxisomes in C l a r a c e l l s a r e s e l d o m found, w h i l e t y p e II p n e u m o cytes contain numerous peroxisomes localized near lamellar bodies. In C l a r a c e l l s p e r o x i s o m e s a p p e a r in t h e n e i g h b o r h o o d of s o - c a l l e d m y e l i n a t e d f i g u r e s . T h i s p r o v i d e s s t r o n g e v i d e n c e t h a t p e r o x i s o m e s of both cells prevent lipid peroxidation. In both cells PATO-positive mat e r i a l s h o w s o n l y l i t t l e m e m b r a n e a s s o c i a t e d a p p e a r a n c e . In l a m e l l a r b o d i e s , w h i c h do not a p p e a r to h a v e r e a c h e d f u l l m a t u r i t y , t h e h o m o g e n e o u s p a r t s g i v e a s t r o n g r e a c t i o n to P A T O , w h i l e l a m e l l a t e d p a r t s g i v e no r e a c t i o n . T h e O s O 4 - Z n I 2 m e t h o d s h o w s t h e i n v e r s e a p p e a r a n c e . T h e r e f o r e it i s s u p p o s e d t h a t m u c o p o l y s a c c h a r i d e s a r e p r e s e n t in i m mature lamellar bodies and that they disappear during maturation.
INTRODUCTION Phagocytizing activity of their macrophages is an essential point in the defense mechanism of the lung. Possibly type II pneumocytes, which also synthesize surfactant, belong to macrophages. The role of the Clara cell in the defense mechanism, although not clear up to this time, according to the experimental work of Schiller (1964) lies in its secretory activity. According to Azzopardi and Pneumonologie
152,
1 2 3 - 1 2 9 (1975)
124
Fig. 1 (A) Type II pneumocyte with eatalase-positive peroxisomes (arrows) in the neighborhood of lamellar bodies (L). Uncontrasted, X 37 400. (B) Part of a Clara cell with a peroxisome (arrow) in the neighborhood of a so-called myelinated figure (MY). M =mitochondrium. Uranylacetate, lead citrate, X 71 300.
125
Fig. 1 (C) T y p e II p n e u m o c y t e with f i n e - g r a n u l a t e d i m p r e g n a t i o n of the ER. L a m e l l a r b o d i e s (L} show a s t r o n g i m p r e g n a t i o n of the l a m e l l a t e d p a r t s . O s O 4 - Z n I 2 , unfixed t i s s u e , u n c o n t r a s t e d , X 11 500. ( D ) C l a r a cell with f i n e - g r a n u l a t e d ER and n e g a t i v e s e c r e t i o n v a c u o l e s (arro%vs). O s O 4 - Z n I 2 , unfixed t i s s u e , u n c o n t r a s t e d , X 11 500.
127
T h u r l b e c k (1969) and N i d e n (1967) it is s u s p e c t e d that C l a r a c e l l s s y n thesize surfactant. To obtain a broad methodological basis for e x p e r i m e n t a l investigat i o n s on s e c r e t o r y a c t i v i t y , s e c r e t i o n p r o d u c t , and s y n t h e s i s of s u r f a c t a n t we p e r f o r m e d s o m e h i s t o c h e m i c a l r e a c t i o n s on n o r m a l l u n g s of W i s t a r r a t s .
R E S U L T S AND CONCLUSIONS Acid p h o s p h a t a s e (lead s a l t m e t h o d , m o d i f i e d G o m o r i m e d i u m pH 5 . 0 , a c c o r d i n g to B a r k a and A n d e r s o n (1963)) is d e l i v e r e d f r o m the Golgi a p p a r a t u s of the C l a r a c e l l up to p r i m a r y l y s o s o m e s and a p p e a r s in the a p i c a l s e c r e t i o n v a c u o l e s . T h e y c a n s h o w a p o s i t i v e o r n e g a t i v e r e a c t i o n and t h e r e f o r e t h e y a r e of l y s o s o m a l o r i g i n . T y p e II p n e u m o c y t e s show a m u c h s t r o n g e r a c t i v i t y of a c i d p h o s p h a t a s e . R e a c t i o n is l o c a l i z e d in l a m e l l a r b o d i e s , t y p i c a l l y s o s o m e s , and Golgi a p p a r a t u s . E n d o g e n o u s p e r o x i d a s e ( c a t a l a s e ) , r e a c t i o n a c c o r d i n g to B e a r d and Novikoff (1969) in H 2 0 2 - c o n t a i n i n g m e d i u m pH 9 . 0 , s h o w s p a r t i c l e bound r e a c t i o n in t h e n u m e r o u s p e r o x i s o m e s of t y p e II p n e u r n o c y t e s . Peroxisomes lie nearest the lamellar bodies (Fig. IA). Peroxisomes are seldom found in Clara cells. If so-called myelinated figures are produced, peroxisomes can be found in their neighborhood (Fig. IB). The osmium-zinc-iodide method according to Maillet (1963), solution prepared after Niebauer et al. (1969), appears to impregnate surfactant (Blfimcke et al. 1973). In unfixed tissue only Clara cells (Fig. 1 D) and type II pneumocytes (Fig. IC) show a fine-granulated reaction. After pre-fixation with glutaraldehyde impregnation is much stronger. Both cell types (demonstrated in a Clara cell, (Fig. 2A) reveal a strong reaction in cisternae and perinuclear cleft of the ER. Secretion vacuoles of Clara cells are never impregnated in both unfixed and fixed tissue. The silverhydroxamate reaction for phosphoglyceride (Weller et al., 1965) s h o w s a p o s i t i v e a p p e a r a n c e in t h e l a m e l l a t e d b o d i e s of t y p e II p n e u m o c y t e s (Fig. 2B). S e c r e t i o n v a c u o l e s of C l a r a c e l l s , in c o n t r a s t to the f i n d i n g s of Niden (1967), give no r e a c t i o n . F i n e d i s t r i b u t e d m a t e r i a l c a n o n l y be found to s o m e e x t e n t in a s s o c i a t i o n with E R m e m branes.
F i g . 2. (A) C l a r a c e l l with s t r o n g i m p r e g n a t e d p e r i n u c l e a r c l e f t and c i s t e r n a e of the E R . S e c r e t i o n v a c u o l e s ( a r r o w s ) not i m p r e g n a t e d . N = n u c l e u s . O s O 4 - Z n I 2 , p r e f i x a t i o n , u n c o n t r a s t e d , X 7 600. (B) S i l v e r h y d r o x a m a t e r e a c t i o n of a l a m e l l a r b o d y (L) in t y p e II p n e u m o c y t e . U n c o n t r a s t e d , X 71 300. (C) P a r t of a t y p e II p n e u m o c y t e with not f u l l y m a t u r a t e d l a m e l l a r b o d i e s (L). P A T O - p o s i t i v e m a t e r i a l s ( a r r o w s ) in the m o r e h o m o g e n e o u s p a r t s . U n c o n t r a s t e d , X 31 200. (D) P a r t of a t y p e II p n e u m o c y t e with not f u l l y m a t u r a t e d l a m e l l a r b o d i e s (L). O s O 4Z n I 2 - p o s i t i v e m a t e r i a l in the m o r e l a m e l l a t e d p a r t s ( a r r o w s ) . N = n u c l e u s . P r e f i x e d t i s s u e , u n c o n t r a s t e d , X 31 200.
128
The periodic acid-thiocarbazide-OsO 4 (PATO-) method (Hanker et al. (1964) is adequate for the light-microscopical PAS reaction. Clara cells show little PATO-positive material in membrane associated position to ER and secretion vacuoles. Type II pneumocytes also give scanty reaction in association to the outer membranes of !amellar bodies. On the other hand, particles with a more homogeneous appearance, which can be interpreted as non-fully maturated lamellar bodies, show a s t r o n g r e a c t i o n of t h e i r m a t r i x and m a r g i n (Fig. 2C). S i m i l a r p a r t i c l e s in the o s m i u m - z i n c - i o d i d e m e t h o d show t h e i n v e r s e r e a c t i o n (Fig. 2D). O u r f i n d i n g s l e a d to t h e f o l l o w i n g c o n c l u s i o n s : 1) S e c r e t i o n v a c u o l e s of C l a r a c e l l s a g r e e o n l y with r e g a r d to acid p h o s p h a t a s e c o n t e n t with l a m e l l a t e d b o d i e s of t y p e II p n e u m o c y t e s . No OsO 4 - Z n I 2 p o s i t i v e m a t e r i a i n o r p h o s p h o g l y c e r i d e s c o u l d b e d e m o n s t r a t e d . C o r r e s p o n d i n g to t h e p o s i t i v e r e a c t i o n of t h e E R we p r o p o s e a p a r t i c i p a t i o n of E R in the s y n t h e s i s of s u r f a e t a n t . P e r f o r m a n c e of t h e r e a c t i o n f o r c h o l i n e c o n t a i n i n g t i p i d s , a f t e r L a n d i n g et al. (1952), s u p p o r t e d t h i s s t a t e m e n t ( A c h t e r r a t h , u n p u b l i s h e d observations). 2. In a c c o r d a n c e with the p e r o - i s o m e c o n c e p t (De Duve and Baudhin, 1966), f r o m the n e i g h b o r h o o d of p e r o x i s o m e s to l a m e l l a r b o d i e s and s o - c a l l e d m y e l i n a t e d f i g u r e s we s u p p o s e t h a t an e s s e n t i a l point of t h e i r r o t e in t h e s e c e l l s is the p r e v e n t i o n of lipid p e r o x i d a t i o n . 3. P A T O - p o s i t i v e m a t e r i a l is s y n t h e s i z e d in o n l y i n s i g n i f i c a n t a m o u n t s in n o r m a l t y p e II p n e u m o c y t e s and in C l a r a c e l l s , T h e f i n d i n g s in non-fulIy maturated lamellar bodies are interesting. PATO-positive m a t e r i a l is p o s s i b l y p r o d u c e d in p r e m a t u r e l a m e l l a t e d b o d i e s and disappears during maturation.
REFERENCES Achterrath, U. : In preparation Azzopardi, A., Thurlbeck, W. M, • The histochemistry of the noneiliated bronchiolar epithelical cell. Amer. Rev, Resp. Dis, 99, 516-525 (1969) Barka, I., Anderson, P. J. : Histochemistry. Theory, Practice and Bibliography. pp. 240 (New York-Evanston-London. Harper & Row, 1963) Beard, M. E. , Novikoff, A. B. : Distribution of peroxisomes (microbodies) in the nephron of the rat. A cytochemical study. J. Cell Biol. 4__~2, 501-518 (1969) B1/imcke, S . , K e s s l e r , W. D . , N i e d o r f , H. R . , B e c k e t , N. H . , Veith, P . J. : U l t r a s t r u c t u r e of l a m e l l a r b o d i e s of t y p e II p n e u m o c y t e s a f t e r o s m i u m - z i n c i m p r e g n a t i o n . J. U l t r a s t r u c t . R e s . 42, 4 1 7 - 4 3 3 (1973) De Duve, C . , B a u d h i n , P . : P e r o x i s o m e s ( m i c r o b o d i e s and r e l a t e d p a r t i e l e s ) . P h y s i o l . Rev. 46, 323-357 (1966) H a n k e r , J. S . , S e a m a n , A. R . , W e i s s , L . P . , Ueno, H . , B e r g m a n , R. A . , S e l i g m a n , A. M. : O s m i o p h i l i c r e a g e n t s : New c y t o c h e m i c a t p r i n c i p l e f o r light and e l e c t r o n m i c r o s c o p y . S c i e n c e 146, 1039-1043 (1964) L a n d i n g , B . H . , U z m a n , L . L . , Whipple, A . : P h o s p h o m o l y b d i c acid as a s t a i n i n g r e a g e n t f o r lipidE, L a b . I n v e s t . 1, 456 (1952)
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M a i l l e t , M. : L e r e a c t i f au t e t r a o x y d e d ' o s m i u m - i o d u r e du z i n c . Z. m i k r . anat. F o r s c h . 76, 397-425 (1963) Niden, A. H. : B r o n c h i o l a r and l a r g e a l v e o l a r c e l l in p u l m o n a r y p h o s p h o l p i d m e t a b o l i s m . S c i e n c e 158, 1323-1324 (1967) N i e b a u e r , G . , K r a w c z y k , W. S . , Kidd, R. L . , W i l l g r a m , G. F . : O s m i u m z i n c i o d i d e r e a c t i v e s i t e s in the e p i d e r m a l L a n g e r h a n s c e l l . J. C e l l ]3iol. 43, 8 0 - 8 9 (1969) S c h i l l e r , E. : W e i t e r e U n t e r s u c h u n g e n f i b e r die s e k r e t o r i s c h e T~itigkeit d e s B r o n e h i o l a r e p i t h e l s . V e r h . Anat. G e s . 58, 232-241 (1964) W e l l e r , R. O . , B a y l i s s , O. ]3., Abdulla, Y. H . , A d a m s , C. W. M. : The e l e c t r o n - h i s t o c h e m i c a l d e m o n s t r a t i o n of p h o s p h o g l y c e r i d e . J. H i s t o c h e m . C y t o c h e m . 13_..., 6 9 0 - 6 9 2 (1965) D r . Ute A c h t e r r a t h Pathologisches Institut U n i v e r sit~itsklinikum E s s e n D-43 E s s e n H u f e l a n d s t r . 55 F e d e r a l R e p u b l i c of G e r m a n y
Pathologisches
and S. B l i ~ m c k e
Institut, Universit~tsklinikum
Essen,
Fed. Rep. Germany
Ab s t r act. C l a r a c e l l s and t y p e II p n e u m o c y t e s of n o r m a l r a t l u n g s w e r e i n v e s t i g a t e d w i t h an e l e c t r o n m i c r o s c o p e a f t e r i n c u b a t i o n f o r acid phosphatase, endogenous peroxidase (catalase), osmium-zinciodide positive material, phosphoglycerides, and periodic acid-thiocarbazide-OsO 4 positive material. Comparison of the two cells revealed that secretion vacuoles like lamellar bodies are secondary lysosomes. Whereas lamellated bodies show a strong reaction with osmium-zinciodide and phosphoglycerides, no r e a c t i o n of C l a r a c e l l s e c r e t i o n v a c u o l e s c o u l d b e o b s e r v e d . In b o t h c e l l s t h e O s O 4 - Z n I 2 p r o c e d u r e r e v e a l e d a s t r o n g r e a c t i o n of E R m e m b r a n e s a n d a f t e r f i x a t i o n a r e a c t i o n of E R c i s t e r n a e and p e r i n u c l e a r c l e f t w a s o b s e r v e d . S e c r e t i o n v a c u o l e s of C l a r a c e l l s t h e r e f o r e do not a p p e a r to c o n t a i n s u r f a c e a c t i v e m a t e r i a l but it i s s u p p o s e d t h a t t h i s m a t e r i a l i s s y n t h e s i z e d and s t o r e d b y t h e ER. Peroxisomes in C l a r a c e l l s a r e s e l d o m found, w h i l e t y p e II p n e u m o cytes contain numerous peroxisomes localized near lamellar bodies. In C l a r a c e l l s p e r o x i s o m e s a p p e a r in t h e n e i g h b o r h o o d of s o - c a l l e d m y e l i n a t e d f i g u r e s . T h i s p r o v i d e s s t r o n g e v i d e n c e t h a t p e r o x i s o m e s of both cells prevent lipid peroxidation. In both cells PATO-positive mat e r i a l s h o w s o n l y l i t t l e m e m b r a n e a s s o c i a t e d a p p e a r a n c e . In l a m e l l a r b o d i e s , w h i c h do not a p p e a r to h a v e r e a c h e d f u l l m a t u r i t y , t h e h o m o g e n e o u s p a r t s g i v e a s t r o n g r e a c t i o n to P A T O , w h i l e l a m e l l a t e d p a r t s g i v e no r e a c t i o n . T h e O s O 4 - Z n I 2 m e t h o d s h o w s t h e i n v e r s e a p p e a r a n c e . T h e r e f o r e it i s s u p p o s e d t h a t m u c o p o l y s a c c h a r i d e s a r e p r e s e n t in i m mature lamellar bodies and that they disappear during maturation.
INTRODUCTION Phagocytizing activity of their macrophages is an essential point in the defense mechanism of the lung. Possibly type II pneumocytes, which also synthesize surfactant, belong to macrophages. The role of the Clara cell in the defense mechanism, although not clear up to this time, according to the experimental work of Schiller (1964) lies in its secretory activity. According to Azzopardi and Pneumonologie
152,
1 2 3 - 1 2 9 (1975)
124
Fig. 1 (A) Type II pneumocyte with eatalase-positive peroxisomes (arrows) in the neighborhood of lamellar bodies (L). Uncontrasted, X 37 400. (B) Part of a Clara cell with a peroxisome (arrow) in the neighborhood of a so-called myelinated figure (MY). M =mitochondrium. Uranylacetate, lead citrate, X 71 300.
125
Fig. 1 (C) T y p e II p n e u m o c y t e with f i n e - g r a n u l a t e d i m p r e g n a t i o n of the ER. L a m e l l a r b o d i e s (L} show a s t r o n g i m p r e g n a t i o n of the l a m e l l a t e d p a r t s . O s O 4 - Z n I 2 , unfixed t i s s u e , u n c o n t r a s t e d , X 11 500. ( D ) C l a r a cell with f i n e - g r a n u l a t e d ER and n e g a t i v e s e c r e t i o n v a c u o l e s (arro%vs). O s O 4 - Z n I 2 , unfixed t i s s u e , u n c o n t r a s t e d , X 11 500.
127
T h u r l b e c k (1969) and N i d e n (1967) it is s u s p e c t e d that C l a r a c e l l s s y n thesize surfactant. To obtain a broad methodological basis for e x p e r i m e n t a l investigat i o n s on s e c r e t o r y a c t i v i t y , s e c r e t i o n p r o d u c t , and s y n t h e s i s of s u r f a c t a n t we p e r f o r m e d s o m e h i s t o c h e m i c a l r e a c t i o n s on n o r m a l l u n g s of W i s t a r r a t s .
R E S U L T S AND CONCLUSIONS Acid p h o s p h a t a s e (lead s a l t m e t h o d , m o d i f i e d G o m o r i m e d i u m pH 5 . 0 , a c c o r d i n g to B a r k a and A n d e r s o n (1963)) is d e l i v e r e d f r o m the Golgi a p p a r a t u s of the C l a r a c e l l up to p r i m a r y l y s o s o m e s and a p p e a r s in the a p i c a l s e c r e t i o n v a c u o l e s . T h e y c a n s h o w a p o s i t i v e o r n e g a t i v e r e a c t i o n and t h e r e f o r e t h e y a r e of l y s o s o m a l o r i g i n . T y p e II p n e u m o c y t e s show a m u c h s t r o n g e r a c t i v i t y of a c i d p h o s p h a t a s e . R e a c t i o n is l o c a l i z e d in l a m e l l a r b o d i e s , t y p i c a l l y s o s o m e s , and Golgi a p p a r a t u s . E n d o g e n o u s p e r o x i d a s e ( c a t a l a s e ) , r e a c t i o n a c c o r d i n g to B e a r d and Novikoff (1969) in H 2 0 2 - c o n t a i n i n g m e d i u m pH 9 . 0 , s h o w s p a r t i c l e bound r e a c t i o n in t h e n u m e r o u s p e r o x i s o m e s of t y p e II p n e u r n o c y t e s . Peroxisomes lie nearest the lamellar bodies (Fig. IA). Peroxisomes are seldom found in Clara cells. If so-called myelinated figures are produced, peroxisomes can be found in their neighborhood (Fig. IB). The osmium-zinc-iodide method according to Maillet (1963), solution prepared after Niebauer et al. (1969), appears to impregnate surfactant (Blfimcke et al. 1973). In unfixed tissue only Clara cells (Fig. 1 D) and type II pneumocytes (Fig. IC) show a fine-granulated reaction. After pre-fixation with glutaraldehyde impregnation is much stronger. Both cell types (demonstrated in a Clara cell, (Fig. 2A) reveal a strong reaction in cisternae and perinuclear cleft of the ER. Secretion vacuoles of Clara cells are never impregnated in both unfixed and fixed tissue. The silverhydroxamate reaction for phosphoglyceride (Weller et al., 1965) s h o w s a p o s i t i v e a p p e a r a n c e in t h e l a m e l l a t e d b o d i e s of t y p e II p n e u m o c y t e s (Fig. 2B). S e c r e t i o n v a c u o l e s of C l a r a c e l l s , in c o n t r a s t to the f i n d i n g s of Niden (1967), give no r e a c t i o n . F i n e d i s t r i b u t e d m a t e r i a l c a n o n l y be found to s o m e e x t e n t in a s s o c i a t i o n with E R m e m branes.
F i g . 2. (A) C l a r a c e l l with s t r o n g i m p r e g n a t e d p e r i n u c l e a r c l e f t and c i s t e r n a e of the E R . S e c r e t i o n v a c u o l e s ( a r r o w s ) not i m p r e g n a t e d . N = n u c l e u s . O s O 4 - Z n I 2 , p r e f i x a t i o n , u n c o n t r a s t e d , X 7 600. (B) S i l v e r h y d r o x a m a t e r e a c t i o n of a l a m e l l a r b o d y (L) in t y p e II p n e u m o c y t e . U n c o n t r a s t e d , X 71 300. (C) P a r t of a t y p e II p n e u m o c y t e with not f u l l y m a t u r a t e d l a m e l l a r b o d i e s (L). P A T O - p o s i t i v e m a t e r i a l s ( a r r o w s ) in the m o r e h o m o g e n e o u s p a r t s . U n c o n t r a s t e d , X 31 200. (D) P a r t of a t y p e II p n e u m o c y t e with not f u l l y m a t u r a t e d l a m e l l a r b o d i e s (L). O s O 4Z n I 2 - p o s i t i v e m a t e r i a l in the m o r e l a m e l l a t e d p a r t s ( a r r o w s ) . N = n u c l e u s . P r e f i x e d t i s s u e , u n c o n t r a s t e d , X 31 200.
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The periodic acid-thiocarbazide-OsO 4 (PATO-) method (Hanker et al. (1964) is adequate for the light-microscopical PAS reaction. Clara cells show little PATO-positive material in membrane associated position to ER and secretion vacuoles. Type II pneumocytes also give scanty reaction in association to the outer membranes of !amellar bodies. On the other hand, particles with a more homogeneous appearance, which can be interpreted as non-fully maturated lamellar bodies, show a s t r o n g r e a c t i o n of t h e i r m a t r i x and m a r g i n (Fig. 2C). S i m i l a r p a r t i c l e s in the o s m i u m - z i n c - i o d i d e m e t h o d show t h e i n v e r s e r e a c t i o n (Fig. 2D). O u r f i n d i n g s l e a d to t h e f o l l o w i n g c o n c l u s i o n s : 1) S e c r e t i o n v a c u o l e s of C l a r a c e l l s a g r e e o n l y with r e g a r d to acid p h o s p h a t a s e c o n t e n t with l a m e l l a t e d b o d i e s of t y p e II p n e u m o c y t e s . No OsO 4 - Z n I 2 p o s i t i v e m a t e r i a i n o r p h o s p h o g l y c e r i d e s c o u l d b e d e m o n s t r a t e d . C o r r e s p o n d i n g to t h e p o s i t i v e r e a c t i o n of t h e E R we p r o p o s e a p a r t i c i p a t i o n of E R in the s y n t h e s i s of s u r f a e t a n t . P e r f o r m a n c e of t h e r e a c t i o n f o r c h o l i n e c o n t a i n i n g t i p i d s , a f t e r L a n d i n g et al. (1952), s u p p o r t e d t h i s s t a t e m e n t ( A c h t e r r a t h , u n p u b l i s h e d observations). 2. In a c c o r d a n c e with the p e r o - i s o m e c o n c e p t (De Duve and Baudhin, 1966), f r o m the n e i g h b o r h o o d of p e r o x i s o m e s to l a m e l l a r b o d i e s and s o - c a l l e d m y e l i n a t e d f i g u r e s we s u p p o s e t h a t an e s s e n t i a l point of t h e i r r o t e in t h e s e c e l l s is the p r e v e n t i o n of lipid p e r o x i d a t i o n . 3. P A T O - p o s i t i v e m a t e r i a l is s y n t h e s i z e d in o n l y i n s i g n i f i c a n t a m o u n t s in n o r m a l t y p e II p n e u m o c y t e s and in C l a r a c e l l s , T h e f i n d i n g s in non-fulIy maturated lamellar bodies are interesting. PATO-positive m a t e r i a l is p o s s i b l y p r o d u c e d in p r e m a t u r e l a m e l l a t e d b o d i e s and disappears during maturation.
REFERENCES Achterrath, U. : In preparation Azzopardi, A., Thurlbeck, W. M, • The histochemistry of the noneiliated bronchiolar epithelical cell. Amer. Rev, Resp. Dis, 99, 516-525 (1969) Barka, I., Anderson, P. J. : Histochemistry. Theory, Practice and Bibliography. pp. 240 (New York-Evanston-London. Harper & Row, 1963) Beard, M. E. , Novikoff, A. B. : Distribution of peroxisomes (microbodies) in the nephron of the rat. A cytochemical study. J. Cell Biol. 4__~2, 501-518 (1969) B1/imcke, S . , K e s s l e r , W. D . , N i e d o r f , H. R . , B e c k e t , N. H . , Veith, P . J. : U l t r a s t r u c t u r e of l a m e l l a r b o d i e s of t y p e II p n e u m o c y t e s a f t e r o s m i u m - z i n c i m p r e g n a t i o n . J. U l t r a s t r u c t . R e s . 42, 4 1 7 - 4 3 3 (1973) De Duve, C . , B a u d h i n , P . : P e r o x i s o m e s ( m i c r o b o d i e s and r e l a t e d p a r t i e l e s ) . P h y s i o l . Rev. 46, 323-357 (1966) H a n k e r , J. S . , S e a m a n , A. R . , W e i s s , L . P . , Ueno, H . , B e r g m a n , R. A . , S e l i g m a n , A. M. : O s m i o p h i l i c r e a g e n t s : New c y t o c h e m i c a t p r i n c i p l e f o r light and e l e c t r o n m i c r o s c o p y . S c i e n c e 146, 1039-1043 (1964) L a n d i n g , B . H . , U z m a n , L . L . , Whipple, A . : P h o s p h o m o l y b d i c acid as a s t a i n i n g r e a g e n t f o r lipidE, L a b . I n v e s t . 1, 456 (1952)
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M a i l l e t , M. : L e r e a c t i f au t e t r a o x y d e d ' o s m i u m - i o d u r e du z i n c . Z. m i k r . anat. F o r s c h . 76, 397-425 (1963) Niden, A. H. : B r o n c h i o l a r and l a r g e a l v e o l a r c e l l in p u l m o n a r y p h o s p h o l p i d m e t a b o l i s m . S c i e n c e 158, 1323-1324 (1967) N i e b a u e r , G . , K r a w c z y k , W. S . , Kidd, R. L . , W i l l g r a m , G. F . : O s m i u m z i n c i o d i d e r e a c t i v e s i t e s in the e p i d e r m a l L a n g e r h a n s c e l l . J. C e l l ]3iol. 43, 8 0 - 8 9 (1969) S c h i l l e r , E. : W e i t e r e U n t e r s u c h u n g e n f i b e r die s e k r e t o r i s c h e T~itigkeit d e s B r o n e h i o l a r e p i t h e l s . V e r h . Anat. G e s . 58, 232-241 (1964) W e l l e r , R. O . , B a y l i s s , O. ]3., Abdulla, Y. H . , A d a m s , C. W. M. : The e l e c t r o n - h i s t o c h e m i c a l d e m o n s t r a t i o n of p h o s p h o g l y c e r i d e . J. H i s t o c h e m . C y t o c h e m . 13_..., 6 9 0 - 6 9 2 (1965) D r . Ute A c h t e r r a t h Pathologisches Institut U n i v e r sit~itsklinikum E s s e n D-43 E s s e n H u f e l a n d s t r . 55 F e d e r a l R e p u b l i c of G e r m a n y
