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Comparison between locomotory behavior of lymphocytes of WWV mutant and normal house-mouse Wong, S. Y. 1969

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C O M P A R I S O N B E T W E E N L O C O M O T O R Y B E H A V I O R O F L Y M P H O C Y T E S O F W W V M U T A N T A N D / N O R M A L , H O U S E ^ M O U S E . J> b y S , Y . W o n g B . S c . N a n y a n g U n i v e r s i t y 1 9 6 7 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E i n t h e D e p a r t m e n t o f Z o o l o g y W e a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d . T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a S e p t e m b e r , 1 9 6 9 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada Table of Contents page Abstract i L i s t of Tables i i L i s t of Diagrams i i i L i s t of Figures i v Introduction 1 Materials and Methods 6 (a) Preparation of mouse kidney-monolayer 7 (b) Preparation of lymphocytes 10 (c) Staining of whole cultures 11 (d) Photography 12 Results I. Morphological observations 14 (a) Modes of movement 14 (b) C e l l structures and th e i r functional d i f f e r e n t i a t i o n 18 (c) Transformation of lymphocytes 20 (d) C e l l u l a r associations 22 II. Quantitative analysis of the locomotion of lymphocytes 24 Discussion 30 Summary and Conclusion 42 Appendix 44 Lite r a t u r e c i t e d 48 Figures 1 to 7 with explanations 58 Acknowledgement I am grat e f u l to Dr. A. B. Acton for h i s assistance and advice during the course of t h i s study and i n the f i n a l thesis preparation. I wish to thank Dr. C. V. Finnegan and Dr. C. J . Anastasiouj., for allowing me to use the f a c i l i t i e s in t h e i r laboratories and also for t h e i r advice. Thanks also extended to Dr. P. A. Larkin for h i s -suggestion on the s t a t i s t i c a l analysis. I am very much appreciate to the help given by Mrs. M. Douglas in the course of t h i s study and the photographic assistance given by Mr. L. Veto. ABSTRACT The rate of m o t i l i t y of lymphocytes from anemic mice of the WWV genotype and t h e i r normal ++ littermates was determined jLn v i t r o by time-lapse cinephotomicrography. A comparison of t h e i r speeds on an isogenic kidney monolayer or feeder layer suggests that the speed of locomotion of lymphocytes from the WWV mice may be somewhat reduced. If subsequently v e r i f i e d , t h i s woulid suggest that c e l l s produced i n the lymphatic tissue show, in addition to the well known abnormalities i n the erythrocytes, pigmented., c e l l s of the neural crest and the primordial germ c e l l s , a further defect caused by mutation at the W locus. - i i -L i s t o f T a b l e s T a b l e P a c J e I E f f e c t s o f c u l t u r e m e d i u m a n d a g e s o f m i c e i n t h e e s t a b l i s h i n g o f u n i f o r m m o n o l a y e r 8 a I I C e l l s c o u n t s h o w i n g t h e n u m b e r o f m o v i n g l y m p h o c y t e s p e r c u l t u r e c h a m b e r i n c u l t u r e s o f v a r i o u s a g e s 2 2 a I I I M e a s u r e m e n t s o f t i m e s o f l o c o m o t i o n a n d n o n - l o c o m o t i o n i n d i f f e r e n t s u c c e s s i v e p h a s e s f o r s e l e c t e d l y m p h o c y t e s 2 4 a I V T h e m e a n s p e e d o f l o c o m o t i o n o f l y m p h o c y t e s o f v a r i o u s s i z e s i n d i f f e r e n t c o m b i n a t i o n s i n c u l t u r e s o f d i f f e r e n t a g e s 2 5 a V B i o m e t r i c c o n s t a n t s o f t h e r a t e o f l o c o m o t i o n ' . . > f o r t h e 4 t y p e s o f l y m p h o c y t e - m o n o l a y e r c o m b i n a t i o n s 2 6 a - i i i -L i s t o f D i a g r a m s D i a g r a m p a g e A E x p e r i m e n t a l d e s i g n f o r c i n e p h o t o m i c r o g r a p h i c s t u d y o f t h e l o c o m o t i o n o f + + a n d W W V l y m p h o -c y t e s o n + + a n d W W V k i d n e y m o n o l a y e r s 6 a B T h e m e a n s p e e d , t h e m a x i m u m a n d t h e m i n i m u m s p e e d o f t h e l o c o m o t i o n o f + + a n d W W V l y m p h o -c y t e s o n t h e + + a n d W W V m o n o l a y e r s 2 6 b - i v -L i s t o f F i g u r e s F i g u r e p a g e 1 P h o t o g r a p h s o f a s m a l l l y m p h o c y t e a n d t r a c e o f i t s p a t h s i n a t i s s u e c u l t u r e o f W W V / W W V c o m b i n a t i o n 5 8 2 A m e d i u m l y m p h o c y t e a s i n F i g u r e 1 5 9 3 A l a r g e l y m p h o c y t e a s i n F i g u r e 1 6 0 4 A l a r g e l y m p h o c y t e a s i n F i g u r e 1 s h o w i n g c h a n g e s o f s h a p e s 6 1 5 A ' h y p e r t r o p h i e d ' + + l y m p h o c y t e i n a + + m o n o l a y e r 6 2 6 P h o t o g r a p h s o f c l o s e a s s o c i a t i o n o f 2 W W V l y m p h o c y t e s 6 3 7 P h o t o g r a p h s o f c l o s e a s s o c i a t i o n o f 8 W W V l y m p h o c y t e s 6 4 -1-INTRODUCTION Mutations at the dominant-spotting (W) locus i n the house mouse produce in the homozygous condition a t r i a d of p l e i o t r o p i c e f fects that are seen i n the hematopoietic tissue, the coat color and the gonadal development (Russell, 1949, 1954). A number of a l l e l e s have been i d e n t i f i e d at t h i s locus: these are W ( L i t t l e , 1915); Wv ( L i t t l e and Cloudman, 1937); W11 (Russell, et a l . , 1957); and W° (Ballantyne, et a l . , 1962). Because of t h e i r superior v i a b i l i t y , mice of the WWV genotype have been the most extensively studied. Substitution of the gene-pair WWV for the normal ++ a l l e l e s produces a severe, though not l e t h a l , macrocytic anemia. Mice having t h i s WWV genotype are the so-called black-eyed whites which are completely devoid of fur pigmentation and are i n f e r t i l e . However, though the severe anemia of these mice has been extensively studied morphologically (Russell and Fondal, 1951; Russell, et a l . , 1953: Russell, e t l a l . , 1963) and biochemically (Altman, et a l . , 1953; Singer and Russell, 1954; Altman and Russell, 1964), the precise nature of t h i s mutation i s s t i l l obscure, although some evidence has been accumulated regarding the action of t h i s gene. • The gene-action•leading to the hematopoietic defects has been shown to occur i n the hematopoietic c e l l s themselves, rather than the defects being imposed by the c e l l u l a r , environment or by the effects of other parts of the body -2-(McCulloch, et a l . , 1964; Lewis, et a l . , 1967; Russell, et a l . , 1956; Bernstein and Russell, 1959; Russell and Bernstein, 1968). This has been demonstrated repeatedly by successful implantation of hematopoietic c e l l s from the adult marrow or the f e t a l l i v e r of normal donors into adult WWV, juvenile WWV, and l e t h a l l y anemic WWV mice (Bernstein and Russell, 1959; Russell, _et al., 1956; Bernstein, 1963). These workers concluded that the injected normal c e l l s multiply i n response to d i f f e r e n t i a t i n g factors provided by the host and l i t e r a l l y outgrow the indigenous hematopoietic c e l l s of the anemic r e c i p i e n t . Tests of hemoglobin electrophoretic type, inherited independently of the W locus, have shown that implanted normal erythroid precursor c e l l s produce a l l , or nearly a l l , of the c i r c u l a t i n g erythrocytes of WWV mice, whose anemia has been cured following injections of coisogenic normal ++ hematopoietic c e l l s . Thus, there i s excellent evidence that the defect produced by the WWV gene resides within the hematopoietic c e l l s themselves and i s not mediated by humoral or toxic factors. Furthermore, WWV mice have been reported to respond very poorly to injections of large doses of exogenous erythropoietin, although normal littermate, mice (++) respond strongly to very small doses of the same batch of erythropoietin (Keighley, et a l . , 1962; 1966). This defective capacity to respond to erythropoietin has proved to be an inherent character of WWV blood-forming tissues, since ++/WWV chimeras produced by transplantation of normal ++ bone marrow into WWV mice respond well to the administration of t h i s factor (Keighley, et _al., 1962). -3-The pigment defect caused by the W mutation has been l o c a l i z e d i n the neural crest (Silvers, 1961) and recently has been confirmed to reside within the neural crest c e l l s (Mayer and Green, 1968). Markert and S i l v e r s (as reported by S i l v e r s , 1961) transplanted embryonic tissue containing neural crest from mice destined to be completely white into the anterior chamber of the eye, an environment that i s known to be favorable for melanoblast d i f f e r e n t i a t i o n and melanin synthesis. However, they were unable to detect any pigment c e l l s in the grafts; thus, a defect of the neural crest was suggested rather than a defect in t h e i r environment. Mayer and Green (1968) l a t e r investigated t h i s problem by gra f t i n g normal ++ and WW mutant embryonic skin and neural crest in appropriate combinations into the coelom of host chick embryos. Grafts produced by combining ++ neural tubes with WW white skin resulted i n 100% pigment production, through migration of pigmented c e l l s from the neural crest, whereas only 39% of the WW neural tube and ++ skin combination grafts formed pigment. These re s u l t s were interpreted as demonstrating a defect in the melanocytes of the black-eyed whites. Thus, the pigment-cell defect and anemia i n the W mutants are the results of factors acting within the pigment-and blood-forming c e l l s themselves. The deficiency in number of melanoblasts which underlies the lack of pigment i s not a consequence of the anemia because t h i s abnormality i s already evident at the tenth day of gestation (Russell, 1963), that i s , p r i o r to the development of anemia. -4-Although i t i s not yet known how the dominant-spotting gene acts to produce the defect i n the primordial • germ c e l l s , the s t e r i l i t y of the W homozygotes i s evidently an inherent property of the gonad i t s e l f . Ovaries from isologous ++ mice were transplanted to the ovarian capsule of the homozygous mutants whose own ovaries had been removed; these anemic mice were able to support the transplanted ovaries and successfully concluded pregnancies: the o f f s p r i n g showed the genetic characters of the donors of the ovaries (Russell and Russell, 1948). Gonads removed from 12 to 16 day-old mutant embryos and grafted to the spleen of normal adult castrate mice (Russell, et a l . , 1956), or explantation of 12-day WW gonads to a favorable organ culture medium (Borghese, 1956), do not mitigate the germ c e l l defect which i s f u l l y expressed at 9 days. These experiments suggest that the defect i n the primordial germ c e l l s occurs within the c e l l s themselves and i s therefore u n l i k e l y to be secondary to anemia, since the defect i n the gonads appears p r i o r to the development of the f i r s t manifestation of disordered hematopoiesis (Russell, et al., 1956). This suggestion was confirmed by Borghese, who was able to d i s t i n g u i s h i n t i s s u e culture between f e r t i l e and s t e r i l e gonads from 12-day embryos (Borghese, 1955; 1956; 1957). Further confirmation of the defective development of the germ c e l l s and i t s occurrence before the defective hematopoiesis were reported by Mintz and Russell (Mintz, 1957; Mintz and - 5 -R u s s e l l , 1 9 5 5 , 1 9 5 7 ; R u s s e l l , 1 9 6 3 ) , w h o s h o w e d t h a t t h e n u m b e r a n d l o c a t i o n o f t h e p r i m o r d i a l g e r m c e l l s w a s a b n o r m a l i n t h e W W e m b r y o s . T h u s , i t i s q u i t e e v i d e n t f r o m t h e s e t h r e e l i n e s o f e v i d e n c e t h a t t h e p r i m a r y a c t i o n o f t h e W s e r i e s o f g e n e s , t h a t i s , t h e s i t e o f t h e p e r t i n e n t e f f e c t o f W g e n e s , i s l o c a t e d w i t h i n t h e a f f e c t e d c e l l s t h e m s e l v e s . H o w e v e r , t h e i n t r i n s i c n a t u r e o f t h e s e c e l l u l a r d e f e c t s r e m a i n s a n u n s o l v e d p r o b l e m , a l t h o u g h m a n y s u g g e s t i o n s h a v e b e e n p r o p o s e d i n t h e p a s t f e w y e a r s . I t i s k n o w n t h a t t h e r e i s i m p a i r m e n t o f c e l l d i f f e r e n t i a t i o n a n d p r o l i f e r a t i o n i n t h e h e m a t o p o i e t i c t i s s u e s ( R u s s e l l a n d B e r n s t e i n , 1 9 6 6 ) , f a i l u r e o f m u l t i p l i c a t i o n d u r i n g t h e m i g r a t i o n o f t h e p r i m o r d i a l g e r m c e l l s ( M i n t z , 1 9 5 7 , 1 9 6 0 ) , a n d f a i l u r e o f m e l a n o b l a s t s t o a r r i v e a t t h e h a i r f o l l i c l e s ( M a r k e r t a n d S i l v e r s , 1 9 5 6 ) , b u t t h e r e l a t i o n s h i p b e t w e e n t h e s e t h r e e d e f e c t s p r o d u c e d b y t h e W m u t a t i o n a p p e a r s t o b e o b s c u r e . I t i s n o t e w o r t h y , h o w e v e r , t h a t t h e t h r e e t y p e s o f c e l l s a f f e c t e d b y t h i s l o c u s s h a r e t h e c h a r a c t e r -i s t i c s o f b e i n g m i g r a t o r y a n d p r o l i f e r a t i v e . I t i s , t h e r e -f o r e , q u i t e t e m p t i n g t o s u g g e s t t h a t e i t h e r , o r b o t h , o f t h e s e f u n c t i o n s i s a b n o r m a l i n t h e a f f e c t e d c e l l s o f t h e W m u t a n t . I n a d d i t i o n t o t h e t h r e e d e f e c t i v e t i s s u e s , l y m p h o c y t e s a l s o h a v e t h e c h a r a c t e r i s t i c s o f b e i n g m i g r a t o r y a n d p r o -l i f e r a t i v e , a n d i t i s t h e p u r p o s e o f t h i s i n v e s t i g a t i o n t o f i n d o u t w h e t h e r t h e m u t a t i o n a t t h e W l o c u s h a s a n y e f f e c t o n t h e s e c e l l s . - 6 -M A T E R I A L S A N D M E T H O D S F o r t h e s t u d y o f t h e l o c o m o t o r y b e h a v i o r o f t h e l y m p h o c y t e s , n o r m a l + + a n d W W V m u t a n t m i c e o f C 5 7 B 1 b a c k -g r o u n d w e r e u s e d . T h e + + m i c e w e r e o b t a i n e d f r o m t h e o f f s p r i n g o f a n i n b r e d s t r a i n o f C 5 7 B 1 . T h e + + m i c e a r e c h a r a c t e r i z e d b y a c o m p l e t e l y b l a c k h a i r c o a t , b l a c k e y e s , a n d i n g e n e r a l a p i g m e n t e d t a i l a n d f e e t . T h e W W V m i c e , w h i c h a r e s t e r i l e , w e r e o b t a i n e d b y c r o s s i n g t h e m i c e o f W + g e n o t y p e t o t h o s e w i t h t h e W v + g e n o t y p e . T h i s c r o s s g i v e s a b o u t 2 5 % b l a c k - e y e d w h i t e s o f W W V g e n o t y p e . T h e m i c e w i t h W + a n d W v + g e n o t y p e s w e r e m a i n t a i n e d b y b a c k c r o s s i n g t o t h e + + . W v + a n i m a l s a r e g r e y w i t h a w h i t e b e l l y s p o t a n d W + a n i m a l s a r e b l a c k w i t h a w h i t e b e l l y s p o t a n d a w h i t e s t r e a k o n t h e i r f o r e h e a d s . K i d n e y s o f t h e s e m i c e w e r e u s e d t o e s t a b l i s h a u n i f o r m l a y e r o f c e l l s , t h a t i s a s o - c a l l e d m o n o l a y e r , o n a c o v e r s l i p . T h e m o n o l a y e r a c t s b o t h a s a f e e d e r l a y e r , w h i c h c o n d i t i o n s t h e c u l t u r e f o r t h e s u r v i v a l o f t h e - l y m p h o c y t e s , a n d t o p r o v i d e a s u b s t r a t e f o r t h e m o t i l e l y m p h o c y t e s . A t f i r s t , m o n o l a y e r s o f + + a n d W W V g e n o t y p e s w e r e u s e d t o s t u d y t h e l o c o m o t o r y b e h a v i o r o f l y m p h o c y t e s o f t h e s a m e g e n o t y p e . H o w e v e r , a d i f f e r e n c e i n g e n e t i c b a c k g r o u n d o f t h e m o n o l a y e r f r o m t h a t o f t h e l y m p h o c y t e s m a y a f f e c t t h e i r a b i l i t y t o s u p p o r t t h e s u r v i v a l a n d l o c o m o t i o n o f t h e l y m p h o c y t e s . T h e r e f o r e , f o u r s e r i e s o f e x p e r i m e n t s ( s e e D i a g r a m A ) w e r e N e o n a t a l m i c e -6a- A d u l t s ++ W W I 00 J I k i d n e y s Q 0 k i d n e y c e l l s s u s p e n s i o n ua • c u l t u r e . } c h a m b e r s J ++ o°0oo l y m p h n o d e s W W J - Oo°0o l y m p h n o d e c e l l s s u s p e n s i o n c u l t u r e c h a m b e r s w i t h t h e e s t a b l i s h e d m o n o l a y e r s o n t h e c o v e r s l i p s ++/++ + + / W W v W W V / + + W W V / W W V D i a g r a m A . E x p e r i m e n t a l d e s i g n f o r c i n e m i c r o g r a p h i c s t u d y o f t h e l o c o m o t i o n o f + ± a n d W W l y m p h o c y t e s o n + + a n d W W m o n o l a y e r s , - 7 -c a r r i e d o u t t o a v o i d t h e p o s s i b i l i t y o f t h i s b i a s . I n t h e f i r s t s e r i e s , b o t h l y m p h o c y t e s a n d m o n o l a y e r w e r e f r o m + + m i c e . I n t h e s e c o n d s e r i e s , t h e l y m p h o c y t e s w e r e f r o m + + i n d i v i d u a l s a n d t h e m o n o l a y e r f r o m W W V . I n t h e t h i r d s e r i e s , l y m p h o c y t e s w e r e o b t a i n e d f r o m + + m i c e a n d t h e m o n o l a y e r f r o m W W V m u t a n t s . I n t h e f o u r t h s e r i e s , b o t h l y m p h o c y t e s a n d m o n o l a y e r w e r e f r o m W W V m i c e . I n a l l c a s e s , l y m p h o c y t e s w e r e o b t a i n e d f r o m t h e l y m p h n o d e s o f f u l l y m a t u r e d a n i m a l s a n d m o n o l a y e r s w e r e c u l t u r e d f r o m t h e k i d n e y s o f n e o n a t a l m i c e . ( a ) P r e p a r a t i o n o f m o u s e k i d n e y m o n o l a y e r P r i m a r y m o n o l a y e r s w e r e p r e p a r e d b y t h e m e t h o d , w i t h s l i g h t m o d i f i c a t i o n s , d e s c r i b e d b y D u l b e c c o a n d V o g t ( 1 9 5 4 ) . B r i e f l y , a n e o n a t a l m o u s e w a s k i l l e d b y c e r v i c a l d i s l o c a t i o n a n d t h e t w o k i d n e y s w e r e e x c i s e d a s e p t i c a l l y . T h e o r g a n s w e r e w a s h e d w i t h H a n k s ' s o l u t i o n , f i n e l y m i n c e d w i t h s c i s s o r s , a n d t h e n i n c u b a t e d w i t h 0 . 2 5 % t r y p s i n s o l u t i o n . T h e f i r s t s u p e r n a t a n t w a s u s u a l l y d i s c a r d e d a f t e r i n c u b a t i n g f o r 1 5 m i n u t e s a n d t h e s e c o n d a f t e r a f u r t h e r h o u r o f i n c u b a t i o n . T h e t h i r d s u p e r n a t a n t w a s o b t a i n e d a f t e r p i p e t t i n g u p a n d d o w n w i t h a s t e r i l e p a s t e u r p i p e t t e f o r 2 - 3 m i n u t e s a n d s h a k i n g o n a r o t a t o r f o r 1 5 m i n u t e s . T h e c e l l s i n t h e s u p e r n a t a n t s w e r e w a s h e d t w i c e w i t h H a n k s ' s o l u t i o n u s i n g l o w s p e e d c e n t r i f u g a t i o n a n d w e r e f i n a l l y s u s p e n d e d i n t h e s t a n d a r d m e d i u m . T h e c e l l c o n c e n t r a t i o n w a s e s t i m a t e d u s i n g a h e m o c y t o m e t e r a n d a d j u s t e d t o a f i n a l c o n c e n t r a t i o n o f a b o u t -8-5 3x10 c e l l s per ml. The culture chambers were prepared by s t i c k i n g one y rim of a glass r i n g (inner diameter 15 mm and height 5 mm) to a 75x25 mm glass s l i d e with high-vacuum s i l i c o n e grease. Approximately 1 ml. of the kidney-cell suspension was introduced into each chamber u n t i l i t was f u l l . A coverslip (diameter 22 or 25 mm) was used to cover the open face of the chamber by pressing i t gently down, taking care to exclude a i r bubbles, to s t i c k on the greased rim of the chamber. The chambers were inverted to allow the c e l l s to s e t t l e onto the cover s l i p , and were incubated at 37°C for 3-4 days. After t h i s time, a uniform primary monolayer was obtained which was then ready for the introduction of the lymphocytes. In the course of monolayer c u l t i v a t i o n , i t was found that the age of the kidney and the type of medium used for establishing of the monolayer are very c r i t i c a l . A series of experiments was undertaken to test the effects of these va r i a b l e s . The results are tabulated (see Table I) and are summarized as follows: The preliminary observations and comparisons indicate that kidneys obtained from mice of ages varying from one to about twenty days are suitable for establishing monolayers, although the s u i t a b i l i t y decreases as the age of the mouse increases. Kidneys obtained from adult individuals, or from young mice of ages greater than t h i r t y days, give very inconsistent results and usually are T a b l e I . E f f e c t s o f c u l t u r e m e d i u m a n d a g e s o f m i c e i n t h e e s t a b l i s h i n g o f u n i f o r m k i d n e y - m o n o l a y e r s . ^ * s " - * > « * . A g e s M e d i u m ^"^-^^ 1 5 1 0 1 5 2 0 2 5 3 0 A d u l t M i n i m i a l E a g l e ' s ++++ ++++ ++++ ++++ +++ +++ + poor and inconsistant W a y m o u t h M B 7 5 2 / 1 ++ +++ +++ ++ p o o r poor and inconsistant D u l b e c c o ' s m o d i f i e d E a g l e ' s +++ ++ ** ++ ++ ** poor and inconsistant T C 1 9 9 ++ ++ + ++ p o o r p o o r poor and inconsistant M K ++ p o o r ++ ++ + p o o r poor and inconsistant M K w i t h 0 . 5 % y e a s t o l a t e ** + p o o r p o o r p o o r poor and inconsistant M K w i t h 0 . 1 % y e a s t o l a t e + ** p o o r + p o o r + p o o r poor and inconsistant N o t e s : ** m e a n s e i t h e r c u l t u r e s a r e c o n t a m i n a t e d o r t h e m e d i u m i s t o o a l k a l i n e . - S i -unsuitable for monolayer c u l t i v a t i o n , no matter what kind of medium i s used. Five d i f f e r e n t kinds of medium were t r i e d i n i t i a l l y i n the attempt to establish a uniform monolayer; they are (1) Minimal Eagle's Medium supplemented with 10% or 20% c a l f serum; (2) Waymouth's Medium supplemented with 10% f e t a l c a l f serum; (3) Dulbecco's modified Eagle's Medium supplemented with 10% or 20% f e t a l c a l f serum or horse serum; (4) TC 199 medium plus 10% or 20% f e t a l c a l f serum; and (5) MK medium supplemented with 10% f e t a l c a l f serum alone, or with the addition of 0.1% or 0.5% yeastolate. In addition, a l l the media include 100 units/ml. of p e n i c i l l i n G, 0.1 mg/ml. of streptomycin su l f a t e and 0.005 mg/ml. of phenol red as pH indicator. Minimal Eagle's Medium was found to be the most suitable one for growing the neonatal kidneys, but unfortunately none of the f i v e media was suitable for growing the adult kidneys. Consequently, a l l the monolayers used i n the subsequent experiments were established using neonatal kidney tissues i n Minimal Eagle's Medium. The neonatal klctney tissues have the further advantages of being easier to break down, digest, and separate into single c e l l s , or clumps of c e l l s , by either mechanical treatment or with trypsin solution, and the separated c e l l s take a shorter time to adhere to and spread on the coverslip to form a uniform sheet. The preliminary observations seem to show that one kind of medium i s more favorable for the growth and maintenance of ce r t a i n c e l l types from the mouse kidneys. - 1 0 -F o r e x a m p l e , M i n i m a l E a g l e ' s M e d i u m a n d W a y m o u t h ' s M e d i u m a p p e a r t o s u p p o r t t h e g r o w t h o f e p i t h e l i o i d - l i k e c e l l s b e t t e r t h a n t h e y d o t h e f i b r o b l a s t - l i k e c e l l s u n d e r t h e p r e s e n t c o n d i t i o n s . O n t h e o t h e r h a n d , T C 1 9 9 m e d i u m s e e m s t o s u p p o r t t h e g r o w t h o f f i b r o b l a s t - l i k e c e l l s b e t t e r t h a n M i n i m a l E a g l e ' s M e d i u m , a l t h o u g h a u n i f o r m m o n o l a y e r w a s n e v e r e s t a b l i s h e d w i t h t h i s m e d i u m . T h e s e r e s u l t s s u g g e s t t h a t m o s t o f t h e m o u s e k i d n e y c e l l s i n c u l t u r e a r e e p i t h e l i o i d i n n a t u r e a n d t h a t t h e y p r o b a b l y c o r r e s p o n d t o t h e e p i t h e l i a l c e l l s l i n i n g t h e r e n a l t u b u l e s . I t i s , t h e r e f o r e , t e m p t i n g t o s u g g e s t t h a t t h e v a r i o u s c l a s s e s o f c e l l s p r e s e n t i n t h e m o u s e k i d n e y m a y b e i s o l a t e d j L n v i t r o b y r e p e a t e d c u l t i v a t i o n i n a s p e c i f i c m e d i u m s o t h a t t h e i r p h y s i o l o g i c a l r o l e s _ i n v i v o m a y b e s t u d i e d . ( b ) P r e p a r a t i o n o f l y m p h o c y t e s f o r b e h a v i o r a l s t u d i e s L y m p h o c y t e s u s p e n s i o n s w e r e p r e p a r e d f r o m l y m p h n o d e s o f b o t h m a l e a n d f e m a l e + + a n d W W V a d u l t m i c e . T h e m i c e w e r e k i l l e d a n d t h e a x i l l a r y , b r a n c h i a l , i n g u i n a l a n d m e s e n t e r i c l y m p h n o d e s w e r e r e m o v e d w i t h f i n e f o r c e p s t o a P e t r i d i s h c o n t a i n i n g H a n k s ' s o l u t i o n . T h e f a t t y t i s s u e s s u r r o u n d i n g t h e n o d e s w e r e r e m o v e d c a r e f u l l y a n d t h e n o d e s w e r e t h e n w a s h e d t w i c e w i t h H a n k s ' s o l u t i o n . T h e l y m p h o c y t e s w e r e r e l e a s e d f r o m t h e n o d e s b y t e a s i n g i n s a l t s o l u t i o n w i t h t w o f i n e f o r c e p s . T w o t o t h r e e s u c h c e l l s u s p e n s i o n s w e r e c o l l e c t e d a n d w a s h e d t w i c e w i t h H a n k s ' s o l u t i o n u s i n g l o w s p e e d c e n t r i f u g a t i o n . T h e c e l l p e l l e t w a s t h e n s u s p e n d e d i n culture medium (Dulbecco's modified Eagle's medium supplemented with 20% horse serum). The c e l l concentration was f i n a l l y adjusted to give a c e l l count of approximately 1-2x 10 6 c e l l s / m l . The coverslip, when i t was covered with a f u l l y established monolayer, was removed from the culture chamber; the culture medium used to grow and maintain the monolayer was discarded and the culture chamber was r e f i l l e d with the f r e s h l y prepared lymph-node c e l l suspension. The coverslip with i t s monolayer was then replaced and the culture inverted to allow the lymphocytes to s e t t l e so that they would penetrate the sheet of e p i t h e l i o i d c e l l s to move between them and the cov e r s l i p . The cultures were incubated at 37°C and l a t e r used for microscopic observations and time-lapse c inephotomicrography. (c) Staining of whole cultures After each observation, or taking of photographs, the coverslip carrying the monolayer and moving lymphocytes was removed from the chamber and washed rapidl y i n warm (37°C) normal s a l i n e . The whole culture was then fixed r a p i d l y i n absolute methyl alcohol for about 10 minutes. The rapid washing and f i x i n g are very c r i t i c a l in order to r e t a i n the o r i g i n a l contour of the moving c e l l s . The culture was stained with Wright's and Giemsa stains for 10 minutes, d i f f e r e n t i a t e d i n acetone, dried by evaporation of the acetone i n a i r , and mounted with Permount. This technique has been found to preserve the shapes of the lymphocytes moving between coverslip and the monolayer very s a t i s f a c t o r i l y , although a s l i g h t shrinkage in size usually occurs as compared to the c e l l s in the l i v i n g culture. The d i f f e r e n t shapes of lymphocytes may conveniently be described as oval, round, worm-like or hand-mirror-like. The preservation of the contours of moving lymphocytes has been reported to be d i f f i c u l t (De Bruyn, 1945). However, Berman (1942) has preserved the amoeboid forms of rabbit lymphocytes i n t h i n - f i l m tissue cultures. The present finding seems to suggest that rapid f i x a t i o n i s necessary to preserve the amoeboid shape of the moving c e l l s , since slow f i x a t i o n u s u a l l y causes the c e l l s to round up. (d) Photography A time-lapse cinephotomicrographic apparatus was used to trace the movement of each individual lymphocyte. A Zeiss microscope equipped with phase-contrast objectives and a Nikon movie camera were used throughout the experiment. A time-lapse device was connected to the camera and the speed was adjusted to 30 frames/minute. The f i l m (Double X Negative, Kodak Eastman) was taken at a magnification of 320 (Objective x40, Eyepiece x8 and Column factor x l ) . This figure was confirmed by measuring the magnified image of a known scale on the negative. A long-distance condenser was used because of the thick chambers. These chambers were maintained at 37°C + 0.2°C on the microscope stage with a -13-'Sage Curtain Incubator'. A thermister probe connected to the surface of the culture chamber was used to regulate the temperature of the culture. The films were developed in Kodak D l l for 8-12 minutes at 20°C and fixed in high speed 'Amfix' for 5-10 minutes. The outlines of individual moving lymphocytes were traced on paper with the aid of a f i l m editor. In t h i s way the paths of d i f f e r e n t c e l l s were constructed and the speed of each lymphocyte was calculated. -14-RESULTS I. Morphological observations Many studies have been reported in the l i t e r a t u r e concerning the locomotion of lymphocytes _Ln v i t r o . However, a review of the l i t e r a t u r e has f a i l e d to disc l o s e any q u a l i t a t i v e or quantitative study of the m o t i l i t y of either normal or WWV lymphocytes in mice, although some studies have been made on leukemic lymphocytes (De Bruyn, 1949; Pulvertaft, 1959). This i s probably explained by the d i f f i c u l t y i n getting mouse lymphocytes to survive and become active i n tissue culture, unless they are cultured with some other type of c e l l as feeder layer (De Bruyn, 1949; Bichel, 1939;1952; K i e l e r and K i e l e r , 1954; Ginsburg, 1965). I t i s therefore necessary f i r s t to give a b r i e f account of the modes of movement of mouse lymphocytes, though i t may be found that t h e i r movements are somewhat s i m i l a r to those of the other species reported. The following account applies equally well for the locomotion of normal and WWV lymphocytes, since no differences were detected. (a) Modes of movement C e l l s of various sizes were observed to move between the c overslip and the c e l l s of the kidney monolayer, even as early as the f i r s t hour of incubation of the lymphocytes in the culture chamber. The c e l l s continue to move and can be seen s t i l l i n active migration aft e r four to f i v e days of -15-c u l t i v a t i o n , although the nurriber, kind, and probably speed, of the c e l l s in motion have changed by t h i s time. The locomotory behavior of lymphocytes _in v i t r o has been described for man, rabbit, guinea p i g and rat by a number of investigators (Lewis, 1921, 1931, 1933; De Bruyn, 1944, 1945, 1946; Rich, et a l . , 1939; Robineaux, 1963) i n both normal and pathological conditions. The present observations for ++ and WWV mice agree very well with the c l a s s i c descriptions. The locomotion of lymphocytes in tissue culture was described by Lewis (1931). as movement with a 'hand-mirror' shape. This shape i s characterized by an active anterior pseudopodial region, a r e l a t i v e l y inactive c e l l body containing the nucleus, and a posterior t a i l which usually appears to be passively dragged along. The locomotory phase, which i s never continuous, was regarded by De Bruyn (1945) as being highly polarized, and i s interrupted now and then by a non-locomotory phase which was described by Lewis as a 'period of rest* and by De Bruyn as a 'depolarized phase'. During the non-locomotory phase, the anterior pseudopodial region and the t a i l are withdrawn and the c e l l assumes a more or less spherical shape. However, the c e l l s in t h e i r non-locomotory phase are not e n t i r e l y inactive, but move around without showing any d i r e c t i o n a l movement by pushing out temporary pseudopodia from a l l sides of the c e l l body. This i s i n contrast to the moving lymphocytes whose pseudopodia only ari s e from a li m i t e d area at the anterior part of the c e l l . -16-The: locomotory behavior of mouse lymphocytes has never been studied i n d e t a i l i n tissue culture. The present studies show that mouse lymphocytes may exhibit, i n addition to the so-called 'hand-mirror' shape (Figs. l a , d , f ; 2j; 3c, i,m), an oval (Figs. 6b,c; 7a,b,c), a somewhat rectangular (Figs. lh,k; 2b; 3b,h), or sometimes even a very elongated shape'(Figs. 4c,d,g; 5f-i) i n t h e i r locomotory phase. These shapes may be seen, either in d i f f e r e n t lymphocytes, or even in the same ind i v i d u a l at d i f f e r e n t times. Lymphocytes which show an elongated and a more or less c y l i n d r i c a l shape have been termed 'worm-like' i n motion (Lewis, 1931; De Bruyn, 1945). This type of locomotion was regarded by Lewis as the natural movement of lymphocytes inside plasma-c l o t cultures. Figure 4 shows a moving lymphocyte with a 'hand-mirror' shape as i t moves on a f l a t surface that i s not delimited l a t e r a l l y by any obstacle. However, t h i s hand-mirror form i s l a t e r found to elongate and assumes a worm-like' form as the c e l l squeezes i t s way through a limited space between the coverslip and the overlying c e l l . The degree of elongation varies from time to time depending on the space available; the nucleus i s deformed by t h i s 'gap' or 'tunnel' to give r i s e to one or more cons t r i c t i o n s . ' The t a i l of the c e l l becomes less prominent owing to the elongation of the c e l l body as a whole. I t is therefore quite evident that the .locomotory behavior of lymphocytes i n tissue culture depends very much on the type of substratum over which they move. -17-There are many 'factors which may account for the variations i n shape seen in moving lymphocytes: (1) the heterogeneity of the lymphocyte population; (2) t h e i r speed; (3) the space i n which they move that i s available between the coverslip and the monolayer; (4) the age of the culture; and (5) the interaction between the moving c e l l s themselves. However, the exact nature of these factors s t i l l cannot be determined at the present moment, although i t seems that each has i t s e f f e c t on d i f f e r e n t occasions. Tracings of the migratory pathways have revealed, i n general, that lymphocytes move i n a sinuous manner and frequently may repeat the same pattern, either in the same, or i n the opposite d i r e c t i o n . Though the d i r e c t i o n of the movement of lymphocytes was regarded by De Bruyn (1945) as ramdon, the present observations seem to suggest a d e f i n i t e path for the moving lymphocytes. The difference between these two observations may be explained by the fact that the present observations were made of lymphocytes moving between the e p i t h e l i o i d c e l l s and the coverslip so that they were constrained by a limited space. The lymphocytes usually move more or less r a p i d l y for a few minutes in an approximately st r a i g h t l i n e , then come to rest, assume a rounded form, and then pass into a non-locomotory phase for a varying period of time. When the lymphocytes begin to move again, they do not always move o f f in the previous d i r e c t i o n ; they may move in any, including exactly the opposite d i r e c t i o n . In the course of locomotion, a lymphocyte -18-may make a complete c i r c l e (Fig. 3), or come almost to rest and turn and twist about i n a small space (Fig. 1); they may cross one another's paths without apparent deflection, or come into contact with one another without adhering. The bending and turning of a c e l l i n locomotion i s very common and i s always associated with bending or 'constriction' of the nucleus. Sometimes, a lymphocyte may be seen to show a rhythmic 'pulsation' of the c e l l body, giving the impression that the c e l l advances and retreats over a short distance that i s a f r a c t i o n of the length of the cell.body. I t may then seem as i f the c e l l i s i n a non-locomotory phase, but the lymphocyte i s a c t u a l l y advancing against an inpenetrable obstacle. During t h i s period of constraint, the lymphocyte does not round up completely, but maintains i t s hand-mirror form with a somwhat compressed body and a prominent t a i l . A f ter moving i n t h i s way for a short while, a lymphocyte usually stops moving i n this d i r e c t i o n and returns to i t s normal path. This suggests that normally a moving lymphocyte can only penetrate where a gap i s available for i t to squeeze through. (b) C e l l structures and t h e i r functional d i f f e r -e n t i a t i o n The various components are very constant in t h e i r l o cation i n a moving lymphocyte. The nucleus i s the most conspicuous of these, constituting almost the whole of the -19-body, and constantly changing in form during locomotion. This suggests that an important r o l e i s played by the nucleus during the period of active movement. The nucleus has been shown to play an active part in the locomotion of amoeba by nuclear transplantation between various strains (Jeon, 1968). The morphology of the cytoplasm of a moving lymphocyte i s very consistent in i t s unequal d i s t r i b u t i o n between the anterior pseudopodial region, the t a i l , and the region between the nucleus and the t a i l . A considerable amount of cytoplasm i s located at the anterior end only during locomotion, and i t i s t h i s part of the c e l l that i s a c t u a l l y i n close contact with the substratum on which i t moves. The mitochondria within the c e l l are concentrated in the bulk of the cytoplasm which probably acts as the power house to supply energy required in active locomotion. The cytoplasm present in the t a i l i s very scanty, but i t s r i g i d i t y i s c l e a r l y seen as a c e l l , changes i t s d i r e c t i o n of motion. Though the t a i l can be of considerable length, i t swings without showing any tendency to bend. Almost always the same part of the cytoplasm i s observed to develop into the posterior t a i l as the mass of the cytoplasm i s shifted during the process of d i r e c t i o n a l change. However, the s i g n i f i c a n c e of t h i s r i g i d or permanent t a i l i s s t i l l not known, although McFarlane, et a l . (1965, 1966) have suggested that t h i s 'uropod' may function in immunological interchange " of material between the lymphocyte and the -20-substratum, such as a f i b r o b l a s t c e l l . Neither the present, nor the previous observations, (Lewis, 1931; De Bruyn, 1946) support t h i s hypothesis because the t a i l of the moving lymphocyte is never observed to make close contact with the substratum, but appears to be simply a passive part of the moving c e l l . (c) Transformation of lymphocytes The above description of the locomotory behavior of lymphocytes _in v i t r o applies equally well to small, medium, or large lymphocytes i n the untransformed stage in either new or aged cultures (see Figs. 1, 2, 3 and 4). As the cultures become older, the locomotion of some lymphocytes appears to change (see F i g . 5), and t h i s phenomenon was referred to by De Bruyn (1945) as hypertrophy. The present studies also show such transformation, but the time sequence and. the f i n a l fate of the transformed c e l l s do not appear to be the same as those reported by De Bruyn. Hypertrophy of lymphocytes appears to s t a r t at about 6-8 hours of incubation, by which time a few c e l l s are seen to contain one or more vacuoles that are probably associated with t h i s hypertrophy, most c e l l s i n t h i s 6-8 hours period s t i l l show the t y p i c a l motion of lymphocytes. Those that do not, d i f f e r only s l i g h t l y in that the cytoplasm of the c e l l has become more mobile, and occasionally pseudopodia may be seen around the c e l l body and sometimes even on the t a i l . In the non-locomotory phase, the changes are even more conspicuous, -21-since the pseudopodia a r i s i n g from the c e l l body are usually larger and more numerous, giving an impression of an 'undulating' movement. This transformation seems to involve a breakdown of the membrane surrounding the c e l l s , which res u l t s in a more extensive spreading of the cytoplasm and an increase i n the s i z e of the c e l l . The pseudopodial a c t i v i t y of the hypertrophied lymphocytes i s quite s t r i k i n g when contrasted to that of t y p i c a l lymphocytes. The degree of t h i s a c t i v i t y i s d i r e c t l y related to the degree of hypertrophy of the c e l l s . In the 6-8 hour culture, there are many c e l l s which d i f f e r from the t y p i c a l lymphocytes so s l i g h t l y that i t i s impossible to draw a sharp l i n e between them. At about 12 hours of incubation, there are more c e l l s which exhibit the conspicuous pseudopodial a c t i v i t y i n both phases, e s p e c i a l l y in the non-locomotory phase. They continually throw out large pseudopodia from a l l sides of the c e l l and the amount of cytoplasm i s d e f i n i t e l y greater than that of t y p i c a l lymphocytes. The loss of firm consistency by the cytoplasm i s even greater i n cert a i n hypertrophied c e l l s whose cytoplasm becomes somewhat s t i c k y and usually drags along, or sometimes even leaves cytoplasmic material behind on the substratum. The moving lymphocytes, including both the t y p i c a l and hypertrophied types, reach t h e i r maximal a c t i v i t y at about 12 hours of incubation, although the t o t a l number of c e l l s a c t u a l l y moving between the monolayer and the coverslip -22-i s less than 1% of the number of c e l l s introduced (Table I I ) . The number of c e l l s in motion starts to decrease a f t e r about 24 hours of incubation. At t h i s time, there are s t i l l a large number of t y p i c a l lymphocytes and hypertrophied types s t i l l seen, but by now some wandering c e l l s of highly hypertrophied type are encountered. At about 48 hours of incubation, almost a l l the c e l l s are of the highly hypertrophied type, although some t y p i c a l ones can be located. The movement of these highly hypertrophied wandering c e l l s resembles very c l o s e l y that of macrophages in that they continually throw out large pseudopodia from a l l sides of the body without showing any d i r e c t i o n a l movement. However, i t i s doubtful whether th i s macrophage-like movement of the highly hypertrophied lymphocytes i s act u a l l y that of transformed macrophages, as has been claimed by De Bruyn (1945). The morphology of the nucleus of certain transformed lymphocytes was found to be either a complete on incomplete ring, and t h i s type of c e l l was found to increase i n number as the cultures aged. (d) C e l l u l a r associations An association between normal lymphocytes and a va r i e t y of c e l l s , among which are megakaryocytes,, malignant c e l l s , macrophages and c e l l s in mitosis, has been recorded by several investigators (Humble, et a l . , 1956; Pulvertaft, 1959; Sharp and Burwell, 1960). The present observations do not reveal any sign of a t t r a c t i o n of moving lymphocytes to c e l l s - 2 2 a -T a b l e I I . C e l l s c o u n t s h o w i n g n u m b e r o f m o v i n g l y m p h o c y t e s p e r c u l t u r e c h a m b e r i n c u l t u r e s o f d i f f e r e n t t i m e s . A g e o f c u l t u r e s i n h o u r s N u m b e r o f c e l l s i n m o v e m e n t # 1 # 2 3 8 9 8 3 2 4 9 - 3 6 5 1 2 1 1 7 7 -2 4 6 5 0 4 5 6 4 8 - 1 3 8 T o t a l c e l l s i n t r o d u c e d p e r c h a m b e r 6 . 1 x 1 0 5 c e l l s / m l . 4 . 5 x 1 0 5 c e l l s / m l . N o t e s : T h e n u m b e r o f m o v i n g l y m p h o c y t e s p e r c u l t u r e c h a m b e r o f 1 m l . c a p a c i t y i s a l w a y s l e s s t h a n 1 % o f t h e t o t a l n u m b e r o f c e l l s i n t r o d u c e d . -23-u n d e r g o i n g m i t o s i s , a l t h o u g h many d i v i d i n g c e l l s were p r e s e n t i n t h e c u l t u r e s . I n a d d i t i o n , lymphocytes themselves were ne v e r o b s e r v e d t o undergo m i t o s i s , o r t o a s s o c i a t e w i t h macrophages o r o t h e r c e l l t y p e s . On t h e o t h e r hand, a c l o s e a s s o c i a t i o n between lymphocytes t h e m s e l v e s was ob s e r v e d i n c e r t a i n c u l t u r e s i n wh i c h t h e lymphocytes were o b t a i n e d from an a n i m a l t h a t seemed t o have been s e n s i t i z e d b y a wound. In t h e s e c u l t u r e s , lymphocytes appeared t o a t t r a c t one a n o t h e r and t o move around each o t h e r i n c l o s e c o n t a c t . Such an a s s o c i a t i o n may c o n s i s t o f two o r more c e l l s (see F i g s . 6 and 7). They show t h e i r normal m o t i l i t y d u r i n g t h e a s s o c i a t i o n , b u t u s u a l l y w i t h an o v a l o r even rounded o u t l i n e . T h i s may s u g g e s t a change i n shape i n r e l a t i o n t o t h e phenomenon o f immunity. The c o n t i g u o u s lymphocytes c o n t i n u e t o move around one a n o t h e r f o r a c o n s i d e r a b l e p e r i o d o f tim e , and even i f t h e y then move away from one a n o t h e r , t h e y move back a g a i n i n a s h o r t w h i l e . D u r i n g most o f t h i s t i m e , t h e c o n t a c t between t h e lymphocytes appears t o be o f a s u r f a c e - t o - s u r f a c e n a t u r e . Such p r o l o n g e d c o n t a c t s a r e n o t seen i n c u l t u r e s o f lymphocytes o b t a i n e d from an a n i m a l t h a t has no s i g n o f i n j u r y . Though ") random c o n t a c t between normal o r p o s s i b l y t r a n s f o r m e d lymphocytes may be o b s e r v e d , such c o n t a c t s never p e r s i s t e d f o r more t h a n a few m i n u t e s . They sometimes appeared even t o r e p e l each o t h e r . The i n t e r a c t i o n o r a s s o c i a t i o n between lymphocytes and macrophages has been termed ' p e r i p o l e s i s ' b y Sharp and B u r w e l l (1960) and t h e movement o f lymphocytes w i t h i n a c e l l ' e m p e r i p o l e s i s ' by -24-P u l v e r t a f t (1959). The present observation of the association of presumably 'sensitized' lymphocytes with each other i s not an example of p e r i p o l e s i s since they do not i n t e r a c t with another type of c e l l . The same kind of association has been reported by Robinaux (1963) among immune lymphocytes from guinea-pig lymph nodes. II . Quantitative analysis of the locomotion of lymphocytes The locomotion of lymphocytes i s intermittent, with alternating phases of locomotion and non-locomotion. Table III shows a number of measurements of the duration of these two phases i n d i f f e r e n t lymphocytes. The locomotory phase i s measured from the end of one non-locomotory phase to the beginning of the next; and the duration of the non-locomotory phase s i m i l a r l y i s measured from the end of one locomotory phase to the beginning of the next. The data show that the durations of these phases vary a great deal from c e l l to c e l l , and even within the same ind i v i d u a l observed during successive phases. For example, c e l l #3 was stationary for 10 seconds i n one phase and moved for 1080 seconds i n the next. However, c e l l #8 had 5 stationary phases of 58, 152, 42, 22 and 22 seconds successively and 4 locomotory phases of 378, 188, 254 and 256 seconds during a t o t a l time nearly equal to that of c e l l #3. Owing to the great v a r i a t i o n among these measurements, an estimation of the average duration of these two phases i s of doubtful value and an attempt to compare the durations of these two phases i n ++ T a b l e I I I . M e a s u r e m e n t s o f t i m e s o f l o c o m o t i o n a n d n o n - l o c o m o t i o n i n d i f f e r e n t s u c c e s s i v e p h a s e s f o r s e l e c t e d l y m p h o c y t e s . L y m p h o c y t e # N o n - l o c o m o t o r y p h a s e L o c o m o t o r y p h a s e T i m e ( s e c o n d s ) A v e r a g e T i m e ( s e c o n d s ; ) ; A v e r a g e 1 8 3 - 3 2 8 -2 - - 7 6 0 -3 1 0 - 1 0 8 0 -4 1 6 , 3 0 2 3 2 5 9 , 2 9 2 , 1 4 9 4 6 8 2 5 5 8 , 3 1 0 , 1 7 4 , 7 4 , 1 4 1 2 6 8 4 , 3 3 2 , 3 8 8 , 3 3 0 , 6 2 , 6 8 2 1 1 6 1 8 , 2 8 0 , 3 4 1 1 1 3 5 2 , 9 0 6 6 4 9 7 1 8 , 2 2 2 0 5 5 2 , 2 9 4 4 2 3 8 5 8 , 1 5 2 , 4 2 , 2 2 , 2 2 5 9 3 7 8 , 1 8 8 , 2 5 4 , 2 5 6 2 6 9 -25-and WWV lymphocytes was therefore abandoned. An estimation of the locomotion of lymphocytes i s more r e l i a b l e , and the mean rate of locomotion has already been determined i n various laboratories for the rat, rabbit and human (Lewis, 1933; De bruyn, 1945; McCutcheon, 1924; Schrek, 1963). Table IV presents the estimates of the mean speed of locomotion of 30 lymphocytes of d i f f e r e n t genotypes and sizes, i n cultures of d i f f e r e n t ages, and with d i f f e r e n t combinations of lymphocyte and monolayer. The rate of locomotion was estimated from measurements of the distances traversed i n a series of successive inte r v a l s of 20 seconds, that i s for every 10 frames of the time-lapse f i l m . The measurements were made from the posterior end of the c e l l , rather than from the anterior end, because the t a i l of the lymphocyte i s more r i g i d and constant i n form than the anterior transparent area. However, because the c e l l does not always move in a st r a i g h t l i n e , the estimate of the net distance covered during the 20 second i n t e r v a l i s only a nearest approximation. An analysis of the locomotory behavior during t h i s i n t e r v a l reveals that there i s great v a r i a t i o n i n the instantaneous speed of the lymphocytes. The distance traversed i n 20 seconds i s d i f f e r e n t f o r ' d i f f e r e n t lymphocytes at approximately the same time in the same culture and even for the same ind i v i d u a l in successive periods, and from moment to moment. The v a r i a t i o n in the distances traversed may be attributed to the nature of lymphocyte movement; the non-linear movement of lymphocytes, the squeezing of - 2 5 a -T a b l e I V . T h e m e a n s p e e d o f l o c o m o t i o n o f l y m p h o c y t e s o f v a r i o u s s i z e s i n d i f f e r e n t c o m b i n a t i o n s i n c u l t u r e s o f d i f f e -r e n t a g e s . T y p e o f A g e o f S i z e s o f N u m b e r o f M e a n s p e e d c o m b i n a t i o n c u l t u r e l y m p h o c y t e s o b s e r v e d fyu/min.) .; ( h o u r ) i n t e r v a l s ( 2 0 s e c o n d s e a c h ) 5 s m a l l 3 9 9 . 1 6 s m a l l 3 6 1 3 . 9 n o r m a l + + 2 4 s m a l l - m e d i u m 5 2 9 . 1 l y m p h o c y t e s 2 5 s m a l l - m e d i u m 3 1 1 1 . 7 o n n o r m a l 2 6 s m a l l - m e d i u m 3 9 1 3 . 4 + + m o n o l a y e r 4 8 m e d i u m - b i g 4 3 1 1 . 2 ( + + / + + ) 4 9 s m a l l - m e d i u m 9 5 1 0 . 9 8 m e d i u m - b i g 6 6 1 0 . 9 ih s m a l l - m e d i u m 3 9 7 . 4 A v e r a g e m e a n s p e e d f o r + + / + + c o m b i n a t i o n : 1 0 . 8 ja/min. n o r m a l + + 4 s m a l l - m e d i u m 4 0 9 . 5 l y m p h o c y t e s 5 m e d i u m - b i g 7 5 1 3 . 1 o n W W m u t a n t 8 s m a l l 3 8 8 . 2 m o n o l a y e r &i s m a l l 8 0 1 1 . 5 ( + + / W W ) m e d i u m 6 4 9 . 7 A v e r a g e m e a n s p e e d f o r + + / W W V c o m b i n a t i o n : 1 0 . 4 ^ u / m i n . 8h s m a l l - m e d i u m 9 0 9 . 9 9 b i g 3 6 7 . 1 W W m u t a n t 9k s m a l l - m e d i u m 1 0 6 1 2 . 1 l y m p h o c y t e s 8 s m a l l 6 3 1 0 . 5 o n n o r m a l 4 ^ s m a l l - m e d i u m 1 1 9 1 0 . 9 + + r ' j m o n o l a y e r 5 s m a l l 3 7 1 1 . 2 ( W W - / + + ) 4h m e d i u m 4 4 1 2 . 2 5 m e d i u m 3 6 1 1 . 3 9 m e d i u m 3 4 8 . 5 9h m e d i u m - b i g 4 5 9 . 7 A v e r a g e m e a n s p e e d f o r W W V / + + c o m b i n a t i o n : 1 0 . 3 l a / m i n . W W V m u t a n t 4 m e d i u m - b i g 3 6 8 . 6 l y m p h o c y t e s s m a l l - m e d i u m 6 7 7 . 8 o n m u t a n t 2 4 m e d i u m 5 9 8 . 4 W W m o n o l a y e r 1 1 s m a l l 1 0 1 9 . 9 ( W W / W W ) 6 m e d i u m - b i g 6 0 6 . 8 5 s m a l l 7 4 1 0 . 5 A v e r a g e m e a n s p e e d f o r W W V / W W V c o m b i n a t i o n : 8 . 7 y u / m i n . lymphocytes through gaps, and the change of speed just after movement sta r t s , or before i t ceases, a l l contribute to t h i s v a r i a t i o n . However, the mean speed calculated from many such measurements can reasonably be used as a measure for purposes of comparison. In t h i s study, the mean speed was obtained from at le a s t 30 measurements. Taken o v e r a l l , the average speed of the 30 lymphocytes that were studied, taking no account of genotype i s 10.1 + 0.3 u/min. The range i s from 6.8 u/min. to 13.9 u/min (see Table IV). I f a comparison i s made of the rate of locomotion of lymphocytes in mice with others, i t i s quite evident that mouse lymphocytes move much more slowly than do those of man, rat or rabbit. The average rate of movement has been found to be 16.5 u/min. for r a t lymphocytes (Lewis, 1933), 33u/min. for rabbit lymphocytes (De Bruyn, 1945), and from 6 to 20 u/min., with an average of about 15 u/min., for human lymphocytes (Lewis and Webster, 1921; McCutcheon, 1924; Henderson, 1928; Schrek, 1963). Since the culture systems used in the above determination are not i d e n t i c a l , differences in rate of locomotion of lymphocytes from various species must be accepted with caution. For the analysis and evaluation of the e f f e c t of the WWV gene-pair on the locomotory behavior of lymphocytes in  v i t r o , the data presented i n Table IV are subdivided into 4 subsets, each with a d i f f e r e n t lymphocyte-monolayer combination. Table V shows the biometric data and Diagram B summarizes the mean speeds of lymphocytes from ++ and WW^  lymph nodes moving T a b l e V . B i o m e t r i c c o n s t a n t s o f t h e r a t e o f l o c o m o t i o n f o r t h e 4 t y p e s o f l y m p h o c y t e -m o n o l a y e r c o m b i n a t i o n s . K i n d s o f c o m b i n a t i o n s I t e m ++/++ ++/wwv wwv/++ wwv/wwv T o t a l R a n g e i n j u / m i n u t e -F a s t e s t r a t e 1 3 . 9 1 3 . 1 1 2 . 2 1 0 . 5 1 3 . 9 S l o w e s t r a t e 7 . 4 8 . 2 7 . 1 6 . 8 6 . 8 M e a n r a t e o f l o c o m o t i o n i n u / m i n u t e ± s t a n d a r d e r r o r 1 0 . 8 + 0 . 7 1 0 . 4 + 0 . 8 1 0 . 3 + 0 . 5 8 . 7 + 0 . 5 1 0 . 1 ± 0 . 3 S t a n d a r d d e v i a t i o n i n u / m i n u t e 2 . 1 1 . 9 1 . 6 1 . 4 1 . 9 * C o e f f i c i e n t o f v a r i a t i o n ( p e r c e n t ) 1 9 . 3 1 8 . 4 1 5 . 5 1 5 . 5 1 8 . 3 - 2 6 b -+ + / + + + + / W W W W / + + W W / W W K i n d s o f l y r a p h o c y t e - m o n o l a y e r c o m b i n a t i o n s D i a g r a m B . T h e m e a n s p e e d , t h e m a x i m u m a n d t h e m i n i m u m s p e e d o f t h e l o c o m o t i o n o f + ± a n d W W V l y m p h o c y t e s o n t h e + + a n d W W V m o n o l a y e r s . on ++ and W W renal monolayers. The mean speed i n the ++/++ combination i s found to be 10.9 + 0.7 u/min., with a v a r i a t i o n from 7.4 u/min. to 13.9 ]a/min. The mean speed i n the + + / W W V combination varies from 8.2 u/min. to 13.2 u/min., with an average of 10.4 + 0.8 u/min. Similarly, the mean speed i n the W W V / + + combination i s determined to be 10.3 + 0.5 u/min., with a minimum value of 7.1 u/min. and a maximum of 12.2 u/min. F i n a l l y , the mean speed in the W W V / W W V combination i s 8.7 + 0.5 u/min. with a range from 6.8 u/min. to 10.5 u/min. These results suggest that both the mean rate of locomotion and the maximum rate of locomotion may be highest for the 4+ lymphocytes moving on the ++ monolayer, and the lowest for the W W V lymphocytes moving on the W W V monolayer. This means that the m o t i l i t y of lymphocytes of W W V mice i s somewhat impaired when compared to the control groups. The difference i n t h e i r mean speeds i s s t a t i s t i c a l l y s i g n i f i c a n t i f a t - t e s t i s used (P<C 0.025; see Appendix A), but the data are unfortunately too few to allow conclusions to be drawn from any of the other comparisons. Since the term * W W V lymphocytes' has been used to cover a l l lymphocytes from the W W V mice that have a s i m i l a r morphology, a defect i n the lymphocytes with respect to t h e i r rate of locomotion may therefore not apply to a l l . Morphologically s i m i l a r lymphocytes have been shown to be heterogeneous with respect to t h e i r function and l i f e cycle (Gowans and McGregor, 1965). ' W W V lymphocyte* i s used merely -28-for d e s c r i p t i v e purposes and does not imply any q u a l i t a t i v e or quantitative abnormality i n a l l , or even i n any, of the lymphocytes of the mutant mice. Indeed, heterogeneity among morphologically s i m i l a r lymphocytes from the same source has been found to exis t i n the present study by s t a t i s t i c a l analysis of rates of locomotion. When an analysis of variance for the four••'combinations of lymphocyte and monolayer i s made i n a one-way c l a s s i f i c a t i o n of h i e r a c h i c a l design with unequal sample size (for calculation, see Appendix B ) , a s i g n i f i c a n t F-value was obtained for variance between the ind i v i d u a l lymphocytes tested. This leads to the conclusion that lymphocytes from a single mouse are heterogeneous with respect to t h e i r rate of locomotion, and therefore are derived from d i f f e r e n t sub-populations. Recognition of the p a r t i c u l a r c e l l with the impaired m o t i l i t y i s not possible. Furthermore, a close examination of the data for the movement of lymphocytes on a coisogenic renal monolayer shows that the WWV renal monolayer i s somewhat less able than the isogenic ++ renal monolayer to support growth and a c t i v i t y of the ++ lymphocytes and, as might be expected from t h i s , a ++ monolayer supports the a c t i v i t y of WWV lymphocytes better than the isogenic WWV monolayer (see Diagram B ). Thus, the mean rate of locomotion of ++ lymphocytes on an isogenic monolayer was found to be 10.8 + 0.7 ij/min., with a maximum of 13.9 ju/min., whereas the rate recorded for t h e i r movement on the coisogenic WWV monolayer was only 10.4 + 0.8 ju/min., with a maximum va l u e o f 13.1 yu/min. T h i s i n d i c a t e s t h a t both the mean and the maximum speed are somewhat lower f o r the ++, c e l l s moving on a WWV mutant monolayer. S i m i l a r l y , the WWV lymphocytes move f a s t e r on the ++ monolayer, w i t h a mean r a t e o f 10.3 + 0.5 ;u/min., and a maximum o f 12.2 ;u/min. Although these v a l u e s are somewhat s m a l l e r than those f o r ++ lymphocytes moving on the WWV monolayer, they :• are v e r y much g r e a t e r than the r a t e s recorded f o r the WWV lymphocytes moving on the i s o g e n i c WWV monolayer which have a mean r a t e of 8.7 + 0.5 ^ u/min., and a maximum v a l u e of o n l y 10.5 ^ u/min. The b e s t e x p l a n a t i o n o f t h i s v a r i a t i o n i s t h a t the WWV r e n a l monolayer has a somewhat impaired a b i l i t y to support the a c t i v i t y o f e i t h e r normal ++ o r WWV lymphocytes, though the sample s i z e s are too srna'll t o g i v e s t a t i s t i c a l l y s i g n i f i c a n t p r o b a b i l i t i e s (see Appendix B ) . -30-DISCUSSION There have previously been no descriptions of the locomotory behavior of mouse lymphocytes. I t was therefore necessary f i r s t to f i n d out to what extent t h e i r movement resembles, or d i f f e r s from, those of other mammals. The motile lymphocytes i n the present culture system are r e a d i l y recognized when they move on a f l a t surface by t h e i r 'hand-mirror' shape. However, they may change to the so-called 'worm-like' shape when they squeeze through gaps between the c e l l s composing the substratum. The 'worm-like' and 'hand-mirror' configurations of locomotion were described in other species by both Lewis (Lewis and Webster, 1921; Lewis, 1931, 1933) and Rich, et al., (1939) as being the normal shapes of lymphocytes moving _in v i t r o , and i n t h i s respect the lymphocytes of the mouse resemble those of the other species that have been described. In the study of migration from a lymph node fragment into a thick plasma c l o t , Lewis and Webster (1921) noted that each lymphocyte as i t moved through a plasma c l o t gave the impression that i t was being squeezed through a ring, with alternating phases of rest and movement, though even in the rest i n g phase numerous small pseudopodia were continually thrown out and retracted on a l l sides of the c e l l . Locomotion began with the extrusion of a single, large, sausage-shaped, pseudopodium from what now became the anterior end of the -31-moving c e l l . A groove, which was termed 'constriction ring', then developed at the base of the pseudopodium where i t joined the c e l l body. The 'constriction ring' remained fixed i n space and the rest of the c e l l now flowed through i t into the pseudopodium. In the process, the nucleus became very deformed and might even be twisted during i t s passage through the 'ring'; t h i s was the so-called 'worm-l i k e ' configuration. Once the nucleus had passed through the 'ring' i t regained i t s 'hand-mirror' configuration, with the round nucleus i n front and a t a i l of cytoplasm behind. The t a i l was then slowly taken up and the c e l l returned to i t s rounded re s t i n g configuration, and the whole 'ring' cycle was completed. The hand-mirror configuration was described by them as only one phase in the cycle of locomotion. Rich, ^ t a l . (1939) also studied the the movement of lymph node lymphocytes in plasma c l o t cultures, but claimed that a l l the lymphocytes, regardless of t h e i r sizes, always moved with the hand-mirror configuration. The round anterior end of the c e l l displayed a fringe of small pseudopodia, while most of the cytoplasm formed a t a i l t r a i l i n g at the posterior end. The whole c e l l glided forwards s t e a d i l y without the formation of any 'constriction rings' as described by Lewis. This apparent discrepancy between the two accounts was l a t e r resolved by De Bruyn (1944),,, who also studied lymph node c e l l s i n a plasma c l o t . He found that the lymphocytes moving within the c l o t assumed a -32-'worm-like' configuration, as claimed by Lewis, but that a l l those moving between the coverglass and the plasma c l o t exhibited the slow g l i d i n g and constant hand-mirror form described by Rich et a l . De Bruyn thought the 'constriction rings' to be external, and i n fact to be holes i n the f i b r i n network. The present observations agree very well with De Bruyn 1 s, and further speak against Lewis's conclusion:? that 'since lymphocytes have been so accustomed, for thousands of generations, to squeezing through t i g h t places they go on acting i n t h i s way when there are no small holes to go through' since many lymphocytes have been seen i n the present study to stop and change t h e i r d i r e c t i o n of locomotion i f there i s no space available between coverslip and the monolayer, although they may push t h e i r way through for a short distance. Harris (1953), i n hi s study of the movements of thoracic duct^lymphocytes i n a d i l u t e plasma c l o t , claimed that the type of movement i n h i s culture system was very variable: one and same lymphocyte might move, at d i f f e r e n t times, with the hand-mirror configuration, or worm-like configuration, or i n a non-polarized amoeboid fashion. This des c r i p t i o n agrees well with what has been observed i n the present study. Since Harris's culture system was somewhat intermediate between those of Lewis and Rich, et a l . , i t i s not su r p r i s i n g that he found such v a r i a t i o n . Almost a l l the early studies concerning the locomotion of lymphocytes _in v i t r o were made using frames of a cinemicro--33-graphic f i l m of i n d i v i d u a l c e l l s migrating through a plasma c l o t . More recently, the c e l l s were trapped between two coverslips, or between coverslip and agar, and i t i s doubt-f u l whether t h e i r movements in these experimental systems are altogether normal. Previous findings showed that the o p t i c a l conditions for the study of the movement of lympho-cytes i n plasma c l o t s were very poor and the s i t u a t i o n could be improved i f c e l l s were allowed to move on a plane surface such as a glass cov e r s l i p . Unfortunately, lymphocytes w i l l not adhere to a glass surface and crawl on i t (Fich t e l i u s , 1951; Harris, 1953), but they are capable of moving between two c l o s e l y apposed surfaces. The culture system used in the work reported here retains the indispensible conditions, provided by a plasma c l o t , namely the contribution to the s u r v i v a l of the lymphocytes by a feeder layer of other c e l l s , while at the same time dispensing with i t s poor o p t i c a l properties. Both Lewis and De Bruyn have claimed that the movement of lymphocytes i s a purposeless wandering. However, since lymphocytes have the a b i l i t y to move towards certain c e l l types i n v i t r o (Humble, et a l . , 1956; Pulvertaft, 1959) and to regions of inflammation (Conheim,1867; Trowell, 1965) in vivo, and to aggregate in regions of homograft r e j e c t i o n (Wilson, 1965; Mitchison, 1954; Billingham, et a l . , 1954; Gowans, 1965), i t i s doubtful whether the m o t i l i t y of these c e l l s , either _in v i t r o or _in vivo, can be a purposeless wandering. Nevertheless, no one s p e c i f i c stimulus has been -34-reported in the l i t e r a t u r e (McCutcheon, 1946, 1955; Coman, 1940; Harris, 1953; Kass and De Bruyn, 1967). Although there i s s t i l l no evidence of chemotaxis by lymphocytes, the close association of the lymphocytes with other c e l l types i n the phenomena of 'peripolesis' (Sharp and Burwell, 1960) and 'emperipolesis' (Pulvertaft, 1959) has been f u l l y established, though t h e i r exact nature i s s t i l l obscure. The phenomenon of 'peripolesis' was noted e a r l i e r by Pulve r t a f t and Jayne (1953) i n the association between small lymphocytes or megakaryocytes and c e r t a i n tumor c e l l s . The l a t t e r association was also described by Humble, _et aJL. (1956) who concluded that lymphocytes may actually f a c i l i t a t e the spread of cancer. However, the experiment of Sharp and Burwell (1960) suggested that lymphocytes are attracted to c e r t a i n c e l l s , such as macrophages or reticulum c e l l s , in the course of immune reactions. The process of 'emperipolesis' was f i r s t noted by Humble, et al. (1956) when lymphocytes penetrate ce r t a i n megakaryocytes or tumor c e l l s and move f r e e l y round within the host cytoplasm. Later, P u l v e r t a f t (1959) reported the same phenomenon by leukemic lymphocytes of man and mouse within chick f i b r o b l a s t s . Emperipolesis has been observed in v i t r o within macrophages (Lewis, 1925), thymus e p i t h e l i a l c e l l s (Trowell, 1949; Klein, 1958), f i b r o b l a s t s (Fischer and Dolschansky, 1929; Trowell, 1949; Bichel, 1939), and sarcoma c e l l s (Koller and Waymouth, 1953), and by leukemic lymphocytes within histocytes (Shelton and Rice, 1958). -3 5-Though emperipolesis was not seen, the present observations reveal a t h i r d type of association between the presumably sensit i z e d lymphocytes themselves. A sim i l a r type of association has been shown to occur between small and medium lymphocytes by Robineaux (1963) i n h i s study of movements of c e l l s involved i n inflammation and immunity. These re s u l t s seem to suggest an a l t e r a t i o n of the c e l l membrance of lymphocytes associated with disease, inflammation or immune reactions i n the animals concerned. Hypertrophy of lymphocytes, or possibly the trans-formation of lymphocytes into macrophages, was observed i n the present culture system. These investigations show that forms of locomotion exi s t that are intermediate between those of t y p i c a l lymphocytes and macrophages. As the degree of hypertrophy increased with time, the mode of locomotion of . the c e l l s gradually came to resemble more c l o s e l y that of macrophages. The same sequence of hypertrophy has also been reported by De Bruyn (1945) i n h i s studies of the migration of lymphocytes i n plasma-clot cultures. Trowell (1965) has reviewed reports presented in the l i t e r a t u r e concerning the a b i l i t y of lymphocytes to transform into macrophages. He l i s t e d 53 publications, of which 23 claimed that the macrophages developed exclusively from lymphocytes. The a b i l i t y of the monolayer to maintain and prolong the s u r v i v a l and a c t i v i t y of many types of c e l l s , including the lymphocytes, has led many investigators to suggest that -36-th e monolayer may supply necessary metabolites for the coexisting c e l l s , or that i t may remove toxic metabolic products, or that i t may act mechanically (Ginsburg, 1965; Bi c h e l , 1939, 1952; De Bruyn, 1949; K i e l e r and Kieler, 1954; Puck, et a l . , 1956; Brooke and Osgood, 1959; Woodliff, 1964). Therefore the decreased a b i l i t y of the WWV monolayer to support normal a c t i v i t y , as judged by the reduced mean rate of locomotion on the WWV monolayer, may be due either to lack of c e r t a i n metabolites necessary for the maintenance of a balanced condition _in v i t r o , or an a l t e r a t i o n in the surfaces of the c e l l s in the monolayer so that the locomotion i s impeded mechanically. Unfortunately, the difference between the a b i l i t y of normal and WWV monolayer to support the a c t i v i t y of lymphocytes jLn v i t r o may not be s t a t i s t i c a l l y s i g n i f i c a n t , owing to the small sample sizes. In t h i s investigation, i t was found that lymphocytes obtained from the lymph nodes of WWV mutant mice do not d i f f e r i n v i t r o i n any way from those of ++ mice in the manner i n which they move. Their movements are intermittent with alternating locomotory and non-locomotory phases, and they move in a sinuous manner, without abrupt changes i n d i r e c t i o n . However, a quantitative analysis of the reates of locomotion suggests that the c e l l s are heterogeneous in t h i s respect, though t h e i r modes of locomotion are s i m i l a r . C e l l s obtained from lymph nodes are a heterogeneous population with respect to t h e i r functional p o t e n t i a l (Gowans, 1965; Everett, et a l . , 1964; Oort, et a l . , 1965; Good and Finstard, -37-1967; Lance and Taub, 1969). T h i s p r o b a b l y e x p l a i n s why t h e c e l l s t h a t a r e randomly s e l e c t e d f o r exp e r i m e n t s show such a g r e a t v a r i a t i o n . However, assuming t h a t t h e measured r a t e s o f l o c o m o t i o n a r e i n each case o f randomly s e l e c t e d samples from t h e lymp h o i d p o p u l a t i o n s o f ++ and WWV mice, a comparison o f t h e mean speed u s i n g a t - t e s t i s p o s s i b l e . The r e s u l t o f t h e comparison s u g g e s t s , though i t i s n o t c o n c l u s i v e , t h a t a d i f f e r e n c e e x i s t s i n t h e r a t e o f l o c o m o t i o n between lymph-node p o p u l a t i o n s o b t a i n e d from normal and mutant mice, when t h e lymphocytes a r e moving t h r o u g h an i s o g e n i c monolayer. These a r e j u s t t h e c o m b i n a t i o n s t h a t most c l o s e l y resemble t h e n a t u r a l s i t u a t i o n i n t h e mice. However, t h e r e s u l t s may a l s o be i n t e r p r e t e d t o i n d i c a t e t h a t t h e m o t i l i t y o f t h e lymphocytes i s a l s o i n f l u e n c e d by t h e genotype o f t h e monolayer t h r o u g h w h i c h t h e y a r e moving. More measurements a r e needed b e f o r e any a s s e r t i o n s can be made w i t h c o n f i d e n c e . I t t h e r e f o r e seems p o s s i b l e t h a t t h e m u t a t i o n a t t h e W l o c u s i n t h e house mouse, i n a d d i t i o n t o a f f e c t i n g t h e e r y t h r o i d and m y e l o i d t i s s u e o f t h e b l o o d - f o r m i n g t i s s u e , a l s o a f f e c t s t h e c e l l s i n t h e lymp h o i d t i s s u e s . A l t h o u g h th e e x a c t n a t u r e o f t h e d e f e c t i n t h e h e m a t o p o i e t i c t i s s u e i n WWV mutant mice i s s t i l l unknown, i t has, however, been l o c a l i z e d t o an a b n o r m a l i t y i n t h e h e m a t o p o i e t i c c o l o n y -f o r m i n g stem c e l l s (CFU), t h a t may be t h e p r e c u r s o r c e l l s o f th e e r y t h r o i d , m y e l o i d o r even l y m p h o i d t i s s u e s (Wu, e t a l . , 1968). Whether a q u a l i t a t i v e d e f e c t i n t h e CFU c e l l , o r a -38-decrease in t o t a l pool size, i s p r i m a r i l y responsible, remains to be discovered. The defect i n erythropoiesis giving r i s e to the anemia has been shown by spleen cplony studies (McCulloch, et a l . , 1964; Lewis, et a l . , 1967) to reside i n an erythroid stem c e l l . Transplantation of marrow c e l l s from WWV mice into normal ++ i r r a d i a t e d hosts produces colonies that contain almost no erythroid c e l l s and that are fewer in number and smaller i n size than those which occur af t e r transplantation of normal ti s s u e . ++ marrow transplanted into either i r r a d i a t e d or non-irradiated WWV mice re s u l t s i n apparently normal macroscopic colonies and the anemia and r a d i o s e n s i t i v i t y of the WWV mice are eliminated (McCulloch, et a l . , 1964; Lewis, 1967; Russell,'et a l . ( 1956; Bernstein and Russell, 1959; Russell and Bernstein, 1968). These findings suggest a q u a l i t a t i v e defect i n the colony-forming c e l l , or the erythroid precursor c e l l , that r e s u l t s in an impairment of t h e i r d i f f e r e n t i a t i o n into erythroid c e l l s , and perhaps at the same time i n a decrease in pool size of th i s precursor c e l l . Increased c e l l production r e s u l t i n g in improvement i n the anemia, following stimulation of the CFU or the erythroid precursor c e l l s by exposure to hypoxia (Keighley, et a l . , 1966; Fried, et a l . , 1967), or afte r administration of androgens (Fried, et a l . , 1967), together with the c a p a b i l i t y to respond to high doses or erythropoietin (Keighley, et a l . , 1966) i n the WWV mice, suggest that the CFU or the erythroid precursor c e l l of the mutant responds to normal stimulation, but that the s e n s i t i v i t y i s lowered. Studies by Lewis, et _al. (1967) reveal that spleen colonies of a l l c e l l types a r i s i n g from transplanted WWV marrow are smaller and fewer in number than occurs with transplanted normal c e l l s . This suggests a small CFU pool, but on the / other hand, the marrow and spleen may not r e f l e c t the composition of the t o t a l body stem-cell pool (Boggs, et a l . , 1968). Mouse hematopoietic colony-forming stem c e l l s have been shown capable of d i f f e r e n t i a t i n g along both erythropoietic and granulocytic l i n e s (Wu, et a l . , 1967). Studies of the 59 131 incorporation of Fe and IUDR into the spleen colony have suggested that the production of both non-erythroid and erythroid c e l l s i s defective (Bennett and Cudkowicz, 1966). Adult mice of the WWV genotype, i n addition to having a defect in erythropoiesis, also have a defect in t h e i r myeloid and megakaryocytic tissue (Chervenick and Boggs, 1969). This defect i s manifested by a decreased t o t a l nucleated-cell count, as determined in the humerus, as well as by a decreased number of neutrophils and megakaryocytes in the marrow. However, other studies have shown a normal number of v leukocytes and p l a t e l e t s i n the blood of WW mice (Lewis, et a l . , 1967), though there has been a report of a s l i g h t decrease i n both elements (Gruneberg, 1939). The defect i n the myeloid tissue may be due to delayed and reduced p r o l i f e r a t i o n and d i f f e r e n t i a t i o n , as i n the erythropoietic -40-t i s s u e , b u t i t i s l e s s s e v e r e (Bennett, _et _ a l . , 1968). E v i d e n c e has been p r e s e n t e d ( T i l l , ^ t a l . , 1967; F o w l e r , est a l . , 1967; 0'Grady./ je_t aJL., 1968) f o r t h e e x i s t e n c e o f stem-c e l l p o o l more mature t h a n t h e CFU. There i s i n d i r e c t e v i d e n c e t o s u g g e s t t h a t a more d i f f e r e n t i a t e d n e u t r o p h i l s t e m - c e l l p o o l may a l s o e x i s t . S t u d i e s by B e n n e t t , e t a l . , (1968) o f c o l o n y f o r m a t i o n jLn v i t r o i n d i c a t e t h a t the marrow o f WWV mice produces c o l o n i e s s i m i l a r i n s i z e and number t o t h o s e o b s e r v e d when marrow from normal mice i s used. C o l o n i e s i n t h i s _in v i t r o system a r e composed p r i m a r i l y o f n e u t r o p h i l s and macrophages. These o b s e r v a t i o n s , a l o n g w i t h t h e d e c r e a s e i n t o t a l n e u t r o p h i l mass r e p o r t e d by C h e r v e n i c k and Boggs (1969), s u g g e s t t h a t n e u t r o p h i l s a r e n o r m a l l y p roduced beyond t h e CFU c e l l s t a g e and t h a t e i t h e r p r o l i f e r a t i o n or, d i f f e r e n t i a t i o n o f t h e CFU c e l l i s abnormal. F u r t h e r m o r e , t h e CFU c e l l p r e s e n t i n t h e normal bone marrow have been r e p o r t e d t o i n c l u d e c e l l s c a p a b l e o f r e -p o p u l a t i n g n o t o n l y t h e m y e l o i d t i s s u e s b y a l s o t h e lymphoid t i s s u e s o f i r r a d i a t e d mice (Ford, e t a l . , 1966; M i c k l e , e t a l . , 1966). W h i l e i t seems p r o b a b l e t h a t t h e CFU c e l l s o f marrow mig h t be r e s p o n s i b l e f o r r e p o p u l a t i n g b o t h t i s s u e s ( T r e n t i n and F a h l b e r g , 1963; T i l l , e t a l . , 1967; Wu, e t a l . , 1968), p r e s e n t c y t o l o g i c a l e v i d e n c e shows t h a t s i n g l e c l o n e s , whose members a r e i d e n t i f i e d by t h e p r e s e n c e o f unique chromosomal markers, may c o n t a i n n o t o n l y thymus and lymph node c e l l s , b u t a l s o h e m a t o p o i e t i c c o l o n y - f o r m i n g c e l l s . These f i n d i n g s mean, e i t h e r t h a t lymphoid c e l l s a r e descended from CFU c e l l s , -41-or that both classes have a common progenitor. Thus the CFU, erythroblasts, granulocytes, thymic c e l l s , and the c e l l s of the lymph nodes may a l l belong to the same clone. In addition, Shearer and Cudkowicz (1967) have reported that the W mutations also a f f e c t the immune system, since the number of anti-sheep hemolysin-forming c e l l s generated by spleen of mutant mice was only one-third to one-half the number of anti-sheep hemolysin-forming c e l l s generated by spleens of normal littermates. This evidence, together with that mentioned above, strongly indicates that antibody-producing c e l l s and CFU c e l l s may belong to the same clone. Thus the primary target of the genetic defects induced by the W mutations may be either a pluripotent stem c e l l , or a more d i f f e r e n t i a t e d precursor c e l l pool. If the conclusion that has been made from the present study, that there i s a defect i n the locomotory behavior of the small lymphocytes of the WWV mouse, i s confirmed, there w i l l be one more piece of evidence, along with a l l those described above, to show that the W mutations of the mouse have an even more far-reaching e f f e c t than was at f i r s t supposed. Whether t h i s evidence w i l l throw l i g h t on the possible o r i g i n of the b l o o d c e l l l i n e s from a single stem c e l l remains to be seen. -42-SUMMARY AND CONCLUSION Ti m e - l a p s e c i n e p h o t o m i c r o g r a p h y was used t o s t u d y t h e e f f e c t _in v i t r o o f t h e W m u t a t i o n s i n t h e house mouse on t h e l o c o m o t o r y b e h a v i o r o f t h e i r lymphocytes on i s o g e n i c and c o i s o g e n i c | monolayers o f k i d n e y c e l l s . No m o r p h o l o g i c a l l y d e t e c t a b l e d i f f e r e n c e i n t h e modes o f move-ment was o b s e r v e d between WWV and ++ lymph o c y t e s . They b o t h showed p o l a r i z e d movement w i t h o c c a s i o n a l change i n d i r e c t i o n . They move i n a s i n u o u s manner, t h e i r shape c h a n g i n g a l t e r n a t e l y from t h e round r e s t i n g form t o t h e h a n d - m i r r o r o r w o r m - l i k e form o f t h e i r l o c o m o t o r y phase. Many i n t e r -m e d i a t e forms were a l s o o b s e r v e d . A s s o c i a t i o n between pr e s u m a b l y s e n s i t i z e d l y mphocytes, t r a n s f o r m a t i o n o f lymphocytes i n t o m a c r o p h a g e - l i k e c e l l s , and f u n c t i o n a l d i f f e r e n c e s between v a r i o u s p a r t s o f t h e moving lymphocytes were a l s o n o t e d . The movements o f s e l e c t e d lymphocytes were t r a c e d from c i n e m i c r o g r a p h i c f i l m s , and a measurement o f t h e r a t e o f l o c o m o t i o n showed t h a t mouse lymphocytes move a t a p p r o x i m a t e l y 10 /a/minute. A comparison o f t h e mean r a t e s o f l o c o m o t i o n t h r o u g h i s o g e n i c monolayers between t h e WWV and -Hf lymphocytes s u g g e s t s t h a t t h e m o t i l i t y o f t h e WWV lymphocytes i s somewhat re d u c e d . However, h e t e r o g e n e i t y among m o r p h o l o g i c a l l y s i m i l a r lymphocytes o f t h e same genotype w i t h r e s p e c t t o t h e i r r a t e o f l o c o m o t i o n makes i t d i f f i c u l t -43-to draw s a t i s f a c t o r y conclusions. A decrease in the a b i l i t y of a WWV kidney monolayer to support the a c t i v i t y of either ++ or WWV lymphocytes was also found. I t i s tempting to conclude that the lymphocytes, which share with three other known defective tissues the defects in p r o l i f e r a t i o n and migration, caused by the W mutations are s i m i l a r l y defective, as shown by t h e i r impaired m o t i l i t y i n v i t r o . -44-Appendix A. Computation of t: Test of the hypothesis that the difference between the mean speeds of _++ and WWV lymphocytes on t h e i r isologous monolayer i s zero. Speed (u/min.) Item ++/++ wwv/wwv 9.1 8.6 13.9 7.8 9.1 8.4 11.7 9.9 Observations (y) 13.4 11.2 10.9 10.9 7.4 6.8 10. 5 Sums of y 97.6 52.0 Sample size(n) 6 Mean of y (y) 2 (Sums of y) "10.8 5525,:'8i "27 04V 0S - - =2.1 *1 " Y2 2 (Sums of y) /n 1058.4 450.7 2 Sums of y 1092.5 459.8 SS 34.1 9.1 Pooled sums of squares= 43.2 DP 8 5 Pooled degrees of freedom= 13 Variance of Pooled estimate of population 2 sample (s ) 2 variance (S )= 3.3231 P 1/n l / n 1 + l/n 2= 0.2778 S 2 (l/n,+l/n 0)= 0.9232 p i 2 2 Square root of S (l/n,+l/n„)= 0.9608 p l z t , = 2.1857 c a l . t t a b . p=0.025, df=13 = 2 ' 1 6 0 The p r o b a b i l i t y that the hypothesis i s v a l i d i s therefore less than 0.02 5. - 4 5 -A p p e h d i x . B . A n a l y s i s o f v a r i a n c e : N e s t e d ( h i e r a c h i c a l ) d e s i g n i n o n e - w a y c l a s s i f i c a t i o n w i t h u n e q u a l s a m p l e s i z e s . K i n d o f l y m p h o c y t e -m o n o l a y e r c o m b i n a t i o n L y m p h o c y t e # T o t a l d i s t a n c e t r a v e r s e d p e r l y m p h o c y t e ( T ) N u m b e r o f o b s e r v e d i n t e r v a l s (n) T 2 / n + + l y m p h o c y t e o n + + m o n o l a y e r 1 3 C'Tf\ 4 5 " 6 7 8 9 3 4 . 6 0 3 5 . 9 5 2 5 . 4 5 3 3 . 8 5 3 7 . 5 0 7 4 . 5 0 2 5 . 9 5 5 1 . 4 5 2 0 . 6 5 4 3 3 6 3 9 5 2 3 9 9 5 3 1 6 6 3 9 2 7 . 8 4 3 5 . 9 0 1 6 . 6 1 2 2 . 0 4 3 6 . 0 6 5 8 . 4 2 2 1 . 7 2 4 4 . 9 2 1 0 . 9 3 T : m 3 3 9 . 9 0 n : m 4 4 0 T 2 / n : 2 6 2 . m m 5 7 + + l y m p h o c y t e o n V -i W W m o n o l a y e r 1 2 3 4 5 2 7 . 2 5 7 0 . 7 0 2 2 . 2 0 6 5 . 8 5 4 4 . 5 0 4 0 7 5 3 8 8 0 6 4 1 8 . 5 6 6 6 . 6 5 1 2 . 9 7 5 4 . 2 0 3 0 . 9 4 T : m 2 3 0 . 5 0 n : m 2 9 7 T 2 / n : 1 7 8 . m m 9 0 W W V l y m p h o c y t e o n + + m o n o l a y e r 1 2 3 4 5 6 7 8 9 1 0 3 8 . 4 5 2 9 . 1 5 2 0 . 6 5 3 1 . 2 0 4 7 . 5 0 6 3 . 8 0 1 8 . 3 5 9 2 . 3 0 9 2 . 9 5 2 9 . 8 0 4 4 3 6 3 4 4 4 6 3 9 0 3 6 1 0 6 1 1 9 3 7 3 3 . 6 0 2 3 . 6 0 1 2 . 5 4 2 2 . 1 2 3 5 . 8 1 4 5 . 2 3 9 . 3 5 8 0 . 3 7 7 2 . 6 0 2 4 . 0 0 T : m 4 6 4 . 1 5 n : m 6 0 9 T 2 / n : 3 5 3 . m m 7 5 W W V l y m p h o c y t e v o n W W m o n o l a y e r 1 2 3 4 5 6 7 1 . 7 5 3 6 . 1 0 3 5 . 5 0 2 2 . 2 5 5 5 . 8 0 2 9 . 1 0 1 0 1 5 8 5 9 3 6 7 4 6 0 5 0 . 9 7 2 2 . 4 7 2 1 . 3 6 1 3 . 7 5 4 2 . 0 8 1 4 . 1 1 T : m 2 5 0 . 5 0 n : m 3 8 8 T 2 / n : 1 6 1 . r r v m 7 3 - 4 6 -P R E L I M I N A R Y C A L C U L A T I O N S T y p e o f t o t a l T o t a l o f s q u a r e s N u m b e r o f i t e m s s q u a r e d N u m b e r o f o b s e r v a t i o n s p e r s q u a r e d i t e m T o t a l o f s q u a r e s p e r o b s e r v a t i o n G r a n d ( 1 2 8 5 . 0 5 ) 2 1 1 7 3 4 9 5 2 . 3 4 C o m b i n a t i o n s - - - 9 5 6 . 9 5 I n d i v i d u a l c e l l s - - 9 8 1 . 7 2 O b s e r v a t i o n s 1 1 9 0 . 3 5 1 7 3 4 1 1 1 9 0 . 3 5 G r a n d t o t a l o f s q u a r e : ( 3 3 9 . 9 0 + 2 3 0 . 5 0 + 4 6 4 . 1 5 + 2 5 0 . 5 0 ) = ( 1 2 8 5 . 0 5 ) 2 2 G r a n d t o t a l o f s q u a r e p e r o b s e r v a t i o n : ( 1 2 8 5 . 0 5 ) / 1 7 3 4 = 9 5 2 . 3 4 C o m b i n a t i o n s t o t a l o f s q u a r e p e r o b s e r v a t i o n : ( 2 6 2 . 5 7 + 1 7 8 . 9 0 + 3 5 3 . 7 5 + 1 6 1 . 7 3 ) = 9 5 6 . 9 5 I n d i v i d u a l c e l l s t o t a l o f s q u a r e p e r o b s e r v a t i o n i s o b t a i n e d b y 2 a d d i n g a l l t h e v a l u e s i n t h e c o l u m n T / n o f t h e f o u r c o m b i n a t i o n s , O b s e r v a t i o n s t o t a l o f s q u a r e p e r o b s e r v a t i o n i s d e t e r m i n e d b y a d d i n g a l l t h e s q u a r e d v a l u e s o f t h e o r i g i n a l m e a s u r e m e n t s , t h a t i s t h e d i s t a n c e t r a v e r s e d p e r 2 0 s e c o n d s i n t e r v a l , a n d t h e n d i v i d e d b y o n e . A N A L Y S I S O F V A R I A N C E S o u r c e S S D F M S C o m b i n a t i o n s ( 9 5 6 . 9 5 - 9 5 2 . 3 4 ) = 4 . 1 6 3 1 . 5 3 6 7 I n d i v i d u a l c e l l s w i t h i n c o m b i n a t i o n s ( 9 8 1 . 7 2 - 9 5 6 . 9 5 ) = 2 4 . 7 7 2 6 0 . 9 5 2 7 E r r o r w i t h i n i n d i v i d u a l c e l l s ( 1 1 9 0 . 3 5 - 9 8 1 . 7 2 ) = 2 0 8 . 6 3 1 7 0 4 0 . 1 2 2 4 T o t a l ( 1 1 9 0 . 3 5 - 9 5 2 . 3 4 ) = 2 3 8 . 0 1 1 7 3 3 -47-I n a h i e r a c h i c a l c l a s s i f i c a t i o n t h e a p p r o p r i a t e F - r a t i o i s t h e v a r i a n c e e s t i m a t e ( M S ) o f s u c c e s s i v e t i e r s . T h u s , C o m b i n a t i o n s 1 .53 67 F ( : : ) = - 1 . 6 1 3 0 I n d i v i d u a l c e l l s w i t h i n c o m b i n a t i o n s 0 . 9 5 2 7 ( D F = 3 ; 4 ) N o t s i g n i f i c a n t I n d i v i d u a l c e l l s w i t h i n c o m b i n a t i o n s ^ 0 . 9 527 F ( )= •— = 7 . 7 8 3 5 E r r o r w i t h i n i n d i v i d u a l c e l l s 0 . 1 2 24 ( D F = 2 6 ; 1 7 0 4 ) S i g n i f i c a n t , P < D . 0 5 T h e c o n c l u s i o n o f t h e e x p e r i m e n t i s t h e r e f o r e t h a t i n d i v i d u a l c e l l s d i f f e r s i g n i f i c a n t l y b u t t h a t c o m b i n a t i o n s d o n o t , a r e s u l t t h a t s u g g e s t s h e t e r o g e n e i t y i n t h e r a t e o f l o c o m o t i o n o f i n d i v i d u a l l y m p h o c y t e s . F u r t h e r i n v e s t i g a t i o n i s n e c e s s a r y t o a s c e r t a i n t h e c a u s e s o f t h e v a r i a t i o n a m o n g t h e i n d i v i d u a l c e l l s . L i t e r a t u r e c i t e d - 4 8 -A l t m a n K . I . , E . S . R u s s e l l , K . S a l o m o n & J . K . S c o t t 1 9 5 3 C h e m o p a t h o l o g y o f h e m o g l o b i n s y n t h e s i s i n m i c e w i t h a h e r e d i t a r y a n e m i a . F e d . P r o c . 1 2 , 1 6 8 A l t m a n K . I . & E . S R u s s e l l 1 9 6 4 H e m e synthesisjTin n o r m a l a n d g e n e t i c a l l y a n e m i c m i c e . J . C e l l . C o m p . ^ P h y s i o l . 6 4 , 2 9 3 B a l l a n t y n e J . , F . G . B e c k , L . C . S t r o n g & W . C . Q u e v e d o 1 9 6 2 A n o t h e r a l l e l e a t t h e W l o c u s o f t h e m o u s e . J . H e r e d . 5 2 , 2 0 0 B e n n e t t M . & G . C u d k o w i c z 1 9 6 6 D e f i c i e n t p r o d u c t i o n o f n o n - e r y t h r o i d c e l l s b y t r a n s -p l a n t e d m a r r o w o f a n e m i c W W m i c e . F e d . P r o c . 2 5 , 2 9 6 B e n n e t t M . , G . C u d k o w i c z , R . S . F o s t e r , J r . , & D . M e t c a l f 1 9 6 8 H e m o p o i e t i c p r o g e n i t o r c e l l s o f W a n e m i c m i c e s t u d i e d i n v i v o a n d i n v i t r o . J . C e l l P h y s i o l . 7 1 , 2 1 1 B e r m a n L . 1 9 4 2 O b s e r v a t i o n s o n d r y f i l m s o f c u l t u r e o f l y m p h o i d t i s s u e . A r c h , o f P a t h . 3 3 , 2 9 5 B e r n s t e i n S . E . & E . S . R u s s e l l 1 9 5 9 I m p l a n t a t i o n o f n o r m a l b l o o d - f o r m i n g t i s s u e i n g e n e t i -c a l l y a n e m i c m i c e w i t h o u t X - i r r a d i a t i o n o f t h e h o s t . P r o c . S o c . E x p . B i o l . M e d . 1 0 1 , 7 6 9 B e r n s t e i n S . E . 1 9 6 3 M o d i f i c a t i o n o f r a d i o s e n s i t i v i t y o f g e n e t i c a l l y a n e m i c m i c e b y i m p l a n t a t i o n o f b l o o d - f o r m i n g t i s s u e . R a d . R e s . 2 0 , 6 9 5 B i c h e l J . 1 9 3 9 ! O n t h e c u l t i v a t i o n o f a m o u s e l e u k o s i s i n v i t r o . 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A . 1 9 6 5 L y m p h o c y t e s I n " c e l l s a n d t i s s u e s i n c u l t u r e m e t h o d s b i o l o g y a n d p h y s i o l o g y " v o l u m e # 2 e d i . b y W i l l m e r E . N . T r w e l l 0 . -57-Wilson D. B. 1965 Quantitative studies on the behavior of sensitized 1 lymphocytes i n v i t r o . I. Relationship of the degree of destruction of homologous target c e l l s to the number of lymphocytes and to the time of contact i n culture and consideration of the effects of iso -immune serum. J. Exp. Med. 122, 143 Woodliff H. J. 1964 Blood and bone marrow c e l l culture. London, Eyre & Spottiswoode. Wu A. M., J. E. T i l l , L. Siminovitch & E. A. McCulloch 1967 A c y t o l o g i c a l study of the capacity for d i f f e r e n t i a t i o n of normal hemopoietic colony-forming c e l l s . J. C e l l . Physiol. 69, 177 Wu A. M., B. M., J. E. T i l l , L. Siminovitch & E. A. McCulloch 1968 Cytological evidence for a rel a t i o n s h i p between normal hematopoietic colony-forming c e l l s and c e l l s of the lymphoid system. J. Exp. Med. 127, 455 - 5 8 -E x p l a n a t i o n o f F i g u r e s T i s s u e c u l t u r e o f l y m p h n o d e s f r o m e i t h e r + + o r W W V i n d i v i d u a l s . T h e l i n e d r a w i n g s a r e t r a c i n g o f t h e c e l l o u t l i n e s o b t a i n e d b y p r o j e c t i n g t h e t i m e - l a p s e c i n e m i c r o g r a p h i c f i l m o n p a p e r . T h e i n t e r v a l b e t w e e n e a c h t r a c i n g i s 2 0 s e c o n d s . T h e t o t a l m a g n i -f i c a t i o n s o f t h e t r a c e d p a t h w a y s a n d t h e p h o t o g r a p h s a r e 2 1 5 0 x a n d 2 6 5 0 x r e s p e c t i v e l y . T h e l e t t e r s o n e a c h p h o t o g r a p h c o r r e s -p o n d t o t h o s e a l o n g s i d e t h e t r a c i n g o f t h e p a t h o f t h e l y m p h o c y t e . F i g u r e 1 P h o t o g r a p h s o f a s m a l l l y m p h o c y t e a n d t r a c e o f i t s p a t h i n a c u l t u r e i n w h i c h W W V l y m p h o c y t e s a r e c o m b i n e d w i t h a W W V m o n o l a y e r . T w o d e p o l a r i z e d p h a s e s ( d p ) a n d p r o b a b l y t w o r e g i o n s o f ' c o n s t r i c -t i o n ' ( c n ) a r e l o c a t e d i n t h i s t r a c i n g . F i g u r e s l a , b , d , f , i , a n d j s h o w t h e m o v e m e n t w i t h a ' h a n d -m i r r o r ' s h a p e . F i g u r e s l b a n d c s h o w r e g i o n s o f ' c o n s t r i c t i o n ' w i t h d e f o r m a t i o n o f t h e n u c l e u s o n o n e s i d e o f t h e m o v i n g c e l l . F i g u r e s l e a n d g s h o w t h e r o u n d s h a p e o f t h e c e l l i n i t s n o n -l o c o m o t o r y p h a s e . P s e u d o p o d i a l a c t i v i t y c a n b e s e e n o n a l l s i d e s o f t h e c e l l i n F i g u r e l e . F i g u r e s l h a n d k s h o w a s o m e w h a t r e c t a n g u l a r s h a p e o f t h e l y m p h o -c y t e a t a s t a g e w h e n i t h a s j u s t b e g u n t o m o v e . - 5 9 -F i g u r e 2 W W V / W W V c o m b i n a t i o n a s i n F i g u r e 1. ' C o n s t r i c t i o n ' o f t h e n u c l e u s a r e f r e q u e n t l y o b s e r v e d . T h e r e i s p r o b a b l y o n e d e p o l a r i z e d p h a s e ( d p ) . F i g u r e s 2 e - i s h o w a s e r i e s o f ' c o n s t r i c t i o n s ' a s t h e c e l l m o v e s t h r o u g h o b s t a c l e s . F i g u r e s 2a, d , a n d j s h o w t h e ' h a n d - m i r r o r ' f o r m o f l o c o m o t i o n . F i g u r e 2c s h o w s a s o m e w h a t c o m p r e s s e d f o r m o f ' h a n d - m i r r o E ' s h a p e . F i g u r e 2b s h o w s a ' r e c t a n g u l a r ' f o r m w h i c h i s a s s u m e d a f t e r a d e p o l a r i z e d p h a s e . 5 9 - 6 0 -F i g u r e 3 W W V / W W V c o m b i n a t i o n a s i n F i g u r e 1. T h e l y m p h o c y t e m a k e s a c o m p l e t e c i r c l e w h i c h i t r e p e a t s l a t e r ( n o t s h o w n i n t h e t r a c i n g ) . O n l y o n e d e p o l a r i z e d p h a s e ( d p ) i s s e e n i n t h e e n t i r e p e r i o d o f l o c o m o t i o n . • F i g u r e 3a s h o w s t h e l y m p h o c y t e i n t h e r o u n d e d c o n f i g u r a t i o n o f t h e n o n - l o c o m o t o r y p h a s e . F i g u r e 3b s h o w s a s o m e w h a t r e c t a n g u l a r f o r m . F i g u r e s 3 c - i s h o w a s e r i e s o f c h a n g e s f r o m t h e ' h a n d - m i r r o E 1 f o r m t o o n e w h i c h i s c o n s t r i c t e d a s t h e l y m p h o c y t e m o v e s t h r o u g h a l i m i t e d s p a c e . F i g u r e s 3 j - m s h o w i n c l i n a t i o n o f t h e l y m p h o c y t e f r o m s i d e t o s i d e a s i t m o v e s i n a c i r c u l a r p a t h . - 6 1 -F i g u r e 4 L a r g e l y m p h o c y t e i n a W W V / W W V c o m b i n a t i o n a s i n F i g u r e 1 . T h e l y m p h o c y t e c h a n g e s f r o m t h e ' h a n d - m i r r o r ' f o r m t o t h e ' w o r m - l i k e ' f o r m a s i t m o v e s t h r o u g h a . 1 t u n n e l - l i k e ' 1 s p a c e , a n d r e t u r n s t o t h e ' h a n d - m i r r o r ' f o r m a f t e r w a r d s . F i g u r e s 4 b - i s h o w t w o s e r i e s o f s u c h c h a n g e s . F i g u r e 4 a s h o w s t h e l y m p h o c y t e a s i t s t a r t s t o m o v e a f t e r a p e r i o d o f r e s t j ^ F i g u r e s 4 b a n d f s h o w a l y m p h o c y t e w i t h a b e a n - s h a p e d n u c l e u s a s i t b e g i n s t o s q u e e z e t h r o u g h t h e ' t u n n e l - l i k e ' s p a c e . F i g u r e s 4 c a n d g s h o w t h e l y m p h o c y t e w i t h m o s t o f i t s n u c l e u s p u s h e d i n t o t h i s l i m i t e d s p a c e . F i g u r e 4 d s h o w s a ' w o r m - l i k e ' f o r m w i t h a s o m e w h a t t w i s t e d a n d c y l i n d r i c a l n u c l e u s . . F i g u r e 4 e s h o w s t h a t t h e l y m p h o c y t e h a s r e t u r n e d t o i t s ' h a n d -m i r r o r ' f o r m . F i g u r e 4 h i n d i c a t e s t h a t t h e l y m p h o c y t e h a s s t a r t e d t o i n c l i n e t o o n e s i d e a f t e r p a s s i n g t h r o u g h t h e l i m i t e d s p a c e . F i g u r e 4 i s h o w s t h e m o v e m e n t o f t h e l y m p h o c y t e w i t h i n a l i m i t e d s p a c e . N o t e t h e s o m e w h a t e l o n g a t e d s h a p e a n d d e f o r m a t i o n o f t h e n u c l e u s . 6 1 -62-F i g u r e 5 A ' h y p e r t r o p h i e d ' ++ lymphocyte i n a ++ monolayer. The lympho-c y t e becomes 'worm-like' as i t squeezes t h r o u g h a l i m i t e d space. The v a c u o l e s and t h e l a r g e s i z e o f t h e lymphocyte a r e v e r y p r o -minent f e a t u r e s o f t h e h y p e r t r o p h i e d c o n d i t i o n . F i g u r e 5a shows an o v a l c o n f i g u r a t i o n f o r t h e lymphocyte as i t j u s t s t a r t s t o move. F i g u r e s 5b-e show t h e movement o f t h e lymphocyte t h r o u g h a v e r y s m a l l space.The d e f o r m a t i o n o f t h e n u c l e u s i s v e r y prominent; the r e s u m p t i o n o f the 'hand-mirror' form i s seen i n F i g u r e 5e. F i g u r e s 5 f - j show the movement o f t h e lymphocyte t h r o u g h a s l i g h t -l y l i m i t e d space. The e l o n g a t i o n o f the c e l l i s e v i d e n t , as a r e the c o n s t r i c t i o n s and t w i s t i n g o f t h e c e l l body owing t o t h e o b s t a -c l e s a l o n g s i d e t h e p a t h . F i g u r e s 5f and g show t h e 'worm-like' c o n f i g u r a t i o n w i t h a t w i s t e d and c y l i n d r i c a l body. F i g u r e s 5h and i show a e l o n g a t e d form o f 'hand-mirror' shape and F i g u r e 5j shows t h e u s u a l 'hand-mirror' form. F i g u r e 5k shows the round c o n f i g u r a t i o n o f t h e r e s t i n g lymphocyte. -63-Figure 6 Photographs showing close association of two WWV lymphocytes, or possibly plasmacytes, on a WWV monolayer. The nuclei of these two c e l l s show a s t r i k i n g 'spoke-like' appearance. The c e l l s usually have an oval form when either i n close contact (Figure 6b) or separated (Figure 6c). They may twist against each other to form a somewhat elongated shape (Figure 6a). Figure 6d shows the two c e l l s making contact i n t h e i r anterior l a t e r a l regions. 6 3 -64-F i g u r e 7 P h o t o g r a p h s s h o w i n g t h e c l o s e a s s o c i a t i o n a n d s e p a r a t i o n o f 8 W W V p r e s u m a b l y s e n s i t i z e d l y m p h o c y t e s o n a + _ + m o n o l a y e r . T h e c e l l s i n c l o s e c o n t a c t a r e s m a l l a n d m e d i u m - s i z e d l y m p h o c y t e s . T h e y m a y m o v e a r o u n d o n e a n o t h e r i n c l o s e c o n t a c t ( F i g u r e 7 a ) ; t h e y m a y s e p a r a t e i n t o t w o g r o u p s o f a s s o c i a t e d c e l l s ( F i g u r e 7 b ) ; o r t h e y m a y s e p a r a t e f r o m o n e a n o t h e r ( F i g u r e 7 c ) , b u t t h e y e v e n t u a l l y m o v e b a c k t o m e e t o n e a n o t h e r a g a i n . 

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