534
Specialia
Results. The E B P - F C A caused a c c u m u l a t i o n of l y m p h o c y t e s a n d large l y m p h o i d cells in t h e p a r a c o r t e x and in t h e m e d u l l a r y cords w h i c h was m o r e m a r k e d t h a n a f t e r i n j e c t i n g N B P - F C A or FCA alone. The d e v e l o p m e n t a n d a m o u n t of g r a n u l o m a t o u s tissue in t h e hilar area was m o r e p r o m i n e n t in t h e E B P - F C A a n d N B P - F C A groups. I n t h e E B P - F C A group, t h e e n l a r g e m e n t of t h e p a r a c o r t e x was especially p r o m i n e n t ; t h e traffic of l y m p h o c y t e s t h r o u g h t h e h i g h e n d o t h e l i u m venules was v e r y intensive, a n d a h i g h p r o p o r t i o n of cells of t h e p l a s m o cytic series were p r e s e n t in t h e m e d u l l a r y cords w h i c h a l m o s t c o m p r e s s e d t h e sinuses. A u t o r a d i o g r a p h i c estim a t e s of cellular p r o l i f e r a t i v e a c t i v i t y in t h e n o d e s s h o w e d t h a t n u m b e r of p r o l i f e r a t i n g cells significantly increased in b o t h e x p e r i m e n t a l groups. I n t h e c o n t r o l group, L.I. r a n g e d f r o m 1.2% to 1.5%, in t h e N B P - F C A g r o u p f r o m 5.5% to 6.6% a n d in t h e E B P - F C A group it was a p p r o x i m a t e l y 11~ in t h e FCA g r o u p L.I. was 5.8% (table). Discussion. P r e v i o u s w o r k a has s h o w n t h a t p e a k L N w t i n c r e m e n t involves a g r o w t h of t h e l y m p h o i d cell p o p u l a t i o n m a i n l y w i t h i n t h e first 4 days. Following d a y 4, t h e g r o w t h of non-specific g r a n u l o m a prevails. F o r t h i s r e a s o n we c o n c e n t r a t e d our o b s e r v a t i o n s in t h e p r e s e n t w o r k on t h e early stages of disease i n d u c t i o n . A c c o r d i n g to p r e v i o u s findings a, L N g r o w t h a f t e r N B P - F C A was s i g n i f i c a n t l y slower t h a n a f t e r E B P - F C A in t h e s a m e t i m e interval. P r e s e n t results r e v e a l e d t h a t E B P - F C A re-
EXPERIENTIA 33/4
sulted in a m o r e i n t e n s e a c c u m u l a t i o n of l y m p h o i d ceils in t h e p a r a c o r t e x a n d a g r e a t e r d e v e l o p m e n t of g r a n u l o m a t h a n N B P - F C A or FCA alone. M a t u r a t i o n of p l a s m a cells was also a c c e l e r a t e d a f t e r E B P - F C A . N B P - F C A , as c o m p a r e d w i t h F C A alone, i n c r e a s e d s o m e w h a t t h e g r o w t h of t h e l y m p h o i d cell p o p u l a t i o n a n d t h e m e d u l l a r y cords. The significance of t h e l a t t e r m u s t , h o w e v e r , be i n t e r p r e t e d w i t h care, since c o m p r e s s i o n f r o m t h e FCA d e v e l o p i n g l i p o g r a n u l o m a s u b s t a n t i a l l y c h a n g e s t h e circ u l a t i o n and a r c h i t e c t u r e of t h e LN. The l a t t e r m i g h t be e x p r e s s e d m o r e clearly w i t h E B P a n d N B P w i t h o u t t h e a d d i t i o n of FCA. Eliciting E A E w i t h o u t F C A is, however, difficult, if a t all possible. A u t o r a d i o g r a p h i c d a t a i n d i c a t e t h a t t h e n o d a l g r o w t h in b o t h e x p e r i m e n t a l g r o u p s i n v o l v e d p r o l i f e r a t i o n of cells in situ. P r e v i o u s r e p o r t a s h o w e d t h a t g r o w t h of t h e cell p o p u l a t i o n involves also m i g r a t i o n of l y m p h o c y t e s into t h e LN. Following N B P - F C A injection, b o t h processes are therefore a p p a r e n t l y less i n t e n s e and a p p r o x i m a t e values for adm i n i s t r a t i o n of FCA alone. The differences in L N g r o w t h a n d p r o l i f e r a t i o n o b s e r v e d a f t e r a d m i n i s t r a t i o n of E B P FCA and N B P - F C A show t h a t non-specific s t i m u l a t i o n of L N b y basic p r o t e i n w i t h o u t e n c e p h a l i t o g e n i c determ i n a n t (ED) is r e l a t i v e l y small. The p r e s e n c e of E D in t h e basic p r o t e i n molecule ( E B P - F C A ) m a r k e d l y influenced t h e local r e a c t i o n of t h e L N d r a i n i n g t h e site of i n j e c t i o n in t e r m s of increasing m i g r a t i o n of cells into t h e n o d e s a n d p r o l i f e r a t i o n of cells in situ.
Relation between stimulus intensity and neutrophil chemotactic response1 H. U. Keller, J. H. Wissler 2, M. W. Hess a n d H. Cottier
Department o/ Pathology, University o/ Bern, Freiburgstrasse 30, CH-3010 Bern (Switzerland), and Arbeitsgruppe Biochemie, Abteilung /iir experimentelle Kardiologie, Max-Planck-lnstitut /i~r physiologische und klinische Forschung, D-6350 Bad Nauheim (Federal Republic o~ Germany, BRD), 15 December 7976 Summary. The effect of c h e m o t a c t i c p e p t i d e s w h i c h lack c h e m o k i n e t i c a c t i v i t y has been i n v e s t i g a t e d . The n e u t r o p h i l r e s p o n s e is p r o p o r t i o n a l to t h e l o g a r i t h m of t h e s t i m u l u s i n t e n s i t y , or a l t e r n a t i v e l y a p o w e r f u n c t i o n w i t h an e x p o n e n t of 0.3. E q u a l responses are o b t a i n e d for e q u a l ratios b e t w e e n t h e p e p t i d e c o n c e n t r a t i o n in t h e lower c o m p a r t m e n t a n d t h e t h r e s h o l d c o n c e n t r a t i o n . The significance of W e b e r - F e c h n e r ' s law in leucocyte c h e m o t a x i s is discussed. T h e d i r e c t i o n of a c t i v e l y m o v i n g leucocytes can be d e t e r m i n e d b y c h e m i c a l s u b s t a n c e s in t h e e n v i r o n m e n t . Since L e b e r ' s s t u d i e s in 18883, it is generally believed t h a t t h i s process, called c h e m o t a x i s , is i n s t r u m e n t a l in leucocyte a c c u m u l a t i o n a t i n f l a m m a t o r y sites. The r e l a t i o n s h i p b e t w e e n t h e i n t e n s i t y of t h e c h e m o t a c t i c signal and t h e d i r e c t i o n f i n d i n g of leucocytes, as expressed in d i r e c t i o n a l l o c o m o t i o n is still largely u n k n o w n . The i n t e r p r e t a t i o n of earlier d a t a 4-6 was c o m p l i c a t e d b y t h e fact t h a t t h e t e s t m a t e r i a l influenced t h e s p e e d of t h e cells, as well as t h e i r d i r e c t i o n of locomotion. T h u s it h a d c h e m o k i n e t i c as well as c h e m o t a c t i c activity. I t was t h e r e f o r e n e c e s s a r y to e v a l u a t e t h e r e l a t i o n s h i p b e t w e e n s t i m u l u s i n t e n s i t y a n d d i r e c t i o n a l i t y b y m e a n s of purified c y t o t a x i n p r e p a r a t i o n s e x h i b i t i n g c h e m o t a c t i c a c t i v i t y only. P a r t i a l l y purified p e p t i d e p r e p a r a t i o n s c o n t a i n i n g classical a n a p h y l a t o x i n (S-CAT 1.5.1) ~, 8 were p r e p a r e d f r o m d e x t r a n a c t i v a t e d swine s e r u m for such e x p e r i m e n t s . This p r e p a r a t i o n c o n t a i n e d 4% of c h e m o t a c t i c a l l y a c t i v e p e p t i d e s 9. R a n d o m a n d d i r e c t i o n a l l o c o m o t i o n of h u m a n p e r i p h e r a l blood n e u t r o p h i l s was assessed w i t h a m o d i f i e d filter t e c h n i q u e 19, w h i c h p r o v i d e s for s t a b l e g r a d i e n t s (unp u b l i s h e d o b s e r v a t i o n s ) . The b e h a v i o u r o f r e s p o n d i n g ceils ( r a n d o m vs d i r e c t i o n a l locomotion) was also d e t e r m i n e d b y d i r e c t o b s e r v a t i o n . The results o b t a i n e d w i t h
t h e s e 2 t e c h n i q u e s were in a g r e e m e n t . H u m a n s e r u m a l b u m i n (HSA) h a d to be p r e s e n t in t h e t e s t s y s t e m to p e r m i t efficient m o v e m e n t of cells a n d t h e r e b y t h e expression of c h e m o t a x i s in f o r m of d i r e c t i o n a l locomot i o n 11. 1
We thank Miss B. Zanolari, Mrs M. Marti, Mrs Hauck and Mr Dietz for excellent technical assistance. This work was supported by the Swiss National Foundation for Scientific Research. 2 Max-Planck-Institut ffir physiologische und klinische Forschung, D-6350 Bad Nauheim, BRD. 3 Th. Leber, Fortschr. Mecl. 6, 460 (1888). 4 H.U. Keller and 1~. Sorkin, Immunology 70, 409 (1966}. 5 S.H. Zigmond and J. G. Hirsch, J. exp. Med. 137, 387 (1973). 6 P.C. Wilkinson, in: Chemotaxis and Inflammation. Churchill Livingstone, Fdinburgh and London 1974. 7 J . H . Wissler, Eur. J. hnmun. 2i 73 and 84 {1972). 8 J . H . Wissler, V. Stecher and E. Sorkin, Eur. J. Immun. 2, 90 (1972). 9 J . H . Wissler, in: Gram-negative bacterial infections and mode of endotoxin action, p. 91. Ed. B. Urbasehek, R. Urbaschek and E. Neter. Springer, New York 1975. 10 H. U. Keffer, H. Gerber, M. W. Hess and H. Cottier, Agents Actions 6, 326 (1976). 11 H.U. Keller, J. Wissler, M. W. Hess and H. Cottier, in: Movement, Metabolism and Bactericidal Mechanism of Phagocytes (in press).
15.4. 1977
Specialia
W e f o u n d t h a t S - C A T 1.5.1. is s u i t a b l e for t h e a n a l y s i s of t h e c h e m o t a c t i c r e s p o n s e . S - C A T 1.5.1. h a d n o m e a s u r able a c t i v i t y in a b s e n c e of h u m a n s e r u m a l b u m i n . If H S A w a s p r e s e n t in t h e m e d i u m , a p o s i t i v e g r a d i e n t bet w e e n t h e 2 c o m p a r t m e n t s r e s u l t e d in a t t r a c t i o n , a n e g a t i v e g r a d i e n t in c h e m o t a c t i c t r a p p i n g of n e u t r o p h i l s , b u t S - C A T 1.5.1. did n o t i n f l u e n c e r a n d o m l o c o m o t i o n m e a s u r e d in a b s e n c e of a g r a d i e n t (table). T h i s f i n d i n g w a s c o n s i s t e n t o v e r a w i d e c o n c e n t r a t i o n r a n g e of c h e m o t a c t i c p e p t i d e s (1.27 Fg to 0.00127 [~g/ml). T h u s S - C A T 1.5.1. d e t e r m i n e s t h e d i r e c t i o n of t h e cells w i t h o u t affecting t h e r a t e of r a n d o m l o c o m o t i o n , i.e. t h e r a t e of r a n d o m t u r n i n g a n d / o r t h e s p e e d of n e u t r o p h i l s . C o n s e q u e n t l y t h e i n c r e a s e in t h e n u m b e r of cells w h i c h h a v e m o v e d t h r o u g h t h e filter reflects t h e degree of d i r e c t i o n f i n d i n g b y t h i s cell p o p u l a t i o n :
CAT 1.5.1. induces chemotaxis without affecting the rate of random locomotion Test sohltion* (lower compartment)
Cell suspending medi um * (upper compartment)
Percent neutrophils migrated **
2% HSA
2% HSA
0.38 • 0.02
CAT 1.5.1. in 2% HSA
CAT 1.5.1. ill 2% HSA
0 . 3 6 ~ 0.07
CAT 1.5.1. in 2% I[SA
2% HSA
2% HSA
CAT J.5.1. in 2% I-ISA < 0.007
21.8 • 2.9
* The basic medium consisted of 2% human scram albumin (HSA) in f;ey's solution pH 7.2. CAT 1.5.1. (final concentration 1.27 [J.g chemotactic peptides/ml 2% tISA in (;ey's solution) was added to either the test solutiou {positive gradient), the cell suspeusion (uegative gradient) or to b{~th compartme, ts (u~ gradient}. ** V a h l e s r e p r e s e n t t h e p e r c e n t a g e of neutrophils which have migrated through the entire thickuess (140 pun) of the filter ~ SI)K
9 -~
50 o 40 A 3C
,Cells in 2%HSA oCellsin S-Cat 1.5.1. [
~ - - " } ---{
2C "5 7o 10
-o
,,, f=s g
[lower compartment1 [absolute threshold].
2C 1~. 10
Z
10- 2
T h e i n c r e a s e in t h e n u m b e r of n e u t r o p h i l s w h i c h h a v e m o v e d t h r o u g h t h e e n t i r e t h i c k n e s s of t h e filter is p r o p o r t i o n a l to t h e l o g a r i t h m of t h e c o n c e n t r a t i o n of S - C A T 1.5.1. in t h e t e s t c o m p a r t m e n t (figure, A). T h u s t h e r e l a t i v e i n c r e m e n t in e x t e r n a l e n e r g y c o r r e s p o n d s to a c o n s t a n t i n c r e m e n t in t h e r e s p o n s e as e x p r e s s e d b y cell a c c u m u l a t i o n . T h e s e f i n d i n g s are c o m p a t i b l e w i t h o u r earlier h y p o t h e s i s 4 t h a t c h e m o t a x i s as e x p r e s s e d b y d i r e c t i o n a l l o c o m o t i o n is d e t e r m i n e d b y t h e law of W e b e r F e c h n e r . A l t e r n a t i v e l y we h a v e to c o n s i d e r t h a t t h e res p o n s e m a y be d e t e r m i n e d b y S t e v e n s ' p o w e r l a w 1~. i t is in f a c t d i f f i c u l t to d i s t i n g u i s h a s e m i l o g a r i t h m i c r e l a t i o n s h i p (law of W e b e r - F e c h n e r ) b e t w e e n s t i m u l u s i n t e n s i t y a n d m a g n i t u d e of r e s p o n s e f r o m a d o u b l e l o g a r i t h m i c p o w e r f u n c t i o n w i t h a n e x p o n e n t n e a r 0.3 (0.2-0.4), b e c a u s e t h e y m a y h a v e a n a l m o s t i d e n t i c a l c o u r s e o v e r s e v e r a l log u n i t s " . A n e x p o n e n t n e a r 0.3 h a s b e e n f o u n d for h u m a n v i s i o n a n d h e a r i n g , while o t h e r s e n s o r y f u n c t i o n s h a v e d i f f e r e n t e x p o n e n t s for p o w e r f u n c t i o n s r e l a t i n g s u b j e c t i v e m a g n i t u d e to s t i m u l u s int e n s i t y l K S u c h a r e l a t i o n s h i p p r o v i d e s for a c o m p r e s s o r f u n c t i o n to m a t c h t h e s t i m u l u s f r o m o u t s i d e to t h e n e e d s of t h e cells. I n t h e p r e s e n t e x p e r i m e n t s , t h e r a n g e of t h e e x p o n e n t i a l p a r t of t h e r e s p o n s e is, h o w e v e r , q u i t e s m a l l as c o m p a r e d to s e n s o r y f u n c t i o n s of m a c r o o r g a n i s m s vs. T h e r e s p o n s e of n o r m a l n e u t r o p h i l s s u s p e n d e d in 2% H S A r e a c h e s its p l a t e a u a t a dose w h i c h is u p to 50 t i m e s above the threshold concentration. T h e n e u t r o p h i l s c a n also r e s p o n d c h e m o t a c t i c a l l y w h e n S - C A T 1.5.1. is p r e s e n t in b o t h c o m p a r t m e n t s of t h e c h a m b e r , p r o v i d e d t h e r e is a c o n c e n t r a t i o n g r a d i e n t . T h e r e s p o n s e is a g a i n p r o p o r t i o n a l to t h e l o g a r i t h m of t h e a t t r a c t a n t c o n c e n t r a t i o n in t h e lower c o m p a r t m e n t of t h e c h a m b e r u n t i l a p l a t e a u is r e a c h e d . T h e slope of t h e r e g r e s s i o n line is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o b t a i n e d w i t h n e u t r o p h i l s s u s p e n d e d in 2 % H S A o n l y (figure, A). A d d i t i o n of S - C A T 1.5.1. to t h e ceil s u s p e n d i n g m e d i u m , h o w e v e r , a f f e c t s o t h e r f e a t u r e s of t h e n e u t r o p h i I r e s p o n s e . T h e t h r e s h o l d is raised a b o v e tile S - C A T 1.5.1. c o n c e n t r a t i o n in t h e c e l l - s u s p e n d i n g m e d i u m ( u p p e r c o m p a r t m e n t ) , i n d i c a t i n g t h a t t h e cells h a v e r e s p o n d e d to p o s i t i v e c o n c e n t r a t i o n g r a d i e n t s r a t h e r t h a n to t h e a b s o lute test concentration. The 2 curves take a parallel c o u r s e (figure, A), s u g g e s t i n g a g e n e r a l r e l a t i o n s h i p . T h e i n c r e a s e in t h e n e u t r o p h i l r e s p o n s e is p r o p o r t i o n a l to t h e r a t i o b e t w e e n t h e a t t r a c t a n t c o n c e n t r a t i o n in t h e t e s t s o l u t i o n (lower c o m p a r t m e n t ) a n d t h e a b s o l u t e t h r e s h o ] d c o n c e n t r a t i < m c a l c u l a t e d f r o m t h e r e g r e s s i o n line
: Cells preincubated in 2 % HSA ~ preincubated in S-CAT 1.5.1.
g
._~ E
10q
10o
101
535
102
S-CAI 1.5.1 (pg chemotactic peptides/ml test solution)
Relation between imutrophil response and test concentration of S-CAT 1.5.1. A Locomotion (percent neutrophils migrated through the entire thickness [140 btm~ of the filter) of neutrophils suspended in 2% human serum albumin in Gey's solution with or without S-CAT 1.5.1. (0.635 b~g chemotactie peptides/ml) towards test solutions containing increasing concentrations of S-CAT 1.5.1. t3 Response of neutrophils which have been preincubated in either 2% HSA or 2% lISA containing S CAT 1.5.1. (0.635 ~zg chemotactic peptides/inl) for 30 inin at 37~ "lhe cells were then washed extensively and resuspended in 2% HSA in Gey's solution.
E q u a l r a t i o s p r o d u c e e q u a l r e s p o n s e s of a p a r t i c u l a r cell p o p u l a t i o n , e v e n if t h e t h r e s h o l d is s h i f t e d to a d i f f e r e n t level b y t h e a d d i t i o n of S - C A T 1.5.1. to t h e cell s u s p e n d ing m e d i u m . T h i s is c o m p a t i b l e w i t h W e b e r ' s p r i n c i p l e w h i c h s a y s t h a t in o r d e r to p r o d u c e a series of j u s t n o t i c e able d i f f e r e n c e s we m u s t m a k e a fixed r e l a t i v e i n c r e a s e in t h e s t i m u l u s . T h e a d d i t i o n of S - C A T 1.5.1. also lowers t h e p l a t e a u of t h e r e s p o n s e , i n d i c a t i n g t h a t t h e c a p a c i t y for a d i r e c t i o n a l r e s p o n s e to a p o s i t i v e g r a d i e n t is r e d u c e d . C o n s e q u e n t l y t h e l i n e a r r a n g e in t h e s e m i - l o g a r i t h m i c p l o t of s t i m u l u s i n t e n s i t y v e r s u s r e s p o n s e m a g n i t u d e is n a r r o w e d . T h i s m a y , h o w e v e r , be m o r e a p p a r e n t t h a n real, since t h e s e cells are in t u r n in a p o s i t i o n to r e s p o n d to a n e g a t i v e g r a d i e n t b y b e i n g t r a p p e d (table). T h e
12
S. S. Stevens, in: Handbook of Sensory 1)hysiology, vol. I, p. 226. Ed. \V. R. Loewenstein. Springer, Berlin 1971.
536
Specialia
slope of t h e regression lines varied f r o m e x p e r i m e n t to e x p e r i m e n t w h i c h m a y reflect differences in t h e a v e r a g e speed of t h e s e cell p o p u l a t i o n s . I t has b e e n r e p o r t e d t h a t p r e i n c u b a t i o n of n e u t r o p h i l s in a chemotactic medium renders neutrophils unresponsive to f u r t h e r c h e m o t a c t i c s t i m u l a t i o n , e v e n if t h e cells h a v e b e e n w a s h e d e x t e n s i v e l y . This p h e n o m e n o n h a s b e e n called 'deactivation'~a. W e find t h a t n e u t r o p h i l s , w h i c h h a v e b e e n e x p o s e d t o S-CAT 1.5.1. a n d w a s h e d , s h o w n o r m a l r a n d o m l o c o m o t i o n . T h e y b e h a v e as if S-CAT 1.5.1. were still p r e s e n t in t h e cell c o m p a r t m e n t . The t h r e s h o l d for a c h e m o t a c t i c response of such cells is s h i f t e d to a h i g h e r c o n c e n t r a t i o n of S-CAT 1.5.1. a n d a p p r o x i m a t e s t h e c y t o t a x i n c o n c e n t r a t i o n used for pre-
EXPERIENTIA 33/4
i n c u b a t i n g t h e cells. The p l a t e a u of t h e r e s p o n s e is lowered (figure, B). This m a y be c o n s i d e r e d as a t a c h y p h y l a c t i c s t a t e ill l e u c o c y t e c h e m o t a x i s . I t m a y reflect a failure to r e a d a p t efficiently to a n e n v i r o n m e n t w i t h low s t i m u l u s i n t e n s i t y . A d a p t a t i o n , e.g, in visual p e r c e p t i o n 14 or in c h e m o t a x i s of slime m o u l d s 15, is a quite c o m p l e x p h e n o m e n o n . I t s role in leucocyte c h e m o t a x i s r e m a i n s to be f u r t h e r e v a l u a t e d . P.A. Ward and E. L. Becker, J. exp. Med. 127, 693 (1968). P. Gouvas, in: Handbook of Sensory Physiology, voI. VII/2, p. 609. Ed. M, G. F. Fourtes. Springer, New York 1972. 15 G. Gerisch and D. Malchow, in: Biochemistry of Sensory Functions, p. 279. Ed. L. Jaenicke. Springer, New York 1974. 13 14
T h e i n f l u e n c e of l o n g t e r m e s t r o g e n t r e a t m e n t on p l a s m a p r o l a c t i n l e v e l s i n d u c e d b y e t h e r a n e s t h e s i a in o v a r i e c t o m i z e d r a t s 1 G. T. G o o d m a n a n d D. M. L a w s o n
Department o/ Physiology, Wayne State University, School o/Medicine, Detroit (Michigan 48201, USA), 6 September 1976 Summary. 2 m e t h o d s of c o n t i n u o u s e s t r o g e n delivery, p o l y e s t r a d i o l p h o s p h a t e i n j e c t i o n a n d i m p l a n t a t i o n of Silastic capsules of estradiol-17 fl, in o v a r i e c t o m i z e d r a t s i n d u c e d increases in p l a s m a p r o l a c t i n in t h e a f t e r n o o n (15.00-17.00) b e g i n n i n g a t 1 week a n d c o n t i n u i n g for 4-8 weeks. I n a d d i t i o n t h e s e m e t h o d s of e s t r o g e n t r e a t m e n t p o t e n t i a t e d the e t h e r - i n d u c e d increase in p l a s m a p r o l a c t i n in t h e m o r n i n g (9.00-11.00) b e g i n n i n g on week 2 a n d c o n t i n u i n g for 3-8 weeks. T h e s e results i n d i c a t e t h a t e s t r o g e n a c t i v a t e s t h e m e c h a n i s m s t h a t cause an a f t e r n o o n surge in prolactin before p o t e n t i a t i n g a m o r n i n g e l e v a t i o n i n d u c e d b y e t h e r anesthesia. R e p e a t e d injections of large a m o u n t s of e s t r o g e n for several d a y s increases t h e p r o l a c t i n c o n t e n t in t h e r a t p i t u i t a r y 2, a, as well as in t h e s e r u m 4 s. E s t r o g e n m a y p r o m o t e t h e relase of p r o l a c t i n into blood b y affecting h y p o t h a l a m i c neural m e c h a n i s m s g, 1% I n c o n t r a s t to t h e chronic s t i m u l a t o r y effects of estrogen, several t y p e s of stressors induce a c u t e e l e v a t i o n s in p r o l a c t i n secretion. The m o s t w i d e l y i n v e s t i g a t e d r e s p o n s e is t h a t to e t h e r inhalation. E t h e r h a s b e e n s h o w n to increase p l a s m a p r o t a c t i n in o v a r i e c t o m i z e d r a t s n a n d t h e r e s p o n s e is e x a g g e r a t e d in t h e o v a r i e c t o m i z e d a n i m a l t r e a t e d w i t h e s t r o g e n for 1-3 weeks 12. The p u r p o s e of t h e p r e s e n t s t u d y was to elucidate t h e effects of l o n g - t e r m e s t r o g e n t r e a t m e n t on t h e e t h e r i n d u c e d rise of p r o l a c t i n in t h e m o r n i n g w h e n t h e levels are low, a n d in t h e a f t e r n o o n w h e n t h e levels are elevated.
neck. Vaginal s m e a r s were o b t a i n e d daily to m o n i t o r t h e effects of t h e e s t r o g e n t r e a t m e n t s . B e g i n n i n g 1 week a f t e r o v a r i e c t o m y a n d e s t r o g e n t r e a t m e n t a n d c o n t i n u i n g once w e e k l y for 8 weeks, a 2 ml blood s a m p l e was o b t a i n e d f r o m t h e o r b i t a l v e n o u s plexus using h e p a r i n i z e d c a p i l l a r y t u b e s a f t e r 5 rain of c o n t i n u o u s e t h e r a n e s t h e s i a in t h e m o r n i n g (9.00-11.00 a.m.) and again in t h e a f t e r n o o n (15.00 17.00 p . m . ) . A n i m a l s were a n e s t h e s i z e d b y an initial e x p o s u r e to e t h e r v a p o r in a large c o n t a i n e r , followed b y m a i n t e n a n c e w i t h a nose cone. The s a m p l e s were c e n t r i f u g e d a n d t h e p l a s m a was collected a n d s t o r e d a t - 2 0 ~ u n t i l assayed. 1
Materials and methods. 30 m a t u r e , female S p r a g u e - D a w l e y r a t s ( S p a r t a n R e s e a r c h Animals, Inc., H a s l e t t , Michigan) weighing 200-250 g were r a n d o m l y d i v i d e d i n t o 6 groups. A f t e r 7 d a y s of a c c l i m a t i o n t o lighting c o n d i t i o n s (lights on f r o m 6.00 to 20.00 h) all t h e r a t s were b i l a t e r a l l y o v a r i e c t o m i z e d . 1 g r o u p was i n j e c t e d w i t h 0.5 m g polyestradiol p h o s p h a t e ( P E P ; 1.0 m g E s t r a d u r i n | A y e r s t L a b o r a t o r i e s ) s.c., a n d 4 groups received s.c. Silastic imp l a n t s c o n t a i n i n g 12.5 or 25 m g crystalline estradiol-17 /3 a t t h e t i m e of o v a r i e c t o m y . The i m p l a n t s were similar to t h o s e used b y L e g a n et al. ~4. 4 sizes of Silastic t u b i n g were utilized : D o w Corning No. 602-265 1.57 m m i.d., 2.41 m m o.d. 14 a n d 24 m m in l e n g t h a n d D o w Corning No. 602-285 1.57 m m i.d., 3.18 m m o.d. also 14 a n d 24 m m in length. A 2 m m w o o d e n p l u g was i n s e r t e d into 1 end of t h e Silastic t u b i n g a n d c r y s t a l l i n e estradiol-17 /~ was p a c k e d into t h e o p e n e n d of t h e t u b i n g . A n o t h e r 2 m m plug was i n s e r t e d into t h e o p e n end, a n d b o t h ends were c o a t e d w i t h Silastic Medical A d h e s i v e Silicone T y p e A (No. 890 Dow Corning). T h e i m p l a n t s were i n c u b a t e d for 30 min in distilled w a t e r a t 20~ a n d w i p e d w i t h e t h a n o l prior to i n s e r t i o n i n t o a s.c. p o c k e t in t h e n a p e of t h e
2 3 4 5 6 7 8 9 10 11 12 13 14
Acknowledgment. This work was supported by NIH General Research Support, grant No. RR05384-14 to Wayne State University School of Medicine. The authors also wish to express their appreciation to Mrs C. Van De Walle for her expert technical assistance in the prolactin radioimmunoassay, to Dr Richard R. Gala for his advice and support of this project and to the Rat Pituitary Hormone Distribution Program of NIAMDD for the generous gift of Rat Prolactin. R.P. Reece, Proc. Soc. exp. Biol. Med. 39, 77 (1938). J. Meites and C. W. Turner, Proe. Soc. exp. Biol. Med. ,19, 190 (1942). C.L. Chert, H. Miniguchi and J. Meites, Proc. Soc. exp. Biol. Med. 126, 317 (1967). J . D . Neill, Endocrinology 87, 1192 (1970). H . G . Kwa, C. A. Feltkamp, A. A. Van der Gugten and F. Verhofstad, J. Endocr. 48, 299 (1970). A.A. van der Gugten, M. Sala and H. G. Kwa, Acta Endocr. 6d, 265 {1970). L. Caligaris, J. J. Astrada and S. Taleisnik, J. Endocr. 60, 205 (1974). W. Bishop, P. S. Kalra, C. P. Fawcett, L. Krulich and S. M. McCann, Endocrinology 91, 1404 (1972). A. Rather and J. Meites, Endocrinology 75, 377 (1964). D.M. Lawson and R. R. Gala, J. Endocr. 62, 75 (1973). D.M. Lawson and R. R. Gala, J. Endocr. 66, 151 (1974). M. G. Subramanian and R. R. Gala, Endocrinology 98, 842 (1976). S.J. Legan, G. A. Coon and F. J. Karseh, Endocrinology 96, 50 (1975).
Specialia
Results. The E B P - F C A caused a c c u m u l a t i o n of l y m p h o c y t e s a n d large l y m p h o i d cells in t h e p a r a c o r t e x and in t h e m e d u l l a r y cords w h i c h was m o r e m a r k e d t h a n a f t e r i n j e c t i n g N B P - F C A or FCA alone. The d e v e l o p m e n t a n d a m o u n t of g r a n u l o m a t o u s tissue in t h e hilar area was m o r e p r o m i n e n t in t h e E B P - F C A a n d N B P - F C A groups. I n t h e E B P - F C A group, t h e e n l a r g e m e n t of t h e p a r a c o r t e x was especially p r o m i n e n t ; t h e traffic of l y m p h o c y t e s t h r o u g h t h e h i g h e n d o t h e l i u m venules was v e r y intensive, a n d a h i g h p r o p o r t i o n of cells of t h e p l a s m o cytic series were p r e s e n t in t h e m e d u l l a r y cords w h i c h a l m o s t c o m p r e s s e d t h e sinuses. A u t o r a d i o g r a p h i c estim a t e s of cellular p r o l i f e r a t i v e a c t i v i t y in t h e n o d e s s h o w e d t h a t n u m b e r of p r o l i f e r a t i n g cells significantly increased in b o t h e x p e r i m e n t a l groups. I n t h e c o n t r o l group, L.I. r a n g e d f r o m 1.2% to 1.5%, in t h e N B P - F C A g r o u p f r o m 5.5% to 6.6% a n d in t h e E B P - F C A group it was a p p r o x i m a t e l y 11~ in t h e FCA g r o u p L.I. was 5.8% (table). Discussion. P r e v i o u s w o r k a has s h o w n t h a t p e a k L N w t i n c r e m e n t involves a g r o w t h of t h e l y m p h o i d cell p o p u l a t i o n m a i n l y w i t h i n t h e first 4 days. Following d a y 4, t h e g r o w t h of non-specific g r a n u l o m a prevails. F o r t h i s r e a s o n we c o n c e n t r a t e d our o b s e r v a t i o n s in t h e p r e s e n t w o r k on t h e early stages of disease i n d u c t i o n . A c c o r d i n g to p r e v i o u s findings a, L N g r o w t h a f t e r N B P - F C A was s i g n i f i c a n t l y slower t h a n a f t e r E B P - F C A in t h e s a m e t i m e interval. P r e s e n t results r e v e a l e d t h a t E B P - F C A re-
EXPERIENTIA 33/4
sulted in a m o r e i n t e n s e a c c u m u l a t i o n of l y m p h o i d ceils in t h e p a r a c o r t e x a n d a g r e a t e r d e v e l o p m e n t of g r a n u l o m a t h a n N B P - F C A or FCA alone. M a t u r a t i o n of p l a s m a cells was also a c c e l e r a t e d a f t e r E B P - F C A . N B P - F C A , as c o m p a r e d w i t h F C A alone, i n c r e a s e d s o m e w h a t t h e g r o w t h of t h e l y m p h o i d cell p o p u l a t i o n a n d t h e m e d u l l a r y cords. The significance of t h e l a t t e r m u s t , h o w e v e r , be i n t e r p r e t e d w i t h care, since c o m p r e s s i o n f r o m t h e FCA d e v e l o p i n g l i p o g r a n u l o m a s u b s t a n t i a l l y c h a n g e s t h e circ u l a t i o n and a r c h i t e c t u r e of t h e LN. The l a t t e r m i g h t be e x p r e s s e d m o r e clearly w i t h E B P a n d N B P w i t h o u t t h e a d d i t i o n of FCA. Eliciting E A E w i t h o u t F C A is, however, difficult, if a t all possible. A u t o r a d i o g r a p h i c d a t a i n d i c a t e t h a t t h e n o d a l g r o w t h in b o t h e x p e r i m e n t a l g r o u p s i n v o l v e d p r o l i f e r a t i o n of cells in situ. P r e v i o u s r e p o r t a s h o w e d t h a t g r o w t h of t h e cell p o p u l a t i o n involves also m i g r a t i o n of l y m p h o c y t e s into t h e LN. Following N B P - F C A injection, b o t h processes are therefore a p p a r e n t l y less i n t e n s e and a p p r o x i m a t e values for adm i n i s t r a t i o n of FCA alone. The differences in L N g r o w t h a n d p r o l i f e r a t i o n o b s e r v e d a f t e r a d m i n i s t r a t i o n of E B P FCA and N B P - F C A show t h a t non-specific s t i m u l a t i o n of L N b y basic p r o t e i n w i t h o u t e n c e p h a l i t o g e n i c determ i n a n t (ED) is r e l a t i v e l y small. The p r e s e n c e of E D in t h e basic p r o t e i n molecule ( E B P - F C A ) m a r k e d l y influenced t h e local r e a c t i o n of t h e L N d r a i n i n g t h e site of i n j e c t i o n in t e r m s of increasing m i g r a t i o n of cells into t h e n o d e s a n d p r o l i f e r a t i o n of cells in situ.
Relation between stimulus intensity and neutrophil chemotactic response1 H. U. Keller, J. H. Wissler 2, M. W. Hess a n d H. Cottier
Department o/ Pathology, University o/ Bern, Freiburgstrasse 30, CH-3010 Bern (Switzerland), and Arbeitsgruppe Biochemie, Abteilung /iir experimentelle Kardiologie, Max-Planck-lnstitut /i~r physiologische und klinische Forschung, D-6350 Bad Nauheim (Federal Republic o~ Germany, BRD), 15 December 7976 Summary. The effect of c h e m o t a c t i c p e p t i d e s w h i c h lack c h e m o k i n e t i c a c t i v i t y has been i n v e s t i g a t e d . The n e u t r o p h i l r e s p o n s e is p r o p o r t i o n a l to t h e l o g a r i t h m of t h e s t i m u l u s i n t e n s i t y , or a l t e r n a t i v e l y a p o w e r f u n c t i o n w i t h an e x p o n e n t of 0.3. E q u a l responses are o b t a i n e d for e q u a l ratios b e t w e e n t h e p e p t i d e c o n c e n t r a t i o n in t h e lower c o m p a r t m e n t a n d t h e t h r e s h o l d c o n c e n t r a t i o n . The significance of W e b e r - F e c h n e r ' s law in leucocyte c h e m o t a x i s is discussed. T h e d i r e c t i o n of a c t i v e l y m o v i n g leucocytes can be d e t e r m i n e d b y c h e m i c a l s u b s t a n c e s in t h e e n v i r o n m e n t . Since L e b e r ' s s t u d i e s in 18883, it is generally believed t h a t t h i s process, called c h e m o t a x i s , is i n s t r u m e n t a l in leucocyte a c c u m u l a t i o n a t i n f l a m m a t o r y sites. The r e l a t i o n s h i p b e t w e e n t h e i n t e n s i t y of t h e c h e m o t a c t i c signal and t h e d i r e c t i o n f i n d i n g of leucocytes, as expressed in d i r e c t i o n a l l o c o m o t i o n is still largely u n k n o w n . The i n t e r p r e t a t i o n of earlier d a t a 4-6 was c o m p l i c a t e d b y t h e fact t h a t t h e t e s t m a t e r i a l influenced t h e s p e e d of t h e cells, as well as t h e i r d i r e c t i o n of locomotion. T h u s it h a d c h e m o k i n e t i c as well as c h e m o t a c t i c activity. I t was t h e r e f o r e n e c e s s a r y to e v a l u a t e t h e r e l a t i o n s h i p b e t w e e n s t i m u l u s i n t e n s i t y a n d d i r e c t i o n a l i t y b y m e a n s of purified c y t o t a x i n p r e p a r a t i o n s e x h i b i t i n g c h e m o t a c t i c a c t i v i t y only. P a r t i a l l y purified p e p t i d e p r e p a r a t i o n s c o n t a i n i n g classical a n a p h y l a t o x i n (S-CAT 1.5.1) ~, 8 were p r e p a r e d f r o m d e x t r a n a c t i v a t e d swine s e r u m for such e x p e r i m e n t s . This p r e p a r a t i o n c o n t a i n e d 4% of c h e m o t a c t i c a l l y a c t i v e p e p t i d e s 9. R a n d o m a n d d i r e c t i o n a l l o c o m o t i o n of h u m a n p e r i p h e r a l blood n e u t r o p h i l s was assessed w i t h a m o d i f i e d filter t e c h n i q u e 19, w h i c h p r o v i d e s for s t a b l e g r a d i e n t s (unp u b l i s h e d o b s e r v a t i o n s ) . The b e h a v i o u r o f r e s p o n d i n g ceils ( r a n d o m vs d i r e c t i o n a l locomotion) was also d e t e r m i n e d b y d i r e c t o b s e r v a t i o n . The results o b t a i n e d w i t h
t h e s e 2 t e c h n i q u e s were in a g r e e m e n t . H u m a n s e r u m a l b u m i n (HSA) h a d to be p r e s e n t in t h e t e s t s y s t e m to p e r m i t efficient m o v e m e n t of cells a n d t h e r e b y t h e expression of c h e m o t a x i s in f o r m of d i r e c t i o n a l locomot i o n 11. 1
We thank Miss B. Zanolari, Mrs M. Marti, Mrs Hauck and Mr Dietz for excellent technical assistance. This work was supported by the Swiss National Foundation for Scientific Research. 2 Max-Planck-Institut ffir physiologische und klinische Forschung, D-6350 Bad Nauheim, BRD. 3 Th. Leber, Fortschr. Mecl. 6, 460 (1888). 4 H.U. Keller and 1~. Sorkin, Immunology 70, 409 (1966}. 5 S.H. Zigmond and J. G. Hirsch, J. exp. Med. 137, 387 (1973). 6 P.C. Wilkinson, in: Chemotaxis and Inflammation. Churchill Livingstone, Fdinburgh and London 1974. 7 J . H . Wissler, Eur. J. hnmun. 2i 73 and 84 {1972). 8 J . H . Wissler, V. Stecher and E. Sorkin, Eur. J. Immun. 2, 90 (1972). 9 J . H . Wissler, in: Gram-negative bacterial infections and mode of endotoxin action, p. 91. Ed. B. Urbasehek, R. Urbaschek and E. Neter. Springer, New York 1975. 10 H. U. Keffer, H. Gerber, M. W. Hess and H. Cottier, Agents Actions 6, 326 (1976). 11 H.U. Keller, J. Wissler, M. W. Hess and H. Cottier, in: Movement, Metabolism and Bactericidal Mechanism of Phagocytes (in press).
15.4. 1977
Specialia
W e f o u n d t h a t S - C A T 1.5.1. is s u i t a b l e for t h e a n a l y s i s of t h e c h e m o t a c t i c r e s p o n s e . S - C A T 1.5.1. h a d n o m e a s u r able a c t i v i t y in a b s e n c e of h u m a n s e r u m a l b u m i n . If H S A w a s p r e s e n t in t h e m e d i u m , a p o s i t i v e g r a d i e n t bet w e e n t h e 2 c o m p a r t m e n t s r e s u l t e d in a t t r a c t i o n , a n e g a t i v e g r a d i e n t in c h e m o t a c t i c t r a p p i n g of n e u t r o p h i l s , b u t S - C A T 1.5.1. did n o t i n f l u e n c e r a n d o m l o c o m o t i o n m e a s u r e d in a b s e n c e of a g r a d i e n t (table). T h i s f i n d i n g w a s c o n s i s t e n t o v e r a w i d e c o n c e n t r a t i o n r a n g e of c h e m o t a c t i c p e p t i d e s (1.27 Fg to 0.00127 [~g/ml). T h u s S - C A T 1.5.1. d e t e r m i n e s t h e d i r e c t i o n of t h e cells w i t h o u t affecting t h e r a t e of r a n d o m l o c o m o t i o n , i.e. t h e r a t e of r a n d o m t u r n i n g a n d / o r t h e s p e e d of n e u t r o p h i l s . C o n s e q u e n t l y t h e i n c r e a s e in t h e n u m b e r of cells w h i c h h a v e m o v e d t h r o u g h t h e filter reflects t h e degree of d i r e c t i o n f i n d i n g b y t h i s cell p o p u l a t i o n :
CAT 1.5.1. induces chemotaxis without affecting the rate of random locomotion Test sohltion* (lower compartment)
Cell suspending medi um * (upper compartment)
Percent neutrophils migrated **
2% HSA
2% HSA
0.38 • 0.02
CAT 1.5.1. in 2% HSA
CAT 1.5.1. ill 2% HSA
0 . 3 6 ~ 0.07
CAT 1.5.1. in 2% I[SA
2% HSA
2% HSA
CAT J.5.1. in 2% I-ISA < 0.007
21.8 • 2.9
* The basic medium consisted of 2% human scram albumin (HSA) in f;ey's solution pH 7.2. CAT 1.5.1. (final concentration 1.27 [J.g chemotactic peptides/ml 2% tISA in (;ey's solution) was added to either the test solutiou {positive gradient), the cell suspeusion (uegative gradient) or to b{~th compartme, ts (u~ gradient}. ** V a h l e s r e p r e s e n t t h e p e r c e n t a g e of neutrophils which have migrated through the entire thickuess (140 pun) of the filter ~ SI)K
9 -~
50 o 40 A 3C
,Cells in 2%HSA oCellsin S-Cat 1.5.1. [
~ - - " } ---{
2C "5 7o 10
-o
,,, f=s g
[lower compartment1 [absolute threshold].
2C 1~. 10
Z
10- 2
T h e i n c r e a s e in t h e n u m b e r of n e u t r o p h i l s w h i c h h a v e m o v e d t h r o u g h t h e e n t i r e t h i c k n e s s of t h e filter is p r o p o r t i o n a l to t h e l o g a r i t h m of t h e c o n c e n t r a t i o n of S - C A T 1.5.1. in t h e t e s t c o m p a r t m e n t (figure, A). T h u s t h e r e l a t i v e i n c r e m e n t in e x t e r n a l e n e r g y c o r r e s p o n d s to a c o n s t a n t i n c r e m e n t in t h e r e s p o n s e as e x p r e s s e d b y cell a c c u m u l a t i o n . T h e s e f i n d i n g s are c o m p a t i b l e w i t h o u r earlier h y p o t h e s i s 4 t h a t c h e m o t a x i s as e x p r e s s e d b y d i r e c t i o n a l l o c o m o t i o n is d e t e r m i n e d b y t h e law of W e b e r F e c h n e r . A l t e r n a t i v e l y we h a v e to c o n s i d e r t h a t t h e res p o n s e m a y be d e t e r m i n e d b y S t e v e n s ' p o w e r l a w 1~. i t is in f a c t d i f f i c u l t to d i s t i n g u i s h a s e m i l o g a r i t h m i c r e l a t i o n s h i p (law of W e b e r - F e c h n e r ) b e t w e e n s t i m u l u s i n t e n s i t y a n d m a g n i t u d e of r e s p o n s e f r o m a d o u b l e l o g a r i t h m i c p o w e r f u n c t i o n w i t h a n e x p o n e n t n e a r 0.3 (0.2-0.4), b e c a u s e t h e y m a y h a v e a n a l m o s t i d e n t i c a l c o u r s e o v e r s e v e r a l log u n i t s " . A n e x p o n e n t n e a r 0.3 h a s b e e n f o u n d for h u m a n v i s i o n a n d h e a r i n g , while o t h e r s e n s o r y f u n c t i o n s h a v e d i f f e r e n t e x p o n e n t s for p o w e r f u n c t i o n s r e l a t i n g s u b j e c t i v e m a g n i t u d e to s t i m u l u s int e n s i t y l K S u c h a r e l a t i o n s h i p p r o v i d e s for a c o m p r e s s o r f u n c t i o n to m a t c h t h e s t i m u l u s f r o m o u t s i d e to t h e n e e d s of t h e cells. I n t h e p r e s e n t e x p e r i m e n t s , t h e r a n g e of t h e e x p o n e n t i a l p a r t of t h e r e s p o n s e is, h o w e v e r , q u i t e s m a l l as c o m p a r e d to s e n s o r y f u n c t i o n s of m a c r o o r g a n i s m s vs. T h e r e s p o n s e of n o r m a l n e u t r o p h i l s s u s p e n d e d in 2% H S A r e a c h e s its p l a t e a u a t a dose w h i c h is u p to 50 t i m e s above the threshold concentration. T h e n e u t r o p h i l s c a n also r e s p o n d c h e m o t a c t i c a l l y w h e n S - C A T 1.5.1. is p r e s e n t in b o t h c o m p a r t m e n t s of t h e c h a m b e r , p r o v i d e d t h e r e is a c o n c e n t r a t i o n g r a d i e n t . T h e r e s p o n s e is a g a i n p r o p o r t i o n a l to t h e l o g a r i t h m of t h e a t t r a c t a n t c o n c e n t r a t i o n in t h e lower c o m p a r t m e n t of t h e c h a m b e r u n t i l a p l a t e a u is r e a c h e d . T h e slope of t h e r e g r e s s i o n line is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o b t a i n e d w i t h n e u t r o p h i l s s u s p e n d e d in 2 % H S A o n l y (figure, A). A d d i t i o n of S - C A T 1.5.1. to t h e ceil s u s p e n d i n g m e d i u m , h o w e v e r , a f f e c t s o t h e r f e a t u r e s of t h e n e u t r o p h i I r e s p o n s e . T h e t h r e s h o l d is raised a b o v e tile S - C A T 1.5.1. c o n c e n t r a t i o n in t h e c e l l - s u s p e n d i n g m e d i u m ( u p p e r c o m p a r t m e n t ) , i n d i c a t i n g t h a t t h e cells h a v e r e s p o n d e d to p o s i t i v e c o n c e n t r a t i o n g r a d i e n t s r a t h e r t h a n to t h e a b s o lute test concentration. The 2 curves take a parallel c o u r s e (figure, A), s u g g e s t i n g a g e n e r a l r e l a t i o n s h i p . T h e i n c r e a s e in t h e n e u t r o p h i l r e s p o n s e is p r o p o r t i o n a l to t h e r a t i o b e t w e e n t h e a t t r a c t a n t c o n c e n t r a t i o n in t h e t e s t s o l u t i o n (lower c o m p a r t m e n t ) a n d t h e a b s o l u t e t h r e s h o ] d c o n c e n t r a t i < m c a l c u l a t e d f r o m t h e r e g r e s s i o n line
: Cells preincubated in 2 % HSA ~ preincubated in S-CAT 1.5.1.
g
._~ E
10q
10o
101
535
102
S-CAI 1.5.1 (pg chemotactic peptides/ml test solution)
Relation between imutrophil response and test concentration of S-CAT 1.5.1. A Locomotion (percent neutrophils migrated through the entire thickness [140 btm~ of the filter) of neutrophils suspended in 2% human serum albumin in Gey's solution with or without S-CAT 1.5.1. (0.635 b~g chemotactie peptides/ml) towards test solutions containing increasing concentrations of S-CAT 1.5.1. t3 Response of neutrophils which have been preincubated in either 2% HSA or 2% lISA containing S CAT 1.5.1. (0.635 ~zg chemotactic peptides/inl) for 30 inin at 37~ "lhe cells were then washed extensively and resuspended in 2% HSA in Gey's solution.
E q u a l r a t i o s p r o d u c e e q u a l r e s p o n s e s of a p a r t i c u l a r cell p o p u l a t i o n , e v e n if t h e t h r e s h o l d is s h i f t e d to a d i f f e r e n t level b y t h e a d d i t i o n of S - C A T 1.5.1. to t h e cell s u s p e n d ing m e d i u m . T h i s is c o m p a t i b l e w i t h W e b e r ' s p r i n c i p l e w h i c h s a y s t h a t in o r d e r to p r o d u c e a series of j u s t n o t i c e able d i f f e r e n c e s we m u s t m a k e a fixed r e l a t i v e i n c r e a s e in t h e s t i m u l u s . T h e a d d i t i o n of S - C A T 1.5.1. also lowers t h e p l a t e a u of t h e r e s p o n s e , i n d i c a t i n g t h a t t h e c a p a c i t y for a d i r e c t i o n a l r e s p o n s e to a p o s i t i v e g r a d i e n t is r e d u c e d . C o n s e q u e n t l y t h e l i n e a r r a n g e in t h e s e m i - l o g a r i t h m i c p l o t of s t i m u l u s i n t e n s i t y v e r s u s r e s p o n s e m a g n i t u d e is n a r r o w e d . T h i s m a y , h o w e v e r , be m o r e a p p a r e n t t h a n real, since t h e s e cells are in t u r n in a p o s i t i o n to r e s p o n d to a n e g a t i v e g r a d i e n t b y b e i n g t r a p p e d (table). T h e
12
S. S. Stevens, in: Handbook of Sensory 1)hysiology, vol. I, p. 226. Ed. \V. R. Loewenstein. Springer, Berlin 1971.
536
Specialia
slope of t h e regression lines varied f r o m e x p e r i m e n t to e x p e r i m e n t w h i c h m a y reflect differences in t h e a v e r a g e speed of t h e s e cell p o p u l a t i o n s . I t has b e e n r e p o r t e d t h a t p r e i n c u b a t i o n of n e u t r o p h i l s in a chemotactic medium renders neutrophils unresponsive to f u r t h e r c h e m o t a c t i c s t i m u l a t i o n , e v e n if t h e cells h a v e b e e n w a s h e d e x t e n s i v e l y . This p h e n o m e n o n h a s b e e n called 'deactivation'~a. W e find t h a t n e u t r o p h i l s , w h i c h h a v e b e e n e x p o s e d t o S-CAT 1.5.1. a n d w a s h e d , s h o w n o r m a l r a n d o m l o c o m o t i o n . T h e y b e h a v e as if S-CAT 1.5.1. were still p r e s e n t in t h e cell c o m p a r t m e n t . The t h r e s h o l d for a c h e m o t a c t i c response of such cells is s h i f t e d to a h i g h e r c o n c e n t r a t i o n of S-CAT 1.5.1. a n d a p p r o x i m a t e s t h e c y t o t a x i n c o n c e n t r a t i o n used for pre-
EXPERIENTIA 33/4
i n c u b a t i n g t h e cells. The p l a t e a u of t h e r e s p o n s e is lowered (figure, B). This m a y be c o n s i d e r e d as a t a c h y p h y l a c t i c s t a t e ill l e u c o c y t e c h e m o t a x i s . I t m a y reflect a failure to r e a d a p t efficiently to a n e n v i r o n m e n t w i t h low s t i m u l u s i n t e n s i t y . A d a p t a t i o n , e.g, in visual p e r c e p t i o n 14 or in c h e m o t a x i s of slime m o u l d s 15, is a quite c o m p l e x p h e n o m e n o n . I t s role in leucocyte c h e m o t a x i s r e m a i n s to be f u r t h e r e v a l u a t e d . P.A. Ward and E. L. Becker, J. exp. Med. 127, 693 (1968). P. Gouvas, in: Handbook of Sensory Physiology, voI. VII/2, p. 609. Ed. M, G. F. Fourtes. Springer, New York 1972. 15 G. Gerisch and D. Malchow, in: Biochemistry of Sensory Functions, p. 279. Ed. L. Jaenicke. Springer, New York 1974. 13 14
T h e i n f l u e n c e of l o n g t e r m e s t r o g e n t r e a t m e n t on p l a s m a p r o l a c t i n l e v e l s i n d u c e d b y e t h e r a n e s t h e s i a in o v a r i e c t o m i z e d r a t s 1 G. T. G o o d m a n a n d D. M. L a w s o n
Department o/ Physiology, Wayne State University, School o/Medicine, Detroit (Michigan 48201, USA), 6 September 1976 Summary. 2 m e t h o d s of c o n t i n u o u s e s t r o g e n delivery, p o l y e s t r a d i o l p h o s p h a t e i n j e c t i o n a n d i m p l a n t a t i o n of Silastic capsules of estradiol-17 fl, in o v a r i e c t o m i z e d r a t s i n d u c e d increases in p l a s m a p r o l a c t i n in t h e a f t e r n o o n (15.00-17.00) b e g i n n i n g a t 1 week a n d c o n t i n u i n g for 4-8 weeks. I n a d d i t i o n t h e s e m e t h o d s of e s t r o g e n t r e a t m e n t p o t e n t i a t e d the e t h e r - i n d u c e d increase in p l a s m a p r o l a c t i n in t h e m o r n i n g (9.00-11.00) b e g i n n i n g on week 2 a n d c o n t i n u i n g for 3-8 weeks. T h e s e results i n d i c a t e t h a t e s t r o g e n a c t i v a t e s t h e m e c h a n i s m s t h a t cause an a f t e r n o o n surge in prolactin before p o t e n t i a t i n g a m o r n i n g e l e v a t i o n i n d u c e d b y e t h e r anesthesia. R e p e a t e d injections of large a m o u n t s of e s t r o g e n for several d a y s increases t h e p r o l a c t i n c o n t e n t in t h e r a t p i t u i t a r y 2, a, as well as in t h e s e r u m 4 s. E s t r o g e n m a y p r o m o t e t h e relase of p r o l a c t i n into blood b y affecting h y p o t h a l a m i c neural m e c h a n i s m s g, 1% I n c o n t r a s t to t h e chronic s t i m u l a t o r y effects of estrogen, several t y p e s of stressors induce a c u t e e l e v a t i o n s in p r o l a c t i n secretion. The m o s t w i d e l y i n v e s t i g a t e d r e s p o n s e is t h a t to e t h e r inhalation. E t h e r h a s b e e n s h o w n to increase p l a s m a p r o t a c t i n in o v a r i e c t o m i z e d r a t s n a n d t h e r e s p o n s e is e x a g g e r a t e d in t h e o v a r i e c t o m i z e d a n i m a l t r e a t e d w i t h e s t r o g e n for 1-3 weeks 12. The p u r p o s e of t h e p r e s e n t s t u d y was to elucidate t h e effects of l o n g - t e r m e s t r o g e n t r e a t m e n t on t h e e t h e r i n d u c e d rise of p r o l a c t i n in t h e m o r n i n g w h e n t h e levels are low, a n d in t h e a f t e r n o o n w h e n t h e levels are elevated.
neck. Vaginal s m e a r s were o b t a i n e d daily to m o n i t o r t h e effects of t h e e s t r o g e n t r e a t m e n t s . B e g i n n i n g 1 week a f t e r o v a r i e c t o m y a n d e s t r o g e n t r e a t m e n t a n d c o n t i n u i n g once w e e k l y for 8 weeks, a 2 ml blood s a m p l e was o b t a i n e d f r o m t h e o r b i t a l v e n o u s plexus using h e p a r i n i z e d c a p i l l a r y t u b e s a f t e r 5 rain of c o n t i n u o u s e t h e r a n e s t h e s i a in t h e m o r n i n g (9.00-11.00 a.m.) and again in t h e a f t e r n o o n (15.00 17.00 p . m . ) . A n i m a l s were a n e s t h e s i z e d b y an initial e x p o s u r e to e t h e r v a p o r in a large c o n t a i n e r , followed b y m a i n t e n a n c e w i t h a nose cone. The s a m p l e s were c e n t r i f u g e d a n d t h e p l a s m a was collected a n d s t o r e d a t - 2 0 ~ u n t i l assayed. 1
Materials and methods. 30 m a t u r e , female S p r a g u e - D a w l e y r a t s ( S p a r t a n R e s e a r c h Animals, Inc., H a s l e t t , Michigan) weighing 200-250 g were r a n d o m l y d i v i d e d i n t o 6 groups. A f t e r 7 d a y s of a c c l i m a t i o n t o lighting c o n d i t i o n s (lights on f r o m 6.00 to 20.00 h) all t h e r a t s were b i l a t e r a l l y o v a r i e c t o m i z e d . 1 g r o u p was i n j e c t e d w i t h 0.5 m g polyestradiol p h o s p h a t e ( P E P ; 1.0 m g E s t r a d u r i n | A y e r s t L a b o r a t o r i e s ) s.c., a n d 4 groups received s.c. Silastic imp l a n t s c o n t a i n i n g 12.5 or 25 m g crystalline estradiol-17 /3 a t t h e t i m e of o v a r i e c t o m y . The i m p l a n t s were similar to t h o s e used b y L e g a n et al. ~4. 4 sizes of Silastic t u b i n g were utilized : D o w Corning No. 602-265 1.57 m m i.d., 2.41 m m o.d. 14 a n d 24 m m in l e n g t h a n d D o w Corning No. 602-285 1.57 m m i.d., 3.18 m m o.d. also 14 a n d 24 m m in length. A 2 m m w o o d e n p l u g was i n s e r t e d into 1 end of t h e Silastic t u b i n g a n d c r y s t a l l i n e estradiol-17 /~ was p a c k e d into t h e o p e n e n d of t h e t u b i n g . A n o t h e r 2 m m plug was i n s e r t e d into t h e o p e n end, a n d b o t h ends were c o a t e d w i t h Silastic Medical A d h e s i v e Silicone T y p e A (No. 890 Dow Corning). T h e i m p l a n t s were i n c u b a t e d for 30 min in distilled w a t e r a t 20~ a n d w i p e d w i t h e t h a n o l prior to i n s e r t i o n i n t o a s.c. p o c k e t in t h e n a p e of t h e
2 3 4 5 6 7 8 9 10 11 12 13 14
Acknowledgment. This work was supported by NIH General Research Support, grant No. RR05384-14 to Wayne State University School of Medicine. The authors also wish to express their appreciation to Mrs C. Van De Walle for her expert technical assistance in the prolactin radioimmunoassay, to Dr Richard R. Gala for his advice and support of this project and to the Rat Pituitary Hormone Distribution Program of NIAMDD for the generous gift of Rat Prolactin. R.P. Reece, Proc. Soc. exp. Biol. Med. 39, 77 (1938). J. Meites and C. W. Turner, Proe. Soc. exp. Biol. Med. ,19, 190 (1942). C.L. Chert, H. Miniguchi and J. Meites, Proc. Soc. exp. Biol. Med. 126, 317 (1967). J . D . Neill, Endocrinology 87, 1192 (1970). H . G . Kwa, C. A. Feltkamp, A. A. Van der Gugten and F. Verhofstad, J. Endocr. 48, 299 (1970). A.A. van der Gugten, M. Sala and H. G. Kwa, Acta Endocr. 6d, 265 {1970). L. Caligaris, J. J. Astrada and S. Taleisnik, J. Endocr. 60, 205 (1974). W. Bishop, P. S. Kalra, C. P. Fawcett, L. Krulich and S. M. McCann, Endocrinology 91, 1404 (1972). A. Rather and J. Meites, Endocrinology 75, 377 (1964). D.M. Lawson and R. R. Gala, J. Endocr. 62, 75 (1973). D.M. Lawson and R. R. Gala, J. Endocr. 66, 151 (1974). M. G. Subramanian and R. R. Gala, Endocrinology 98, 842 (1976). S.J. Legan, G. A. Coon and F. J. Karseh, Endocrinology 96, 50 (1975).
